Recombinant Desulfovibrio vulgaris 3,4-dihydroxy-2-butanone 4-phosphate synthase (ribB)

What is 3,4-dihydroxy-2-butanone 4-phosphate synthase and what is its role in bacterial metabolism?

3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS) is a critical enzyme in the riboflavin (vitamin B2) biosynthesis pathway of microorganisms and plants. In bacteria such as Desulfovibrio vulgaris, this enzyme catalyzes a key step in the synthesis of riboflavin, which serves as a precursor for flavin coenzymes essential for numerous metabolic processes. The enzyme converts ribulose 5-phosphate to 3,4-dihydroxy-2-butanone 4-phosphate, which is subsequently used in the biosynthesis of riboflavin . This reaction requires Mg²⁺ for catalytic activity, and the enzyme typically functions as a homodimer comprised of two identical subunits of approximately 23 kDa each, forming a 47-kDa complex in solution . Given its absence in the human genome and presence in numerous pathogens, DHBPS represents an attractive target for developing novel antibiotics with minimal side effects.

How does DHBPS from Desulfovibrio vulgaris compare to the same enzyme in other bacterial species?

While specific comparative data for Desulfovibrio vulgaris DHBPS is limited in the provided search results, researchers generally approach such comparative analyses by examining sequence homology, structural conservation, and catalytic efficiency across different bacterial species. The DHBPS enzyme from Escherichia coli has been well-characterized structurally through NMR techniques, revealing important details about its active site and catalytic mechanism . When comparing DHBPS enzymes across bacterial species, researchers typically analyze conserved amino acid residues involved in substrate binding and catalysis, as well as potential structural differences that might affect enzyme activity or stability. Such comparative analyses are essential for understanding species-specific adaptations of the enzyme and can inform the development of species-selective inhibitors. These comparisons might be particularly relevant when considering Desulfovibrio species, which have been associated with Parkinson's disease, potentially making their metabolic enzymes targets for therapeutic intervention .

What expression systems are suitable for recombinant production of Desulfovibrio vulgaris DHBPS?

For recombinant production of Desulfovibrio vulgaris DHBPS, researchers typically consider several expression systems based on the specific requirements of their studies. E. coli remains the most common heterologous expression system due to its well-established protocols, rapid growth, and high protein yields. When expressing DHBPS, researchers often use E. coli strains optimized for protein expression, such as BL21(DE3) derivatives, with expression vectors containing strong inducible promoters like T7 . The expression protocols frequently involve optimization of induction conditions, including temperature, inducer concentration, and duration. For structural studies requiring isotope labeling, as demonstrated with E. coli DHBPS, researchers can employ strategies such as residue-specific isotope labeling and protein deuteration to facilitate NMR analysis of larger proteins like DHBPS (47 kDa) . Alternative expression systems, such as Bacillus subtilis or eukaryotic systems, may be considered if E. coli expression results in insoluble or inactive protein, though each system presents its own advantages and challenges for recombinant protein production.

What is the significance of Desulfovibrio bacteria in human health and disease?

Desulfovibrio bacteria, including D. vulgaris and related species, have emerged as potentially significant contributors to human health and disease, particularly neurodegenerative conditions. Recent research has established a strong association between Desulfovibrio bacteria and Parkinson's disease (PD) . In a comparative study, these sulfate-reducing bacteria were found in 80% of PD patients compared to only 40% of healthy controls, with statistical analysis revealing a strong correlation (P = 0.022, Fisher's exact test, Phi value = 0.408) . Furthermore, all PD patients (100%) tested positive for the Desulfovibrio-specific [FeFe]-hydrogenase gene (hydA), compared to only 65% of healthy controls, with this gene presence strongly correlated with PD (P = 0.008, Phi value = 0.461) . The potential pathogenic mechanism may involve these bacteria's production of hydrogen sulfide (H₂S) and other neurotoxic metabolites that could contribute to neuroinflammation and neurodegeneration. Additionally, researchers have observed that the quantity of Desulfovibrio bacteria in fecal samples correlates with the severity of Parkinson's disease, suggesting a potential dose-dependent relationship between bacterial load and disease progression .

What structural characterization techniques are most effective for studying recombinant Desulfovibrio vulgaris DHBPS?

For comprehensive structural characterization of recombinant Desulfovibrio vulgaris DHBPS, researchers should consider a multi-technique approach. Nuclear Magnetic Resonance (NMR) spectroscopy has proven particularly valuable for studying DHBPS enzymes, as demonstrated with the E. coli homolog . To overcome size limitations of this 47 kDa dimeric enzyme, researchers can employ protein deuteration strategies combined with residue-specific isotope labeling to achieve resonance assignment and solution structure determination . These advanced NMR approaches are particularly useful for investigating ligand binding and identifying active site residues through chemical shift mapping. X-ray crystallography provides complementary high-resolution structural information, though researchers should be aware that crystal structures might not fully capture the dynamic aspects of the enzyme, particularly in loop regions that can be crucial for catalysis . Small-angle X-ray scattering (SAXS) can provide additional insights into the solution behavior of the enzyme, especially regarding oligomeric state and conformational changes upon substrate binding. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers another valuable approach for mapping dynamic regions and conformational changes that might not be apparent in static structural models. When characterizing the enzyme's metal-binding properties, particularly its interaction with Mg²⁺, techniques such as isothermal titration calorimetry (ITC) and electron paramagnetic resonance (EPR) spectroscopy can provide detailed thermodynamic and structural information.

How can directed evolution be applied to optimize the catalytic efficiency of recombinant DHBPS?

Directed evolution represents a powerful approach for optimizing enzymatic properties, including catalytic efficiency, of recombinant DHBPS. This methodology involves iterative rounds of genetic diversification followed by selection or screening for desired properties . For DHBPS optimization, researchers could begin with the design of a high-throughput screening assay that directly measures enzyme activity, possibly through colorimetric or fluorescent detection of product formation or substrate consumption. The genetic diversification phase typically employs methods such as error-prone PCR, DNA shuffling, or site-saturation mutagenesis to generate libraries of enzyme variants . As observed in other enzyme evolution studies, initial rounds of directed evolution often focus on identifying beneficial mutations outside the active site that might reinforce the enzyme's structure without directly affecting substrate binding . Subsequent rounds can then target residues closer to the active site to fine-tune catalytic parameters. Researchers should be aware that evolutionary improvements often follow a pattern of diminishing returns, with early mutations providing large improvements while later mutations primarily reinforce the effects of earlier changes . To maximize the benefits of directed evolution, researchers might consider a combined approach that integrates computational design with experimental evolution, as computational methods can provide a rational starting point with at least minimal activity that can then be optimized through directed evolution . Through this iterative process, researchers have achieved improvements in catalytic efficiency of designed enzymes by factors of hundreds or even thousands .

What are the challenges in determining the exact catalytic mechanism of Desulfovibrio vulgaris DHBPS?

Elucidating the precise catalytic mechanism of Desulfovibrio vulgaris DHBPS presents several significant challenges. Foremost among these is the complexity of the reaction itself, which involves multiple steps including substrate binding, chemical transformation, and product release. The enzyme must effectively coordinate each of these steps while maintaining selectivity and efficiency . Another major challenge involves capturing the dynamic nature of the enzyme during catalysis, as many structural techniques provide only static snapshots that may not fully represent the conformational changes occurring throughout the catalytic cycle. The presence of loop regions, which are often essential for substrate binding and catalysis but can be highly mobile, further complicates structural analysis . Additionally, understanding the precise role of metal cofactors, such as Mg²⁺, requires specialized techniques to determine how they contribute to substrate positioning and activation. Researchers must also consider potential allosteric effects and long-range interactions within the enzyme that might not be immediately apparent from active site analysis alone. These challenges are exemplified by observations in other enzyme systems, where mutations distant from the active site can dramatically alter catalytic efficiency through mechanisms that are difficult to predict or interpret structurally . To overcome these challenges, researchers typically employ an integrated approach combining structural studies (X-ray crystallography, NMR), computational modeling (molecular dynamics, quantum mechanics/molecular mechanics), and functional assays (steady-state and pre-steady-state kinetics, isotope effects) to build a comprehensive understanding of the catalytic mechanism.

How might the function of DHBPS in Desulfovibrio vulgaris relate to the association between Desulfovibrio bacteria and Parkinson's disease?

The potential relationship between DHBPS function in Desulfovibrio vulgaris and the association of these bacteria with Parkinson's disease represents an intriguing research question at the intersection of biochemistry and neurology. While direct evidence linking DHBPS specifically to Parkinson's disease pathogenesis is not presented in the provided search results, several hypothetical mechanisms warrant investigation. As DHBPS catalyzes a critical step in riboflavin biosynthesis, alteration of riboflavin production could potentially impact bacterial metabolism and subsequent production of neurotoxic metabolites. Notably, Desulfovibrio species generate hydrogen sulfide (H₂S) as part of their sulfate-reducing metabolism, and dysregulation of this process has been implicated in neurodegeneration . Researchers might investigate whether inhibition or alteration of DHBPS activity affects H₂S production or other potentially neurotoxic metabolic pathways in these bacteria. Another research avenue involves exploring how riboflavin availability might influence interactions between Desulfovibrio and other gut microbiota, particularly species like Akkermansia muciniphila and Bifidobacterium, which have been found at increased levels in PD patients and are known to degrade mucin . This degradation potentially releases sulfate that can be utilized by Desulfovibrio bacteria, establishing a metabolic interaction network that might contribute to disease pathogenesis . Comprehensive investigation would require integrated approaches combining recombinant enzyme studies, bacterial metabolism analysis, microbiome profiling, and animal models of Parkinson's disease to establish potential causal relationships between DHBPS function and neurodegeneration.

What computational approaches are most effective for predicting inhibitors of Desulfovibrio vulgaris DHBPS?

Developing effective computational approaches for predicting inhibitors of Desulfovibrio vulgaris DHBPS requires integration of multiple computational techniques informed by experimental structural and functional data. Structure-based virtual screening represents a foundational approach, utilizing molecular docking to evaluate interactions between potential inhibitors and the enzyme's active site. This method becomes particularly powerful when informed by detailed structural information about substrate binding and catalytic residues, as has been demonstrated with the E. coli DHBPS homolog through NMR chemical shift mapping and site-directed mutagenesis . Researchers should be mindful that computational design methods often "get in the ballpark, but don't get the details completely right," potentially resulting in orders of magnitude difference in predicted versus actual enzymatic activity or inhibition . To improve prediction accuracy, molecular dynamics simulations can account for protein flexibility, particularly in loop regions that might undergo conformational changes during ligand binding but are often treated as rigid in standard docking approaches . Ensemble docking, which considers multiple protein conformations, may better capture the dynamic nature of enzyme-inhibitor interactions. Quantum mechanics/molecular mechanics (QM/MM) calculations provide additional insights into transition states and reaction intermediates, potentially identifying opportunities for transition state analogs as potent inhibitors. Machine learning approaches trained on experimental enzyme-inhibitor interaction data can further refine prediction accuracy, especially when combined with targeted fragment-based design strategies. For optimal results, researchers should implement an iterative approach that alternates between computational prediction and experimental validation, continuously refining the computational model based on experimental outcomes.

What are the optimal conditions for measuring DHBPS enzymatic activity?

Establishing optimal conditions for measuring DHBPS enzymatic activity requires careful consideration of multiple parameters to ensure reliable and reproducible results. Standard assay conditions typically include maintaining the pH between 7.5-8.0 using buffers such as Tris-HCl or HEPES, which provide stability without interfering with the reaction. Since DHBPS requires Mg²⁺ for catalytic activity, researchers should include this divalent cation at concentrations typically ranging from 1-5 mM in the assay buffer . Temperature optimization is crucial, with most enzymatic assays performed at 25-37°C depending on the specific research question and the thermal stability of the recombinant enzyme. For kinetic analysis, researchers should determine appropriate substrate (ribulose 5-phosphate) concentration ranges, typically spanning from 0.1 × KM to 10 × KM, to accurately determine kinetic parameters such as KM, kcat, and kcat/KM. Activity measurement methods include spectrophotometric assays monitoring either substrate consumption or product formation, with the latter often coupled to additional enzymatic reactions that generate detectable chromogenic or fluorogenic products. High-performance liquid chromatography (HPLC) or mass spectrometry can provide more direct quantification of reaction products for verification of spectrophotometric results. Researchers should also establish appropriate enzyme concentrations that yield linear reaction rates within the measurement timeframe, typically aiming for 5-20% substrate conversion to maintain initial velocity conditions. Control reactions lacking substrate or enzyme should be included to account for background signals, and appropriate statistical analysis should be applied to experimental replicates to ensure data reliability.

How can researchers effectively purify recombinant Desulfovibrio vulgaris DHBPS while maintaining enzymatic activity?

Effective purification of recombinant Desulfovibrio vulgaris DHBPS with preserved enzymatic activity requires a carefully optimized protocol addressing multiple critical factors. After expression in a suitable system such as E. coli, cells should be lysed under gentle conditions, typically using buffer systems containing 20-50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0), 100-300 mM NaCl, and 5-10% glycerol to stabilize the enzyme. Including protease inhibitors and conducting all purification steps at 4°C helps minimize protein degradation. Since DHBPS requires Mg²⁺ for activity, researchers should consider including 1-5 mM MgCl₂ in purification buffers to maintain the enzyme's native conformation and activity . For initial capture, affinity chromatography using tags such as His₆ or GST provides efficient enrichment, though researchers should verify that the chosen tag does not interfere with enzymatic activity or structural integrity. Tag removal using specific proteases may be necessary for certain applications, particularly structural studies. Further purification typically employs ion exchange chromatography to separate the target enzyme from remaining contaminants based on charge differences, followed by size exclusion chromatography to achieve final purification and confirm the enzyme's dimeric state in solution . Throughout the purification process, researchers should monitor both protein purity using SDS-PAGE and enzymatic activity using established assays to track recovery and specific activity. For applications requiring highly concentrated enzyme, such as structural studies, researchers should optimize concentration methods to prevent aggregation, potentially including stabilizing additives such as glycerol or low concentrations of reducing agents. For long-term storage, stability studies comparing different conditions (buffer composition, protein concentration, temperature) are essential to maintain enzymatic activity for extended periods.

What NMR techniques are most suitable for studying the structure and dynamics of recombinant DHBPS?

Advanced NMR techniques offer powerful approaches for elucidating both the structure and dynamics of recombinant DHBPS, though the enzyme's relatively large size (47 kDa as a dimer) presents significant challenges that require specialized strategies . Protein deuteration represents a critical technique for studying such large proteins by NMR, as demonstrated with E. coli DHBPS, where incorporation of deuterium in non-exchangeable positions significantly reduces spin relaxation and improves spectral quality . Researchers can combine this with selective protonation or isotopic labeling of specific amino acids to obtain targeted structural information without spectral crowding. Transverse relaxation-optimized spectroscopy (TROSY) techniques further enhance spectral quality for large proteins by selecting the slowest relaxing component of the NMR signal. For structural determination, researchers typically collect a series of multidimensional experiments (¹⁵N-HSQC, HNCO, HNCA, HNCACB, etc.) on appropriately labeled samples to establish backbone and side-chain resonance assignments . Chemical shift mapping represents a particularly valuable approach for identifying ligand binding sites and active site residues, as demonstrated with E. coli DHBPS, where this technique helped identify residues directly involved in substrate binding and catalysis . To study enzyme dynamics, researchers can employ relaxation dispersion experiments to characterize conformational exchange processes occurring on microsecond to millisecond timescales, potentially revealing catalytically relevant motions. Residual dipolar coupling (RDC) measurements provide additional information about the relative orientation of structural elements, while paramagnetic relaxation enhancement (PRE) can probe longer-range interactions within the enzyme structure. These advanced NMR approaches, when integrated with complementary techniques such as X-ray crystallography and computational modeling, provide comprehensive insights into both the structural and dynamic features that underlie DHBPS function.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of DHBPS?

Site-directed mutagenesis represents a powerful approach for dissecting the catalytic mechanism of DHBPS through systematic alteration of specific amino acid residues followed by detailed functional characterization. Based on structural data and sequence conservation analysis, researchers typically target residues suspected to participate directly in substrate binding, metal coordination, or chemical catalysis . Conservative mutations (e.g., Asp to Glu) help distinguish between structural roles and catalytic functions, while more dramatic substitutions (e.g., Asp to Ala) can completely eliminate specific functional groups. For each mutant enzyme, researchers should conduct comprehensive kinetic analysis comparing kinetic parameters (KM, kcat, kcat/KM) with the wild-type enzyme under identical conditions to quantify the impact of each mutation. Changes in substrate specificity or the pH-activity profile following specific mutations can provide particularly valuable mechanistic insights. Beyond traditional steady-state kinetics, pre-steady-state kinetic analysis using stopped-flow or rapid quench techniques can identify rate-limiting steps affected by specific mutations. Structural characterization of mutant enzymes, using techniques such as X-ray crystallography or NMR, helps distinguish between mutations that alter catalysis through direct participation in chemical steps versus those that induce conformational changes affecting substrate binding or product release . Researchers can also employ chemical rescue experiments, where exogenous compounds functionally substitute for removed side chains, to further validate the specific roles of individual residues. The integrated analysis of multiple mutants often reveals cooperative networks of residues that work together to achieve efficient catalysis, providing a more complete understanding of the enzyme's catalytic mechanism than could be obtained from individual mutations alone.

How might recombinant DHBPS be used in the development of novel antibiotics targeting Desulfovibrio species?

Recombinant DHBPS offers significant potential in the development of novel antibiotics targeting Desulfovibrio species, particularly given the absence of the riboflavin biosynthetic pathway in humans . This fundamental difference provides an opportunity for selective targeting with minimal off-target effects in human cells. Research strategies might begin with high-throughput screening of chemical libraries against purified recombinant Desulfovibrio vulgaris DHBPS to identify lead compounds with inhibitory activity. Structure-based drug design approaches, informed by detailed structural characterization of the enzyme's active site using techniques such as NMR or X-ray crystallography, can guide the rational optimization of these lead compounds . Researchers should prioritize compounds that demonstrate selective inhibition of bacterial DHBPS without affecting human enzymes, potentially focusing on unique structural features or binding pockets identified through comparative analysis of bacterial and human proteins. Promising inhibitors would then undergo evaluation in whole-cell assays to assess their ability to penetrate bacterial membranes and inhibit growth of Desulfovibrio species. Given the association between Desulfovibrio bacteria and Parkinson's disease, such antibiotics might have therapeutic potential beyond traditional infectious disease applications . Development efforts should include assessment of resistance potential and medicinal chemistry optimization to improve pharmacokinetic properties. Combination approaches might also be considered, where DHBPS inhibitors are paired with compounds targeting other essential pathways in Desulfovibrio species to minimize resistance development and enhance therapeutic efficacy.

How might studying DHBPS contribute to understanding the role of Desulfovibrio in the gut microbiome?

Investigating DHBPS from Desulfovibrio vulgaris could provide valuable insights into how these bacteria establish themselves within the gut microbiome and potentially contribute to conditions like Parkinson's disease. Since DHBPS catalyzes a critical step in riboflavin biosynthesis, characterizing this enzyme could help elucidate how Desulfovibrio species meet their nutritional requirements within the competitive gut environment. Researchers might explore how DHBPS activity and riboflavin availability affect Desulfovibrio growth, persistence, and metabolic activity within complex microbial communities through in vitro gut microbiome models. Given the observed correlation between Desulfovibrio abundance and Parkinson's disease severity, understanding factors that regulate DHBPS expression and activity could potentially identify intervention points for modulating these bacteria's presence or metabolism . Comparative genomics approaches examining DHBPS sequence variation across Desulfovibrio species found in healthy individuals versus those with Parkinson's disease might reveal strain-specific differences with potential pathogenic significance. Researchers could also investigate metabolic interactions between Desulfovibrio and other gut microbes, particularly species like Akkermansia muciniphila and Bifidobacterium, which are increased in Parkinson's disease patients and known to degrade mucin, potentially releasing sulfate that can be utilized by Desulfovibrio bacteria . Such cross-feeding relationships might be influenced by riboflavin availability and thus DHBPS activity. Finally, developing specific inhibitors of Desulfovibrio DHBPS could provide tools for selectively manipulating these bacteria within the gut microbiome, potentially offering therapeutic approaches for conditions associated with Desulfovibrio overgrowth, such as Parkinson's disease .