Recombinant Chromobacterium violaceum Serine hydroxymethyltransferase (glyA)

What is the biochemical function of Serine hydroxymethyltransferase in C. violaceum metabolism?

Serine hydroxymethyltransferase in C. violaceum catalyzes the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene tetrahydrofolate (MTHF) . This enzymatic reaction serves as the principal source of cellular glycine, which is essential for protein synthesis and other metabolic processes . The reaction simultaneously generates MTHF, one of the few one-carbon donors in biosynthetic pathways involved in purine, thymidylate, and methionine synthesis .

In C. violaceum, as in many bacteria, SHMT plays a pivotal role at the intersection of amino acid metabolism and one-carbon metabolism . The enzyme's position in these metabolic networks makes it crucial for cellular growth and reproduction, particularly in rapidly dividing cells during infection processes . The metabolic centrality of SHMT is further highlighted by studies showing that SHMT deficiency induces glycine-auxotrophy in various organisms including E. coli .

The importance of SHMT in C. violaceum may be heightened by the organism's thymidylate synthesis pathway . Many bacteria that contain flavin-dependent thymidylate synthase ThyX (rather than the canonical ThyA) appear to rely exclusively on SHMT for MTHF synthesis . This metabolic arrangement places additional importance on SHMT function in these organisms, potentially making it an attractive target for antimicrobial development .

SHMT's role extends beyond basic metabolism, as it provides essential building blocks for nucleic acid synthesis and methylation reactions, processes that are critical during bacterial adaptation to host environments and biofilm formation .

What structural features characterize C. violaceum SHMT?

While the three-dimensional structure of C. violaceum SHMT has not been specifically reported in the literature, insights can be drawn from the highly conserved structural organization of SHMT across bacterial species . Bacterial serine hydroxymethyltransferases typically organize as homodimers composed of two identical subunits, in contrast to mammalian SHMTs that form homotetramers of four identical subunits .

The enzyme structure is highly conserved evolutionarily, with the active site notably positioned at the interface between two monomers . This structural arrangement facilitates the binding of the pyridoxal 5'-phosphate (PLP) cofactor, which forms a Schiff base (internal aldimine) with a conserved lysine residue in the active site . The PLP binding affinity can vary between species; for instance, H. pylori SHMT showed unexpectedly weak binding affinity for its cofactor .

The three-dimensional structure of bacterial SHMT typically reveals several functional domains, including sites for binding PLP, serine/glycine substrates, and tetrahydrofolate . The catalytic mechanism involves a series of conformational changes during substrate binding and product release . In the case of H. pylori SHMT, the apoprotein structure determined at 2.8Å resolution showed a disordered active site, suggesting structural flexibility that might influence cofactor binding .

For C. violaceum SHMT, comparative structural analysis with other bacterial SHMTs would likely reveal both conserved features essential for catalysis and unique aspects that might influence its specific properties or potential as a drug target . Determining the crystal structure of C. violaceum SHMT would be valuable for understanding any species-specific features that could be exploited for selective inhibitor design.

How might quorum sensing regulate glyA expression and function in C. violaceum?

C. violaceum employs N-hexanoyl-L-homoserine lactone (C6-HSL) as its primary quorum sensing autoinducer, which directs morphological differentiation of cells associated with biofilm development . Atomic force microscopy has revealed that QS autoinducers induce unusual morphological changes in C. violaceum, including membrane invaginations that later develop into polymer matrix extrusions during biofilm formation . These significant cellular changes likely involve extensive metabolic remodeling, potentially including SHMT-dependent pathways.

The wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274 (QS-deficient) exhibit distinct morphological features when examined by atomic force microscopy . The mutant strain lacks the invaginations present in wild-type cells, but these can be restored by exogenous C6-HSL addition . This QS-dependent morphological differentiation may require coordinated regulation of metabolic enzymes, including those involved in amino acid metabolism and one-carbon transfer reactions.

Investigating potential regulatory connections would require multiple experimental approaches . Transcriptomic analysis comparing wild-type and QS-deficient C. violaceum strains could reveal whether glyA expression is influenced by QS systems . Proteomics approaches might identify post-translational modifications of SHMT in response to QS activation . Metabolomic analysis could determine if QS activation alters fluxes through SHMT-dependent pathways, particularly during biofilm formation .

What role does C. violaceum SHMT play in biofilm formation and maintenance?

Biofilm formation is a critical virulence determinant for C. violaceum, but the specific role of SHMT in this process remains largely unexplored despite the potential metabolic connections . The development and maintenance of biofilms require substantial metabolic remodeling and coordination, processes in which SHMT might play significant roles.

Atomic force microscopy has revealed that C. violaceum undergoes remarkable morphological differentiation during biofilm formation . Wild-type cells develop numerous invaginations of the external cytoplasmic membrane, which later transform into polymer matrix extrusions as the biofilm matures . In contrast, QS-deficient mutants are covered with diffusely distributed extracellular substance lacking the organized structure observed in wild-type biofilms . These distinct matrix structures suggest differential regulation of metabolic processes involved in extracellular polymer synthesis.

SHMT's potential role in biofilm formation stems from its position at critical metabolic junctions . The enzyme provides glycine needed for protein synthesis and potentially for extracellular matrix components . Additionally, the one-carbon units generated by SHMT are essential for nucleic acid synthesis during the rapid cell division that occurs in developing biofilms . The metabolic requirements of biofilm formation might therefore place increased demands on SHMT activity.

Investigating SHMT's role in biofilm formation would require multiple experimental approaches . Construction of C. violaceum glyA deletion or conditional knockdown mutants would allow direct assessment of the enzyme's contribution to biofilm development . Microscopic techniques including atomic force microscopy and confocal microscopy could reveal structural differences in biofilms formed by wild-type versus SHMT-deficient strains . Metabolomic analysis comparing planktonic and biofilm growth modes might reveal changes in one-carbon metabolism during biofilm development .

How does C. violaceum SHMT activity compare with orthologs from other bacterial species?

Comparative analysis of SHMT enzymatic properties across bacterial species can reveal unique characteristics that might influence metabolic roles or potential as drug targets . While specific comparisons of C. violaceum SHMT with other bacterial orthologs are not detailed in the current literature, several aspects would be important to investigate.

Kinetic parameters, including substrate affinity (Km values), catalytic efficiency (kcat/Km), and reaction rates under varying conditions, can differ significantly between bacterial SHMTs despite their conserved core function . For instance, comparative studies might reveal whether C. violaceum SHMT exhibits preferential directionality (serine to glycine versus glycine to serine) compared to orthologs from other species . Such differences could reflect adaptation to specific metabolic needs or environmental conditions encountered by C. violaceum.

Cofactor binding represents another potentially variable characteristic . H. pylori SHMT exhibits unusually weak binding affinity for its PLP cofactor, which is reflected in its three-dimensional structure showing a disordered active site . Whether C. violaceum SHMT shares this characteristic or displays different cofactor affinity would be important to determine, as it could influence enzyme stability and activity under various conditions .

Regulation mechanisms, including allosteric regulation by metabolites or post-translational modifications, might also differ between bacterial SHMTs . These regulatory features could allow SHMT activity to respond to specific cellular needs or environmental signals encountered during C. violaceum's lifecycle, particularly during infection or biofilm formation .

Experimental approaches for such comparative studies would include expressing and purifying recombinant SHMTs from C. violaceum and other bacterial species, followed by detailed biochemical characterization under standardized conditions . Structural studies using X-ray crystallography or cryo-electron microscopy could reveal differences in active site architecture that might explain functional variations .

What expression systems are optimal for producing recombinant C. violaceum SHMT?

Producing functional recombinant C. violaceum SHMT requires careful selection of expression systems and optimization of conditions . Based on successful approaches with other bacterial SHMTs, several methodological considerations are particularly important.

Expression vectors and host strains represent the foundation of a successful recombinant protein production strategy . E. coli expression systems, particularly BL21(DE3) or similar strains designed for recombinant protein production, are typically suitable hosts for bacterial SHMT expression . The glyA gene from C. violaceum can be cloned into expression vectors such as the pET series (for T7 RNA polymerase-based expression) or pQE series (for T5 promoter-based expression), which provide strong inducible promoters and appropriate fusion tags for purification . As demonstrated with H. pylori SHMT, expression in pQE60 vector allowed functional complementation of an E. coli ΔglyA strain, confirming production of active enzyme .

Induction conditions significantly impact recombinant SHMT yield and activity . For PLP-dependent enzymes like SHMT, lower induction temperatures (16-25°C rather than 37°C) often improve proper folding and cofactor incorporation . IPTG concentration and induction duration should be optimized experimentally, as demonstrated in the H. pylori SHMT studies where IPTG-induced expression successfully produced functional enzyme . Supplementing growth media with pyridoxine or PLP may enhance production of properly folded, active enzyme by ensuring cofactor availability during protein synthesis .

Purification strategies typically involve affinity chromatography using tags such as poly-histidine, followed by additional purification steps if higher purity is required . For SHMT purification, buffers containing PLP (typically 10-50 μM) are recommended to prevent cofactor loss during purification steps . The inclusion of reducing agents and glycerol in purification buffers can enhance enzyme stability by protecting cysteine residues and preventing protein aggregation .

How can one verify the functional activity of purified C. violaceum SHMT?

Verifying the functional activity of purified recombinant C. violaceum SHMT requires multiple complementary approaches to confirm both structural integrity and catalytic function . Several established methods are particularly valuable for characterizing SHMT activity.

Spectroscopic analysis provides a straightforward initial assessment of proper folding and cofactor binding . PLP-containing enzymes like SHMT exhibit characteristic absorption spectra with peaks around 425-435 nm, corresponding to the internal aldimine formed between PLP and a lysine residue in the enzyme active site . Changes in this spectrum upon addition of substrates (serine or glycine) indicate formation of reaction intermediates, confirming a functionally active enzyme . These spectroscopic properties can provide valuable information about enzyme-PLP-substrate complexes, as demonstrated in studies of bacterial SHMTs showing spectroscopic evidence for formation of characteristic enzyme-PLP-glycine-folate complexes .

Enzymatic activity assays directly measure substrate conversion rates . The canonical SHMT reaction (serine to glycine conversion with concurrent formation of MTHF) can be monitored through various coupled assays or direct product quantification . For instance, HPLC-based methods can quantify the formation of glycine from serine . Alternatively, radioactive assays using 14C-labeled substrates can track carbon transfer between metabolites with high sensitivity .

Functional complementation provides an in vivo verification of enzyme activity . As demonstrated with H. pylori SHMT, recombinant enzyme can be tested for its ability to rescue growth of an E. coli ΔglyA strain on minimal medium without glycine supplementation . This approach confirmed that recombinant H. pylori SHMT was functionally active, allowing the glycine-auxotrophic E. coli ΔglyA strain to grow on minimal medium containing only serine as a glycine source . Similar complementation tests would be valuable for confirming C. violaceum SHMT functionality.

What approaches can be used to investigate the role of glyA in C. violaceum pathogenicity?

Investigating the role of glyA in C. violaceum pathogenicity requires a multifaceted approach combining genetic manipulation, phenotypic characterization, and infection models . Several methodological strategies are particularly valuable for such studies.

Genetic manipulation techniques, including gene deletion or inactivation, provide the foundation for functional studies . Construction of a C. violaceum ΔglyA mutant, similar to the H. pylori ΔglyA strain described in the literature, would allow direct assessment of the gene's contribution to virulence . The H. pylori ΔglyA strain was created by replacing the glyA gene with a non-polar kanamycin resistance cassette through homologous recombination . A similar approach in C. violaceum would involve designing flanking regions for homologous recombination, typically 500bp or longer on each side of the glyA gene . If complete deletion proves lethal, conditional knockdown approaches using inducible antisense RNA or CRISPR interference might be necessary .

Phenotypic characterization should compare wild-type and glyA-deficient strains across multiple parameters . Growth rates in various media compositions would establish basic metabolic requirements and potential auxotrophies . The H. pylori ΔglyA strain exhibited a dramatically slowed growth rate (doubling time increased from 4 hours to 21 hours), indicating the importance of this gene even when not essential . Biofilm formation capacity can be quantified using crystal violet staining or microscopic examination . Production of virulence factors, including the characteristic violacein pigment, should be measured to determine if glyA affects their expression .

Microscopic analysis, particularly atomic force microscopy, can reveal morphological changes associated with glyA deficiency . As demonstrated with quorum sensing mutants of C. violaceum, AFM can detect subtle changes in cell surface properties and extracellular matrix production that may correlate with altered virulence . Comparing wild-type and glyA-deficient C. violaceum using this technique could reveal morphological phenotypes associated with SHMT deficiency .

What strategies can be employed to design selective inhibitors of C. violaceum SHMT?

Designing selective inhibitors of C. violaceum SHMT requires understanding both conserved catalytic features and unique structural elements that differentiate it from human orthologs . Several rational approaches can guide inhibitor development with potential antimicrobial applications.

Structure-based design strategies rely on crystallographic data or homology models of C. violaceum SHMT to identify targetable sites . While the specific structure of C. violaceum SHMT has not been reported, the H. pylori SHMT structure determined at 2.8Å resolution provides valuable insights into bacterial SHMT architecture . This structure revealed a disordered active site and provided structural insights into the enzyme's low affinity for the PLP cofactor . If C. violaceum SHMT shares similar features, this could be exploited for inhibitor design. Molecular docking and virtual screening can identify compounds predicted to bind at the active site or at unique allosteric sites .

Mechanism-based inhibitors target specific aspects of the catalytic cycle . SHMT's dependence on PLP creates opportunities for designing inhibitors that interfere with cofactor binding or the formation of reaction intermediates . The observation that H. pylori SHMT has unusually weak binding affinity for PLP suggests that compounds stabilizing an inactive enzyme configuration might be particularly effective . Transition state analogs mimicking the structure of reaction intermediates often show high affinity and specificity .

Substrate-competitive inhibitors represent another valuable approach . Compounds that compete with serine, glycine, or tetrahydrofolate binding can effectively block enzyme activity . The challenge with this approach is achieving selectivity versus human SHMT isoforms, which requires exploiting subtle differences in substrate binding pockets between bacterial and human enzymes .

Fragment-based drug discovery involves identifying small molecular fragments that bind with low affinity to different sites on the enzyme, then linking or growing these fragments to develop higher-affinity inhibitors . This approach is particularly valuable for enzymes like SHMT with complex active sites and multiple substrate binding pockets .

How can one evaluate inhibitor selectivity between bacterial and human SHMT?

Evaluating inhibitor selectivity between bacterial and human SHMT isoforms is crucial for developing antimicrobial compounds with minimal host toxicity . Multiple complementary approaches can assess this critical parameter at biochemical, structural, and cellular levels.

Comparative biochemical assays provide direct quantification of inhibitor potency against different SHMT orthologs . Recombinant C. violaceum SHMT and human cytosolic and mitochondrial SHMT isoforms can be purified and used in parallel inhibition assays under standardized conditions . Determination of IC50 values or inhibition constants (Ki) allows calculation of selectivity indices (ratio of human to bacterial IC50) . Compounds exhibiting at least 100-fold selectivity for bacterial over human enzymes would be considered promising candidates for further development .

Structural biology approaches reveal atomic-level details of inhibitor binding modes . Co-crystal structures of inhibitors bound to bacterial and human SHMTs can identify specific interactions that contribute to selectivity . While the crystal structure of H. pylori SHMT has been determined at 2.8Å resolution, revealing important structural features including a disordered active site, similar structural information for C. violaceum SHMT would greatly facilitate selective inhibitor design . Comparison with human SHMT structures would highlight exploitable differences in binding sites .

Thermal shift assays (differential scanning fluorimetry) provide a medium-throughput method to assess inhibitor binding across multiple protein targets . By measuring changes in protein thermal stability upon inhibitor binding, this technique can quickly evaluate whether compounds preferentially stabilize bacterial versus human SHMT . This approach is particularly valuable for initial selectivity screening before proceeding to more resource-intensive assays .

Cellular assays assess inhibitor activity and selectivity in a more complex environment . Compounds can be tested for their ability to inhibit growth of C. violaceum while showing minimal toxicity to human cell lines . For SHMT inhibitors, assays in glycine-minimal media can enhance the dependency on SHMT activity and increase assay sensitivity . Metabolomic analyses of treated bacteria and human cells can confirm the mechanism of action by measuring changes in glycine, serine, and one-carbon metabolite levels .

What high-throughput screening methods are suitable for identifying C. violaceum SHMT inhibitors?

High-throughput screening (HTS) for C. violaceum SHMT inhibitors requires robust, sensitive assays that can reliably detect inhibitory activity across large compound libraries . Several complementary screening approaches are particularly suitable for this purpose.

Spectrophotometric activity assays can be adapted to microplate formats for HTS campaigns . The SHMT reaction produces MTHF, which can be coupled to secondary enzymes like methylene tetrahydrofolate dehydrogenase that generate detectable signals (e.g., NADPH production measured at 340 nm) . Optimizing assay components including enzyme concentration, substrate concentrations, and buffer conditions is critical for achieving a suitable signal-to-noise ratio and Z' factor (>0.5) for HTS applications .

Thermal shift assays (differential scanning fluorimetry) offer an activity-independent screening approach . This method measures changes in protein thermal stability upon inhibitor binding using fluorescent dyes that bind to exposed hydrophobic regions as the protein unfolds . Compounds that stabilize or destabilize SHMT are identified as potential binders, though follow-up assays are needed to confirm inhibitory activity . The advantage of this approach is that it does not require enzymatic activity and can detect compounds binding at allosteric sites that might be missed by activity-based screens .

Fluorescence-based assays using specifically designed substrate analogs can provide higher sensitivity than traditional spectrophotometric methods . Development of fluorogenic substrates that yield fluorescent products upon SHMT-catalyzed conversion would enable miniaturized, high-sensitivity screening assays . While such specialized substrates would require custom synthesis, they could significantly enhance screening throughput and sensitivity .

Surface plasmon resonance (SPR) or biolayer interferometry enable real-time monitoring of compound binding to immobilized SHMT . These label-free technologies can determine binding kinetics (association and dissociation rates) and affinity constants, providing detailed characterization of hit compounds . While throughput is lower than plate-based assays, these methods are valuable for confirming and characterizing hits from primary screens .

How can recombinant C. violaceum SHMT be utilized for metabolic pathway analysis?

Recombinant C. violaceum SHMT serves as a valuable tool for investigating fundamental aspects of bacterial one-carbon metabolism and its integration with other metabolic pathways . Several innovative research applications highlight its utility in this domain.

Isotope tracing experiments using labeled substrates with recombinant SHMT can map the flow of one-carbon units through metabolic networks . By incorporating stable isotopes (13C, 15N) in serine or glycine and tracking their distribution in downstream metabolites using mass spectrometry, researchers can quantify flux through SHMT-dependent pathways under various conditions . This approach is particularly valuable for understanding how C. violaceum's one-carbon metabolism responds to environmental changes or contributes to pathogenicity .

Comparative enzymology across bacterial species can reveal evolutionary adaptations in one-carbon metabolism . Recombinant SHMT from C. violaceum can be compared with orthologs from other organisms in terms of kinetic properties, substrate preferences, or regulatory mechanisms . These comparisons provide insights into how metabolic pathways have adapted to different ecological niches and pathogenic lifestyles . For instance, comparing C. violaceum SHMT with enzymes from bacteria that use different thymidylate synthesis pathways (ThyA vs. ThyX) could reveal metabolic adaptations related to folate metabolism .

Integration with systems biology approaches enables contextualization of SHMT function within the broader metabolic network . Purified recombinant SHMT can be used to parameterize metabolic models with experimentally determined kinetic constants . These models can then predict how changes in SHMT activity might affect connected pathways and cellular phenotypes, generating testable hypotheses about metabolic regulation and adaptation .

What challenges exist in crystallizing C. violaceum SHMT and how might they be overcome?

Obtaining high-quality crystals of C. violaceum SHMT for structural studies presents several potential challenges based on experiences with related bacterial SHMTs . Understanding these challenges and implementing strategic solutions is essential for successful structural characterization.

Protein stability and homogeneity often present primary challenges in protein crystallization . Recombinant C. violaceum SHMT may exhibit conformational heterogeneity, particularly if PLP occupancy is variable . The H. pylori SHMT structure revealed unexpectedly weak binding affinity for the PLP cofactor, resulting in a disordered active site . If C. violaceum SHMT shares this characteristic, it could complicate crystallization efforts. Solutions include rigorous purification protocols to achieve high homogeneity, addition of excess PLP during purification and crystallization to ensure full cofactor occupancy, and thermal stability screening to identify optimal buffer conditions .

Protein engineering approaches can address intrinsically disordered regions that hinder crystallization . If specific flexible domains prevent crystal formation, targeted modifications based on secondary structure predictions and sequence alignments with successfully crystallized SHMTs might yield more crystallizable variants . Surface entropy reduction, where clusters of high-entropy surface residues (typically lysines and glutamates) are mutated to alanines, has successfully facilitated crystallization of many recalcitrant proteins .

Alternative crystallization techniques may overcome limitations of traditional vapor diffusion methods . Microseeding, where small crystal fragments are used to nucleate new crystals under optimized conditions, can significantly improve crystal quality . Lipidic cubic phase crystallization, typically used for membrane proteins, has occasionally succeeded with soluble proteins resistant to traditional methods . High-throughput screening using robotic systems allows testing of thousands of conditions with minimal protein consumption, increasing the chances of finding suitable crystallization parameters .

How does C. violaceum glyA deletion affect biofilm structure compared to wild-type strains?

Understanding how glyA deletion affects C. violaceum biofilm structure would provide crucial insights into SHMT's role in bacterial community development and pathogenicity . While specific studies on glyA-deficient C. violaceum biofilms are not detailed in the current literature, informed predictions can be made based on related research.

Atomic force microscopy (AFM) has revealed remarkable morphological features in wild-type C. violaceum biofilms that would serve as important comparison points for glyA mutant studies . Wild-type cells develop numerous invaginations of the external cytoplasmic membrane during early biofilm formation . These invaginations later transform into polymer matrix extrusions as the biofilm matures, creating a highly organized extracellular structure . In contrast, quorum sensing-deficient mutants exhibit a diffusely distributed extracellular substance lacking this organized architecture .

The metabolic functions of SHMT suggest several mechanisms by which glyA deletion might affect biofilm structure . First, SHMT generates glycine, which is required for protein synthesis and potentially for certain extracellular matrix components . Limitation of this amino acid could alter the composition and physical properties of the biofilm matrix . Second, SHMT produces one-carbon units necessary for nucleic acid synthesis during the rapid cell division that occurs in developing biofilms . Deficiencies in these metabolic precursors could affect biofilm density and cellular organization .

Methodological approaches for investigating these effects would include comparative microscopic analysis of wild-type and glyA-deficient biofilms . Atomic force microscopy would reveal nanoscale structural features and mechanical properties as demonstrated with quorum sensing mutants . Confocal laser scanning microscopy with appropriate staining would provide information about biofilm thickness, density, and extracellular matrix distribution . Biochemical analysis of extracted biofilm matrix could identify compositional differences that might explain structural alterations .

The anticipated effects of glyA deletion on C. violaceum biofilm structure would likely include reduced biomass due to slower growth rates (as observed with H. pylori ΔglyA, which showed a five-fold increase in doubling time) . The highly organized polymer matrix extrusions characteristic of wild-type biofilms might be altered or absent, potentially resembling the disorganized extracellular material observed in quorum sensing mutants . These structural changes would have significant implications for biofilm resilience and contribution to pathogenicity .