Juodeikis, R., Lee, M., Mayer, M., Mantell, J., Brown, I., Verkade, P., Woolfson, D., Prentice, M., Frank, S. and Warren, M. (2020). Effect of metabolosome encapsulation peptides on enzyme activity, co-aggregation, incorporation and bacterial microcompartment formation. MicrobiologyOpen [Online]. Available at: https://doi.org/10.1002/mbo3.1010.
Metabolosomes, catabolic bacterial microcompartments, are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multi-component protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation prior to the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variation with respect to enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides.
Kieninger, C., Baker, J., Podewitz, M., Wurst, K., Jockusch, S., Lawrence, A., Deery, E., Gruber, K., Liedl, K., Warren, M. and Kräutler, B. (2019). Zinc Substitution of Cobalt in Vitamin B12: Zincobyric acid and Zincobalamin as Luminescent Structural B12-Mimics. Angewandte Chemie International Edition [Online] 131:14710-14714. Available at: https://doi.org/10.1002/anie.201908428.
Replacing the central cobalt ion of vitamin B12 by other metals has been a long‐held aspiration within the B12‐field. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid (Znby) and zincobalamin (Znbl), the Zn‐analogues of the natural cobalt‐corrins cobyric acid and vitamin B12, respectively. The solution structures of Znby and Znbl were studied by NMR‐spectroscopy. Single crystals of Znby were produced, providing the first X‐ray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding CoII‐corrins, making such Zn‐corrins potentially useful for investigations of B12‐dependent processes. The singlet excited state of Znby had a short life‐time, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (ET=190 kJ mol−1) and was found to be an excellent photo‐sensitizer for 1O2 (ΦΔ=0.70).
Dadswell, K., Creagh, S., McCullagh, E., Liang, M., Brown, I., Warren, M., McNally, A., MacSharry, J. and Prentice, M. (2019). Bacterial microcompartment-mediated ethanolamine metabolism in E. coli urinary tract infection. Infection and Immunity [Online]. Available at: https://doi.org/10.1128/IAI.00211-19.
Urinary tract infections (UTIs) are common, in general caused by intestinal Uropathogenic E.coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels competitive overgrowth of intestinal E. coli, both pathogenic enterohaemorrhagic E. coli and commensal strains. During UTI urease negative E. coli thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTI is understudied. We evaluated the role of metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analysed infected urine samples by culture, HPLC, qRT-PCR and genomic sequencing. Ethanolamine concentration in urine was comparable to the most abundant reported urinary amino acid D-serine. Transcription of the eut operon was detected in the majority of urine samples screened containing E. coli. All sequenced UPECs had conserved eut operons while metabolic genotypes previously associated with UTI (dsdCXA, metE) were mainly limited to phylogroup B2. In vitro ethanolamine was found to be utilised as a sole source of nitrogen by UPECs. Metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly, eutE (acetaldehyde dehydrogenase) was required for UPECs to utilise ethanolamine to gain a growth advantage in AUM, suggesting ethanolamine is also utilised as a carbon source. This data suggests urinary ethanolamine is a significant additional carbon and nitrogen source for infecting E. coli.
Kieninger, C., Deery, E., Lawrence, A., Podewitz, M., Wurst, K., Nemoto-Smith, E., Widner, F., Baker, J., Jockusch, S., Kreutz, C., Liedl, K., Gruber, K., Warren, M. and Kräutler, B. (2019). The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B12‐Cofactors for Catalysis. Angewandte Chemie [Online] 58:10756-10760. Available at: https://doi.org/10.1002/ange.201904713.
The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt-ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis of the cobalt-free B12 corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single crystal X-ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt-ions. Consequently, the corrin ligand coordinates cobalt-ions in de-symmetrized ‘entatic’ states, thereby promoting the activation
of B12-cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogs of B12.
Lee, M., Palmer, D. and Warren, M. (2019). Biotechnological advances in bacterial microcompartment technology. Trends in Biotechnology [Online] 37:325-336. Available at: https://doi.org/10.1016/j.tibtech.2018.08.006.
Bacterial microcompartments (BMCs) represent proteinaceous macromolecular
nano-bioreactors that are found in a broad range of bacteria, and which are
associated with either anabolic or catabolic processes. They consist of a semipermeable
outer shell that packages a central metabolic enzyme or pathway,
providing both enhanced flux and protection against toxic intermediates.
Recombinant production of BMCs has led to their repurposing with the incorporation
of altogether new pathways. Deconstruction of BMCs into their component parts has
shown that some individual shell proteins self-associate into filaments that can be
further modified into a cytoplasmic scaffold to which enzymes/proteins can be
targeted. BMCs therefore represent a modular system that is highly suited for the
engineering of biological systems for useful purposes.
Osman, D., Martini, M., Foster, A., Chen, J., Scott, A., Morton, R., Steed, J., Lurie-Luke, E., Huggins, T., Lawrence, A., Deery, E., Warren, M., Chivers, P. and Robinson, N. (2019). Bacterial sensors define intracellular free energies for correct enzyme metalation. Nature Chemical Biology [Online] 15:241-249. Available at: https://doi.org/10.1038/s41589-018-0211-4.
There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: The less competitive the metal, the less favourable the free energy and hence greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt-chelatase for vitamin B12.
Lee, M., Mantell, J., Brown, I., Fletcher, J., Verkade, P., Pickersgill, R., Woolfson, D., Frank, S. and Warren, M. (2018). De novo targeting to the cytoplasmic and luminal side of bacterial microcompartments. Nature Communications [Online] 9:3413. Available at: https://doi.org/10.1038/s41467-018-05922-x.
Bacterial microcompartments, BMCs, are proteinaceous organelles that encase a specific metabolic pathway within a semi-permeable protein shell. Short encapsulation peptides can direct cargo proteins to the lumen of the compartments. However, the fusion of such peptides to non-native proteins does not guarantee encapsulation and often causes aggregation. Here, we report an approach for targeting recombinant proteins to BMCs that utilizes specific de novo coiled-coil protein–protein interactions. Attachment of one coiled-coil module to PduA (a component of the BMC shell) allows targeting of a fluorescent protein fused to a cognate coiled-coil partner. This interaction takes place on the outer surface of the BMC. The redesign of PduA to generate an N-terminus on the luminal side of the BMC results in intact compartments to which proteins can still be targeted via the designed coiled-coil system. This study provides a strategy to display proteins on the surface or within the lumen of the BMCs.
Videira, M., Lobo, S., Silva, L., Palmer, D., Warren, M., Prieto, M., Coutinho, A., Sousa, F., Fernandes, F. and Saraiva, L. (2018). Staphylococcus aureus haem biosynthesis and acquisition pathways are linked through haem monooxygenase IsdG. Molecular Microbiology [Online] 109:385-400. Available at: https://doi.org/10.1111/mmi.14060.
Haem is an essential cofactor in central metabolic pathways in the vast majority of living systems. Prokaryotes acquire haem via haem biosynthesis pathways, and some also utilize haem uptake systems, yet it remains unclear how they balance haem requirements with the paradox that free haem is toxic. Here, using the model pathogen Staphylococcus aureus, we report that IsdG, one of two haem oxygenase enzymes in the haem uptake system, inhibits the formation of haem via the internal haem biosynthesis route. More specifically, we show that IsdG decreases the activity of ferrochelatase and that the two proteins interact both in vitro and in vivo. Further, a bioinformatics analysis reveals that a significant number of haem biosynthesis pathway containing organisms possess an IsdG?homologue and that those with both biosynthesis and uptake systems have at least two haem oxygenases. We conclude that IsdG?like proteins control intracellular haem levels by coupling the two pathways. IsdG is thus a target for the treatment of S. aureusinfections.
Lawrence, A., Nemoto-Smith, E., Deery, E., Baker, J., Schroeder, S., Brown, D., Tullet, J., Howard, M., Brown, I., Smith, A., Boshoff, H., Barry, C. and Warren, M. (2018). Construction of Fluorescent Analogs to Follow the Uptake and Distribution of Cobalamin (Vitamin B 12 ) in Bacteria, Worms, and Plants. Cell Chemical Biology [Online]. Available at: https://doi.org/10.1016/j.chembiol.2018.04.012.
Vitamin B12 is made by only certain prokaryotes yet is required by a number of eukaryotes
such as mammals, fish, birds, worms and Protista, including algae. There is still much to learn
about how this nutrient is trafficked across the domains of life. Herein, we describe ways to
make a number of different corrin analogues with fluorescent groups attached to the main
tetrapyrrole-derived ring. A further range of analogues were also constructed by attaching
similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of
complete and incomplete corrinoids to follow uptake in bacteria, worms and plants. By using
these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis
is able to acquire both cobyric acid and cobalamin analogues, that Caenorhabditis elegans
takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium
sativum are also able to transport B12.
Uddin, I., Frank, S., Warren, M. and Pickersgill, R. (2018). A Generic Self-Assembly Process in Microcompartments and Synthetic Protein Nanotubes. Small [Online] 14:1704020. Available at: https://doi.org/10.1002/smll.201704020.
Bacterial microcompartments enclose a biochemical pathway and reactive intermediate within a protein envelope formed by the shell proteins. Herein, the orientation of the propanediol?utilization (Pdu) microcompartment shell protein PduA in bacterial microcompartments and in synthetic nanotubes, and the orientation of PduB in synthetic nanotubes are revealed. When produced individually, PduA hexamers and PduB trimers, tessellate to form flat sheets in the crystal, or they can self?assemble to form synthetic protein nanotubes in solution. Modelling the orientation of PduA in the 20 nm nanotube so as to preserve the shape complementarity and key interactions seen in the crystal structure suggests that the concave surface of the PduA hexamer faces out. This orientation is confirmed experimentally in synthetic nanotubes and in the bacterial microcompartment produced in vivo. The PduB nanotubes described here have a larger diameter, 63 nm, with the concave surface of the trimer again facing out. The conserved concave surface out characteristic of these nano?structures reveals a generic assembly process that causes the interface between adjacent subunits to bend in a common direction that optimizes shape complementarity and minimizes steric clashes. This understanding underpins engineering strategies for the biotechnological application of protein nanotubes.
Lee, M., Mantell, J., Hodgson, L., Alibhai, D., Fletcher, J., Brown, I., Frank, S., Xue, W., Verkade, P., Woolfson, D. and Warren, M. (2017). Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm. Nature Chemical Biology [Online] 14:142-147. Available at: https://doi.org/10.1038/nchembio.2535.
We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.
Huber, I., Palmer, D., Ludwig, K., Brown, I., Warren, M. and Frunzke, J. (2017). Construction of Recombinant Pdu Metabolosome Shells for Small Molecule Production in Corynebacterium glutamicum. ACS Synthetic Biology [Online] 6:2145-2156. Available at: http://dx.doi.org/10.1021/acssynbio.7b00167.
Bacterial microcompartments have significant potential in the area of industrial biotechnology for the production of small molecules, especially involving metabolic pathways with toxic or volatile intermediates. Corynebacterium glutamicum is an established industrial workhorse for the production of amino acids and has been investigated for the production of diamines, dicarboxylic acids, polymers and biobased fuels. Herein, we describe components for the establishment of bacterial microcompartments as production chambers in C. glutamicum. Within this study, we optimized genetic clusters for the expression of the shell components of the Citrobacter freundii propanediol utilization (Pdu) bacterial compartment, thereby facilitating heterologous compartment production in C. glutamicum. Upon induction, transmission electron microscopy images of thin sections from these strains revealed microcompartment-like structures within the cytosol. Furthermore, we demonstrate that it is possible to target eYFP to the empty microcompartments through C-terminal fusions with synthetic scaffold interaction partners (PDZ, SH3 and GBD) as well as with a non-native C-terminal targeting peptide from AdhDH (Klebsiella pneumonia). Thus, we show that it is possible to target proteins to compartments where N-terminal targeting is not possible. The overproduction of PduA alone leads to the construction of filamentous structures within the cytosol and eYFP molecules are localized to these structures when they are N-terminally fused to the P18 and D18 encapsulation peptides from PduP and PduD, respectively. In the future, these nanotube-like structures might be used as scaffolds for directed cellular organization and pathway enhancement.
Moore, S., Sowa, S., Schuchardt, C., Deery, E., Lawrence, A., Ramos, J., Billig, S., Birkemeyer, C., Chivers, P., Howard, M., Rigby, S., Layer, G. and Warren, M. (2017). Elucidation of the biosynthesis of the methane catalyst coenzyme F430. Nature [Online]:78-82. Available at: http://dx.doi.org/10.1038/nature21427.
Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(II)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen III, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA–CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.
Liang, M., Frank, S., Lünsdorf, H., Warren, M. and Prentice, M. (2017). Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli. Biotechnology Journal [Online] 12:1600415. Available at: http://doi.org/10.1002/biot.201600415.
Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal.
Dailey, H., Dailey, T., Gerdes, S., Jahn, D., Jahn, M., O’Brian, M. and Warren, M. (2017). Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product. Microbiology and Molecular Biology Reviews [Online] 81:e00048-16. Available at: https://doi.org/10.1128/MMBR.00048-16.
The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized.
Mayer, M., Juodeikis, R., Brown, I., Frank, S., Palmer, D., Deery, E., Beal, D., Xue, W. and Warren, M. (2016). Effect of bio-engineering on size, shape, composition and rigidity of bacterial microcompartments. Scientific Reports [Online] 6:36899. Available at: http://dx.doi.org/10.1038/srep36899.
Lobo, S., Videira, M., Pacheco, I., Wass, M., Warren, M., Teixeira, M., Matias, P., Romão, C. and Saraiva, L. (2016). Desulfovibrio vulgarisCbiKPcobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues. Environmental Microbiology [Online] 19:106-118. Available at: http://doi.org/10.1111/1462-2920.13479.
The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiKP and CbiKC, that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiKP confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiKP was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiKP is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiKP, which are absent from all other known bacterial cobaltochelatases, has evolved CbiKP a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter.
Widner, F., Lawrence, A., Deery, E., Heldt, D., Frank, S., Gruber, K., Wurst, K., Warren, M. and Kräutler, B. (2016). Total Synthesis, Structure, and Biological Activity of Adenosylrhodibalamin, the Non-Natural Rhodium Homologue of Coenzyme B12. Angewandte Chemie International Edition [Online] 55:11281-11286. Available at: http://doi.org/10.1002/anie.201603738.
B12 is unique among the vitamins as it is biosynthesized only by certain prokaryotes. The complexity of its synthesis relates to its distinctive cobalt corrin structure, which is essential for B12 biochemistry and renders coenzyme B12 (AdoCbl) so intriguingly suitable for enzymatic radical reactions. However, why is cobalt so fit for its role in B12‐dependent enzymes? To address this question, we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5′‐deoxy‐5′‐adenosylrhodibalamin (AdoRbl). AdoRbl was prepared by de novo total synthesis involving both biological and chemical steps. AdoRbl was found to be inactive in vivo in microbial bioassays for methionine synthase and acted as an in vitro inhibitor of an AdoCbl‐dependent diol dehydratase. Solution NMR studies of AdoRbl revealed a structure similar to that of AdoCbl. However, the crystal structure of AdoRbl revealed a conspicuously better fit of the corrin ligand for RhIII than for CoIII, challenging the current views concerning the evolution of corrins.
Helliwell, K., Lawrence, A., Holzer, A., Kudahl, U., Sasso, S., Kräutler, B., Scanlan, D., Warren, M. and Smith, A. (2016). Cyanobacteria and Eukaryotic Algae Use Different Chemical Variants of Vitamin B12. Current biology : CB [Online] 26:999-1008. Available at: http://doi.org/10.1016/j.cub.2016.02.041.
Eukaryotic microalgae and prokaryotic cyanobacteria are the major components of the phytoplankton. Determining factors that govern growth of these primary producers, and how they interact, is therefore essential to understanding aquatic ecosystem productivity. Over half of microalgal species representing marine and freshwater habitats require for growth the corrinoid cofactor B12, which is synthesized de novo only by certain prokaryotes, including the majority of cyanobacteria. There are several chemical variants of B12, which are not necessarily functionally interchangeable. Cobalamin, the form bioavailable to humans, has as its lower axial ligand 5,6-dimethylbenzimidazole (DMB). Here, we show that the abundant marine cyanobacterium Synechococcus synthesizes only pseudocobalamin, in which the lower axial ligand is adenine. Moreover, bioinformatic searches of over 100 sequenced cyanobacterial genomes for B12 biosynthesis genes, including those involved in nucleotide loop assembly, suggest this is the form synthesized by cyanobacteria more broadly. We further demonstrate that pseudocobalamin is several orders of magnitude less bioavailable than cobalamin to several B12-dependent microalgae representing diverse lineages. This indicates that the two major phytoplankton groups use a different B12 currency. However, in an intriguing twist, some microalgal species can use pseudocobalamin if DMB is provided, suggesting that they are able to remodel the cofactor, whereas Synechococcus cannot. This species-specific attribute implicates algal remodelers as novel and keystone players of the B12 cycle, transforming our perception of the dynamics and complexity of the flux of this nutrient in aquatic ecosystems.
Lee, M., Brown, I., Juodeikis, R., Frank, S. and Warren, M. (2016). Employing bacterial microcompartment technology to engineer a shell-free enzyme-aggregate for enhanced 1,2-propanediol production in Escherichia coli. Metabolic Engineering [Online] 36:48-56. Available at: http://doi.org/10.1016/j.ymben.2016.02.007.
Bacterial microcompartments (BMCs) enhance the breakdown of metabolites such as 1,2-propanediol (1,2-PD) to propionic acid. The encapsulation of proteins within the BMC is mediated by the presence of targeting sequences. In an attempt to redesign the Pdu BMC into a 1,2-PD synthesising factory using glycerol as the starting material we added N-terminal targeting peptides to glycerol dehydrogenase, dihydroxyacetone kinase, methylglyoxal synthase and 1,2-propanediol oxidoreductase to allow their inclusion into an empty BMC. 1,2-PD producing strains containing the fused enzymes exhibit a 245% increase in product formation in comparison to un-tagged enzymes, irrespective of the presence of BMCs. Tagging of enzymes with targeting peptides results in the formation of dense protein aggregates within the cell that are shown by immuno-labelling to contain the vast majority of tagged proteins. It can therefore be concluded that these protein inclusions are metabolically active and facilitate the significant increase in product formation.
Bag, S., Prentice, M., Liang, M., Warren, M. and Roy Choudhury, K. (2016). Classification of polyhedral shapes from individual anisotropically resolved cryo-electron tomography reconstructions. BMC Bioinformatics [Online] 17:234-247. Available at: http://doi.org/10.1186/s12859-016-1107-5.
Cryo-electron tomography (cryo-ET) enables 3D imaging of macromolecular structures. Reconstructed cryo-ET images have a “missing wedge” of data loss due to limitations in rotation of the mounting stage. Most current approaches for structure determination improve cryo-ET resolution either by some form of sub-tomogram averaging or template matching, respectively precluding detection of shapes that vary across objects or are a priori unknown. Various macromolecular structures possess polyhedral structure. We propose a classification method for polyhedral shapes from incomplete individual cryo-ET reconstructions, based on topological features of an extracted polyhedral graph (PG).
We outline a pipeline for extracting PG from 3-D cryo-ET reconstructions. For classification, we construct a reference library of regular polyhedra. Using geometric simulation, we construct a non-parametric estimate of the distribution of possible incomplete PGs. In studies with simulated data, a Bayes classifier constructed using these distributions has an average test set misclassification error of?<?5 % with upto 30 % of the object missing, suggesting accurate polyhedral shape classification is possible from individual incomplete cryo-ET reconstructions. We also demonstrate how the method can be made robust to mis-specification of the PG using an SVM based classifier. The methodology is applied to cryo-ET reconstructions of 30 micro-compartments isolated from E. coli bacteria.
The predicted shapes aren’t unique, but all belong to the non-symmetric Johnson solid family, illustrating the potential of this approach to study variation in polyhedral macromolecular structures.
Gu, S., Sushko, O., Deery, E., Warren, M. and Pickersgill, R. (2015). Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap. Scientific Reports [Online] 5:16943-16952. Available at: http://doi.org/10.1038/srep16943.
CobK catalyzes the essential reduction of the precorrin ring in the cobalamin biosynthetic pathway. The crystal structure of CobK reveals that the enzyme, despite not having the signature sequence, comprises two Rossmann fold domains which bind coenzyme and substrate respectively. The two parallel ?-sheets have swapped their last ?-strands giving a novel sheet topology which is an interesting variation on the Rossmann-fold. The trapped ternary complex with coenzyme and product reveals five conserved basic residues that bind the carboxylates of the tetrapyrrole tightly anchoring the product. A loop, disordered in both the apoenzyme and holoenzyme structures, closes around the product further tightening binding. The structure is consistent with a mechanism involving protonation of C18 and pro-R hydride transfer from NADPH to C19 of precorrin-6A and reveals the interactions responsible for the specificity of CobK. The almost complete burial of the reduced precorrin product suggests a remarkable form of metabolite channeling where the next enzyme in the biosynthetic pathway triggers product release.
Lobo, S., Scott, A., Videira, M., Winpenny, D., Gardner, M., Palmer, M., Schroeder, S., Lawrence, A., Parkinson, T., Warren, M. and Saraiva, L. (2015). Staphylococcus aureus haem biosynthesis: characterisation of the enzymes involved in final steps of the pathway. Molecular Microbiology [Online] 97:472-487. Available at: http://doi.org/10.1111/mmi.13041.
Haem is a life supporting molecule that is ubiquitous in all major kingdoms. In Staphylococcus aureus, the importance of haem is highlighted by the presence of systems both for the exogenous acquisition and endogenous synthesis of this prosthetic group. In this work, we show that in S.?aureus the formation of haem involves the conversion of coproporphyrinogen III into coproporphyrin III by coproporphyrin synthase HemY, insertion of iron into coproporphyrin III via ferrochelatase HemH, and oxidative decarboxylation of Fe-coproporphyrin III into protohaem IX by Fe-coproporphyrin oxidase/dehydrogenase HemQ. Together, this route represents a transitional pathway between the classic pathway and the more recently acknowledged alternative biosynthesis machinery. The role of the haem biosynthetic pathway in the survival of the bacterium was investigated by testing for inhibitors of HemY. Analogues of acifluorfen are shown to inhibit the flavin-containing HemY, highlighting this protein as a suitable target for the development of drugs against S.?aureus. Moreover, the presence of a transitional pathway for haem biosynthesis within many Gram positive pathogenic bacteria suggests that this route has the potential not only for the design of antimicrobials but also for the selective discrimination between bacteria operating different routes to the biosynthesis of haem.
Moore, S., Mayer, M., Biedendieck, R., Deery, E. and Warren, M. (2014). Towards a cell factory for vitamin B12 production in Bacillus megaterium: bypassing of the cobalamin riboswitch control elements. New Biotechnology [Online] 31:553-561. Available at: http://dx.doi.org/10.1016/j.nbt.2014.03.003.
Bacillus megaterium is a bacterium that has been used in the past for the industrial production of vitamin B12 (cobalamin), the anti-pernicious anaemia factor. Cobalamin is a modified tetrapyrrole with a cobalt ion coordinated within its macrocycle. More recently, B. megaterium has been developed as a host for the high-yield production of recombinant proteins using a xylose inducible promoter system. Herein, we revisit cobalamin production in B. megaterium DSM319. We have investigated the importance of cobalt for optimum growth and cobalamin production. The cobaltochelatase (CbiXL) is encoded within a 14-gene cobalamin biosynthetic (cbi) operon, whose gene-products oversee the transformation of uroporphyrinogen III into adenosylcobyrinic acid a,c-diamide, a key precursor of cobalamin synthesis. The production of CbiXL in response to exogenous cobalt was monitored. The metal was found to stimulate cobalamin biosynthesis and decrease the levels of CbiXL. From this we were able to show that the entire cbi operon is transcriptionally regulated by a B12-riboswitch, with a switch-off point at approximately 5 nM cobalamin. To bypass the effects of the B12-riboswitch the cbi operon was cloned without these regulatory elements. Growth of these strains on minimal media supplemented with glycerol as a carbon source resulted in significant increases in cobalamin production (up to 200 ?g L?1). In addition, a range of partially amidated intermediates up to adenosylcobyric acid was detected. These findings outline a potential way to develop B. megaterium as a cell factory for cobalamin production using cheap raw materials.
Lobo, S., Lawrence, A., Romão, C., Warren, M., Teixeira, M. and Saraiva, L. (2014). Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM family member. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics [Online] 1844:1238-1247. Available at: http://dx.doi.org/10.1016/j.bbapap.2014.03.016.
An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5?-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S]2 +/1 + centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.
Bali, S., Rollauer, S., Roversi, P., Raux-Deery, E., Lea, S., Warren, M. and Ferguson, S. (2014). Identification and characterization of the ‘missing’ terminal enzyme for siroheme biosynthesis in ?-proteobacteria. Molecular Microbiology [Online] 92:153-163. Available at: http://dx.doi.org/10.1111/mmi.12542.
It has recently been shown that the biosynthetic route for both the d1-haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1-haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin–ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ?cbiX mutant in P.?denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9?Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in ?-proteobacteria as the terminal enzyme of siroheme biosynthesis.
Lawrence, A., Frank, S., Newnham, S., Lee, M., Brown, I., Xue, W., Rowe, M., Mulvihill, D., Prentice, M., Howard, M. and Warren, M. (2014). Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor. ACS Synthetic Biology [Online] 3:454-465. Available at: http://dx.doi.org/10.1021/sb4001118.
Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.
Palmer, D., Schroeder, S., Lawrence, A., Deery, E., Lobo, S., Saraiva, L., McLean, K., Munro, A., Ferguson, S., Pickersgill, R., Brown, D. and Warren, M. (2014). The structure, function and properties of sirohaem decarboxylase - an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway. Molecular Microbiology [Online] 93:247-261. Available at: http://dx.doi.org/10.1111/mmi.12656.
Bali, S., Palmer, D., Schroeder, S., Ferguson, S. and Warren, M. (2014). Recent advances in the biosynthesis of modified tetrapyrroles: the discovery of an alternative pathway for the formation of heme and heme d 1. Cellular and Molecular Life Sciences [Online] 71:2837-2863. Available at: http://dx.doi.org/10.1007/s00018-014-1563-x.
Hemes (a, b, c, and o) and heme d 1 belong to the group of modified tetrapyrroles, which also includes chlorophylls, cobalamins, coenzyme F430, and siroheme. These compounds are found throughout all domains of life and are involved in a variety of essential biological processes ranging from photosynthesis to methanogenesis. The biosynthesis of heme b has been well studied in many organisms, but in sulfate-reducing bacteria and archaea, the pathway has remained a mystery, as many of the enzymes involved in these characterized steps are absent. The heme pathway in most organisms proceeds from the cyclic precursor of all modified tetrapyrroles uroporphyrinogen III, to coproporphyrinogen III, which is followed by oxidation of the ring and finally iron insertion. Sulfate-reducing bacteria and some archaea lack the genetic information necessary to convert uroporphyrinogen III to heme along the “classical” route and instead use an “alternative” pathway. Biosynthesis of the isobacteriochlorin heme d 1, a cofactor of the dissimilatory nitrite reductase cytochrome cd 1, has also been a subject of much research, although the biosynthetic pathway and its intermediates have evaded discovery for quite some time. This review focuses on the recent advances in the understanding of these two pathways and their surprisingly close relationship via the unlikely intermediate siroheme, which is also a cofactor of sulfite and nitrite reductases in many organisms. The evolutionary questions raised by this discovery will also be discussed along with the potential regulation required by organisms with overlapping tetrapyrrole biosynthesis pathways.
Pang, A., Frank, S., Brown, I., Warren, M. and Pickersgill, R. (2014). Structural Insights into Higher Order Assembly and Function of the Bacterial Microcompartment Protein PduA. Journal of Biological Chemistry [Online] 289:22377-22384. Available at: http://dx.doi.org/10.1074/jbc.M114.569285.
Bacterial microcompartments are large proteinaceous assemblies that are found in the cytoplasm of some bacteria. These structures consist of proteins constituting a shell that houses a number of enzymes involved in specific metabolic processes. The 1,2-propanediol-utilizing microcompartment is assembled from seven different types of shell proteins, one of which is PduA. It is one of the more abundant components of the shell and intriguingly can form nanotubule-like structures when expressed on its own in the cytoplasm of Escherichia coli. We propose a model that accounts for the size and appearance of these PduA structures and underpin our model using a combinatorial approach. Making strategic mutations at Lys-26, Val-51, and Arg-79, we targeted residues predicted to be important for PduA assembly. We present the effect of the amino acid residue substitution on the phenotype of the PduA higher order assemblies (transmission electron microscopy) and the crystal structure of the K26D mutant with one glycerol molecule bound to the central pore. Our results support the view that the hexamer-hexamer interactions seen in PduA crystals persist in the cytoplasmic structures and reveal the profound influence of the two key amino acids, Lys-26 and Arg-79, on tiling, not only in the crystal lattice but also in the bacterial cytoplasm. Understanding and controlling PduA assemblies is valuable in order to inform manipulation for synthetic biology and biotechnological applications.
Lawrence, A., Taylor, S., Scott, A., Rowe, M., Johnson, C., Rigby, S., Geeves, M., Pickersgill, R., Howard, M. and Warren, M. (2014). FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR). Bioscience Reports [Online] 34:345-355. Available at: http://dx.doi.org/10.1042/BSR20140060.
Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I).
Moore, S., Lawrence, A., Biedendieck, R., Deery, E., Frank, S., Howard, M., Rigby, S. and Warren, M. (2013). Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12). Proceedings of the National Academy of Sciences [Online] 110:14906-14911. Available at: http://dx.doi.org/10.1073/pnas.1308098110.
It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification.
Collins, H., Biedendieck, R., Leech, H., Gray, M., Escalante-Semerena, J., McLean, K., Munro, A., Rigby, S., Warren, M. and Lawrence, A. (2013). Bacillus megaterium has both a functional BluB protein required for DMB synthesis and a related flavoprotein that forms a stable radical species. PLoS ONE [Online] 8:e55708. Available at: http://dx.doi.org/10.1371/journal.pone.0055708.
Despite the extensive study of the biosynthesis of the complex molecule B12 (cobalamin), the mechanism by which the lower ligand 5,6-dimethylbenzimidazole (DMB) is formed has remained something of a mystery. However, recent work has identified and characterized a DMB-synthase (BluB) responsible for the oxygen-dependent, single enzyme conversion of FMN to DMB. In this work, we have identified BluB homologs from the aerobic purple, nonsulfur, photosynthetic bacterium Rhodobacter capsulatus and the aerobic soil bacterium Bacillus megaterium and have demonstrated DMB synthesis by the use of a novel complementation assay in which a B12 deficient strain, substituted with the precursor cobinamide is recovered either by the addition of DMB or by the recombinant expression of a bluB gene. The DMB-synthetic activity of the purified recombinant BluB enzymes was further confirmed in vitro by providing the enzyme with FMNH2 and oxygen and observing the formation of DMB by HPLC. The formation of a 4a-peroxyflavin intermediate, the first step in the oxygen dependent mechanism of DMB biosynthesis, is reported here and is the first intermediate in the enzyme catalysed reaction to be demonstrated experimentally to date. The identification and characterization of an FMN-binding protein found on the cobI operon of B. megaterium, CbiY, is also detailed, revealing an FMN-containing enzyme which is able to stabilize a blue flavin semiquinone upon reduction with a 1-electron donor.
Moore, S., Biedendieck, R., Lawrence, A., Deery, E., Howard, M., Rigby, S. and Warren, M. (2013). Characterization of the enzyme CbiH60 involved in anaerobic ring contraction of the cobalamin (vitamin B12) biosynthetic pathway. Journal of Biological Chemistry [Online] 288:297-305. Available at: http://dx.doi.org/10.1074/jbc.M112.422535.
The anaerobic pathway for the biosynthesis of cobalamin (vitamin B(12)) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH(60), that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH(60) was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH(60) demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle.
Azim, N., Deery, E., Warren, M., Erskine, P., Cooper, J., Wood, S. and Akhtar, M. (2013). Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium. Acta Crystallographica Section F Structural Biology and Crystallization Communications [Online] 69:906-908. Available at: http://dx.doi.org/10.1107/S1744309113018526.
The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 22.214.171.124) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution.
Deery, E., Schroeder, S., Lawrence, A., Taylor, S., Seyedarabi, A., Waterman, J., Wilson, K., Brown, D., Geeves, M., Howard, M., Pickersgill, R. and Warren, M. (2012). An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis. Nature Chemical Biology [Online] 8:933-940. Available at: http://dx.doi.org/10.1038/nchembio.1086.
The biosynthesis of many vitamins and coenzymes has often proven difficult to elucidate owing to a combination of low abundance and kinetic lability of the pathway intermediates. Through a serial reconstruction of the cobalamin (vitamin B 12) pathway in Escherichia coli and by His tagging the terminal enzyme in the reaction sequence, we have observed that many unstable intermediates can be isolated as tightly bound enzyme-product complexes. Together, these approaches have been used to extract intermediates between precorrin-4 and hydrogenobyrinic acid in their free acid form and permitted the delineation of the overall reaction catalyzed by CobL, including the formal elucidation of precorrin-7 as a metabolite. Furthermore, a substrate-carrier protein, CobE, that can also be used to stabilize some of the transient metabolic intermediates and enhance their onward transformation, has been identified. The tight association of pathway intermediates with enzymes provides evidence for a form of metabolite channeling.
Saha, K., Webb, M., Rigby, S., Leech, H., Warren, M. and Smith, A. (2012). Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana. Biochemical Journal [Online] 444:227-237. Available at: http://dx.doi.org/10.1042/BJ20111993.
Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.
Moore, S. and Warren, M. (2012). The anaerobic biosynthesis of vitamin B12. Biochemical Society Transactions [Online] 40:581-586. Available at: http://dx.doi.org/10.1042/BST20120066.
Vitamin B12 (cobalamin) is a cobalt-containing modified tetrapyrrole that is an essential nutrient for higher animals. Its biosynthesis is restricted to certain bacteria and requires approximately 30 enzymatic steps for its complete de novo construction. Remarkably, two distinct biosynthetic pathways exist, which are termed the aerobic and anaerobic routes. The anaerobic pathway has yet to be fully characterized due to the inherent instability of its oxygen-sensitive intermediates. Bacillus megaterium, a bacterium previously used for the commercial production of cobalamin, has a complete anaerobic pathway and this organism is now being used to investigate the anaerobic B12 pathway through the application of recent advances in recombinant protein production. The present paper provides a summary of recent findings in the anaerobic pathway and future perspectives.
Kazamia, E., Czesnick, H., Nguyen, T., Croft, M., Sherwood, E., Sasso, S., Hodson, S., Warren, M. and Smith, A. (2012). Mutualistic interactions between vitamin B12 -dependent algae and heterotrophic bacteria exhibit regulation. Environmental Microbiology [Online] 14:1466-1476. Available at: http://dx.doi.org/10.1111/j.1462-2920.2012.02733.x.
Many algae are auxotrophs for vitamin B(12) (cobalamin), which they need as a cofactor for B(12) -dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B(12) -dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M.?loti (MAFF303099), we found that it was able to support the growth of B(12) -dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi-continuous culture, indicating a degree of regulation. However, addition of either vitamin B(12) or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B(12) for growth because it encodes a B(12) -independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B(12) . Co-culturing C.?reinhardtii with M.?loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B(12) -independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom.
Warman, A., Robinson, J., Luciakova, D., Lawrence, A., Marshall, K., Warren, M., Cheesman, M., Rigby, S., Munro, A. and McLean, K. (2012). Characterization of Cupriavidus metallidurans CYP116B1- a thiocarbamate herbicide oxygenating P450-phthalate dioxygenase reductase fusion protein. FEBS Journal [Online] 279:1675-1693. Available at: http://dx.doi.org/10.1111/j.1742-4658.2012.08543.x.
The novel cytochrome P450/redox partner fusion enzyme CYP116B1 from Cupriavidus?metallidurans was expressed in and purified from Escherichia coli. Isolated CYP116B1 exhibited a characteristic Fe(II)CO complex with Soret maximum at 449 nm. EPR and resonance Raman analyses indicated low-spin, cysteinate-coordinated ferric haem iron at both 10 K and ambient temperature, respectively, for oxidized CYP116B1. The EPR of reduced CYP116B1 demonstrated stoichiometric binding of a 2Fe-2S cluster in the reductase domain. FMN binding in the reductase domain was confirmed by flavin fluorescence studies. Steady-state reduction of cytochrome c and ferricyanide were supported by both NADPH/NADH, with NADPH used more efficiently (K(m[NADPH]) = 0.9 ± 0.5 ?M and K(m[NADH]) = 399.1 ± 52.1 ?M). Stopped-flow studies of NAD(P)H-dependent electron transfer to the reductase confirmed the preference for NADPH. The reduction potential of the P450 haem iron was -301 ± 7 mV, with retention of haem thiolate ligation in the ferrous enzyme. Redox potentials for the 2Fe-2S and FMN cofactors were more positive than that of the haem iron. Multi-angle laser light scattering demonstrated CYP116B1 to be monomeric. Type I (substrate-like) binding of selected unsaturated fatty acids (myristoleic, palmitoleic and arachidonic acids) was shown, but these substrates were not oxidized by CYP116B1. However, CYP116B1 catalysed hydroxylation (on propyl chains) of the herbicides S-ethyl dipropylthiocarbamate (EPTC) and S-propyl dipropylthiocarbamate (vernolate), and the subsequent N-dealkylation of vernolate. CYP116B1 thus has similar thiocarbamate-oxidizing catalytic properties to Rhodoccocus erythropolis CYP116A1, a P450 involved in the oxidative degradation of EPTC.
Bali, S., Lawrence, A., Lobo, S., Saraiva, L., Golding, B., Palmer, D., Howard, M., Ferguson, S. and Warren, M. (2011). Molecular hijacking of siroheme for the synthesis of heme and d1 heme. Proceedings of the National Academy of Sciences of the United States of America [Online] 108:18260-18265. Available at: http://dx.doi.org/10.1073/pnas.1108228108.
Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B12, coenzyme F430, and heme d1 underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d1 heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d1 heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.
Lee, M. (2017). Utilisation of Bacterial Microcompartment Technology to Enhance Spatial Organisation in Escherichia Coli.
Enhancing the catalytic efficiency of enzymes has been an aspiration for many years and protein engineering has been quite successful in this area. However, researchers are now turning their attention to the organisation of enzymes within cells to enhance whole pathway productivity. As such, bacterial microcompartments (BMCs) have attracted significant interest due to their ability to not only concentrate enzymes and metabolites but also prevent the release of toxic intermediates into the cell owing to their semi-permeable protein shell. The aim of the research described in this thesis was to develop a number of mechanisms to enhance the spatial organisation of proteins within E. coli and thereby increase metabolic productivity.
The ability of BMC encapsulation peptides to target foreign proteins to a BMC was initially investigated. However, we found that the fusion of BMC encapsulation peptides to the enzymes of a 1,2-propanediol (1,2-PD) synthesis pathway resulted in the aggregation of the tagged enzymes rather than their localisation to a BMC. Nonetheless, the tagged enzymes were found to produce significantly more 1,2-PD than strains producing untagged enzymes, irrespective of the presence of BMCs, suggesting that it is the enzyme aggregate that results in enhanced substrate channelling. Furthermore, we showed that a highly conserved hydrophobic motif is conserved across a wide range of BMC targeting peptides and suggest that it is the amphipathic nature of these peptides that results in aggregation of tagged components.
The ability of PduA, a component of the BMC shell, to form filamentous structures was investigated for its potential as a cytoplasmic scaffold. Targeting proteins to the cytoscaffold was explored through the use of de novo designed coiled-coil peptides. Localising the enzymes pyruvate decarboxylase and alcohol dehydrogenase on this scaffold by tagging them with the cognate coiled-coil peptide resulted in a significant enhancement in ethanol production in comparison to a strain lacking the scaffold. Additionally, we showed that it was possible to locate this intracellular scaffold to the inner membrane of the cell, further demonstrating the flexibility of this system.
Finally, we utilised the same coiled-coil peptide technology to target fluorescent proteins to BMCs, overcoming the aggregation behaviour of the native targeting peptides. Through the re-design of a BMC shell protein we showed targeting occurs to both the external and luminal faces of these BMCs. Taken together, the evidence presented in this thesis provides a number of alternative strategies to not only enhance spatial organisation within a cell but also to increase productivity of engineered metabolic pathways.
Nemoto-Smith, E. (2017). Synthesis of Cobalamin Analogues Using Enzymatic and Chemical Modification Methods, and Subsequent Identification of Cobalamin Localisation in a Variety of Organisms.
Cobalamin, also known as vitamin B12, is an essential nutrient for many different organisms including mammals, fish, birds, nematodes, and a variety of bacteria. However, cobalamin is only synthesised by a few bacteria and archaea. Organisms that cannot synthesise cobalamin de novo must obtain it from their diet. In humans, the cobalamin uptake mechanism has been studied in detail, but in many organisms, such as Caenorhabditis elegans, no method of transport has been defined, and their need for cobalamin is recognised by a cobalamin deficiency phenotype.
Corrin ring modified fluorescent analogues of cobyric acid and ribose conjugated fluorescent analogues of cobalamin were synthesised in order to follow the uptake and localisation of these corrinoids in a variety of organisms. Both the C5 corrin-ring modified and the ribose conjugated analogues were absorbed by Salmonella enterica, using the B12 uptake system (Btu) and could be converted into active coenzyme forms. The imaging of these fluorescent analogues enabled the identification of the coelomocytes in C. elegans as a possible storage cell for cobalamin. However, the C5 cobyric acid analogue was not recognised which suggests that the C. elegans cobalamin transport mechanism is specific for complete corrinoid molecules. Lepidium sativum, garden cress, was shown to take up both cobalamin analogues from the roots and store it in the vacuoles of the cotyledons in seedlings, even though plants have no cobalamin requirement. In contrast, Arabidopsis thaliana did not transport any of the cobalamin analogues.
Cobalamin deficiency has been implicated in impeding disease progression in a number of diseases, such as tuberculosis. The Mycobacterium tuberculosis cobalamin uptake protein, BacA, has only recently been identified, and there is still much to learn about the relationship between M. tuberculosis and cobalamin. Incubations of a cobalamin dependent strain of M. tuberculosis, ?metE, with a selection of cobalamin biosynthesis intermediates showed that cobyric acid is the earliest intermediate to be taken up and converted into the cofactor form. The C5 corrin ring modified cobyric acid fluorescent analogue is also capable of rescuing this ?metE strain, and is taken up faster than the ribose conjugated cobalamin analogue. Overall, the research outlined in this thesis demonstrates that fluorescent corrinoid analogues can be used to follow the journey of cobalamin in a broad range of different organisms and systems.
Juodeikis, R. (2016). Engineering Membranes in Escherichia Coli: The Magnetosome, LemA Protein Family and Outer Membrane Vesicles.
Magnetosomes are membranous organelles found in magnetotactic bacteria (MTB). The organelle consist of ferromagnetic crystals housed within a lipid bilayer chained together by an actin-like filament and allows MTB to orient within magnetic fields. The genetic information required to produce these organelles has been linked to four different operons, encoding for 30 genes. These membranous organelles and the magnetic minerals housed within have various biotechnological applications, therefore enhanced recombinant production of such structures in a model organism holds significant potential. The research described in this thesis is focuses on the production of recombinant magnetosomes in the model organism Escherichia coli.
Cloning the genes involved in the generation of the organelle individually or in various combinations resulted in the construction of over 100 different plasmids, compatible with the model organism. SDS-PAGE and electron microscopy analysis was used to characterise E. coli cells harbouring these constructs. The observation of electron dense particles, arranged in a chain structure, show that magnetosome generation in the model organism is possible, but is highly dependent on the growth conditions used. The need for specific growth conditions is later backed up by the analysis of the maturation of the cytochrome c proteins involved in magnetosome biomineralisation, which can only be correctly processed under certain conditions.
Individual production of two different magnetosome proteins, MamQ or MamY, allowed the generation of various membranous structures in E. coli observed in 48.9% and 56.2% of the whole population of cells respectively. Combinations of these with MamI, MamL or MamB in a variety of combinations led to a variation in the phenotype observed. Bioinformatics analysis of MamQ led to the discovery of a novel membrane restructuring protein family, the LemA protein family, present in a broad range of bacteria. Four different LemA proteins from Bacillus megaterium, Clostridium kluyveri, Brucella melitensis or Pseudomonas aeruginosa were then produced in E. coli and the analysis of the resulting strains revealed the presence of novel intracellular membranous structures which vary in size, form and localisation. Furthermore, when attempts were made to target these proteins for the modification of the outer membrane, a mechanism for increased outer membrane vesicle generation was serendipitously discovered and different effects of these proteins were once again observed.
Together, the results described shows good evidence for recombinant magnetosome production in E. coli and opens a new avenue of membrane engineering in this commonly used organism. Such membranous structures have various biotechnological applications, such as enhanced metabolic engineering potential or specialised lipid vesicle production.
Mayer, M. (2016). A Structural and Biochemical Comparison of Wild-Type and Recombinant Propanediol Utilising Bacterial Microcompartments.
Bacterial microcompartments (BMCs) likely represent the largest protein complex found in bacterial cells (estimated 18,000 subunits). BMCs are associated with specific metabolic processes such as carbon fixation in the case of carboxysomes or carbon utilisation in the case of the 1,2-propanediol utilisation metabolosome. With all BMCs there is still much to learn about the structure and function of these supramolecular assemblies.
In this project the structure and function of the Pdu BMC has been studied through a combination of recombinant DNA technology, proteomics, fluorescence microscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM). Initially, wild type Citrobacter freundii (C. freundii) Pdu BMCs were compared to recombinant BMCs that had been produced in Escherichia coli (E. coli). These were found to have very similar profiles in terms of BMC shell composition, size (120-130 nm on average) and shape. However, recombinant empty BMCs (eBMCs), generated by the coexpression of the genes for just the shell proteins, were found to be distinctly different in that they incorporated more of the first shell protein of the operon, PduA, and were also significantly smaller (55-80 nm on average) than the complete BMCs. This work provides the first recorded application of AFM for the study of BMCs and revealed that the eBMCs were stiffer and less flexible than complete BMCs and it is unclear if differences in shell protein composition or the lack of the internal scaffold is causing the reported biophysical differences.
One key property of the BMCs is their ability to absorb substrate and cofactor molecules to allow the internalised metabolic pathway to operate. Adenosylcobalamin (Ado-B12) is required as a coenzyme for the diol dehydratase conversion of 1,2-propanediol (1,2-PD), but the uptake of cobalamin into the BMC was not shown. By feeding cells exogenous cobalamin, it was possible to demonstrate that cobalamin is specifically partitioned from the cytoplasm into the BMC. Moreover the synthesis of fluorescently labelled versions of cobalamin allowed this partitioning to be followed ex-vivo with purified BMCs.
Palmer, D. (2014). The Alternative Haem Biosynthesis Pathway: Structure, Function and Properties of Sirohaem Decarboxylase.
Haem, a cyclic tetrapyrrole, is found in organisms from all three domains of life. Haem is a prosthetic group for many proteins involved in essential biological processes such as respiration and oxygen transport. Synthesis of haem in eukaryotes and most bacteria follows a well defined route with highly conserved intermediates. However, an alternative haem biosynthesis pathway in Archaea and some bacteria was recently elucidated. This newly discovered pathway utilises sirohaem as a metabolic intermediate rather than a prosthetic group.
The alternative haem biosynthesis pathway is catalysed by the Ahb enzymes A, B, C and D. Initial decarboxylation of sirohaem occurs at the two acetic acid side-chains attached to carbons C12 and C18 to give didecarboxysirohaem, a process catalysed by AhbA and AhbB. Subsequently, the radical SAM enzyme AhbC converts didecarboxysirohaem to Fe-coproporphyrin III. Finally, AhbD catalyses the conversion of Fe-coproporphyrin III into haem in another SAM dependent reaction.
This study focused on the AhbA and AhbB proteins from three sources, Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Methanosarcina barkeri. Purifications of individual recombinantly produced proteins revealed that both AhbA and AhbB are highly unstable. However, low concentrations of D. desulfuricans and D. vulgaris AhbA and AhbB proteins were isolated and were discovered to have decarboxylase activity. Simultaneous overproduction of AhbA and AhbB proteins facilitated the co-purification of stable heteromeric AhbA/B complexes from all three organisms. However, despite their sequence similarities, distinctly different properties were observed between the homologues including differences in oliogmeric state and haem/product binding capabilities. The D. desulfuricans enzyme has been crystallised and its structure has been elucidated in both apo- and product-bound forms. Mutagenesis of the D. desulfuricans complex has provided further information about active site residues and a mechanism of action has been proposed. Finally, novel functional chimeric complexes were produced using the Desulfovibrio proteins.