Recombinant Gluconobacter oxydans Probable GTP-binding protein EngB (engB)

Basic Characterization and Background

• What is the genomic context of engB in Gluconobacter oxydans and how does it differ from other acetic acid bacteria?

The engB gene in Gluconobacter oxydans encodes a probable GTP-binding protein that belongs to the Der/EngB subfamily of GTPases. In the G. oxydans 621H strain, comprehensive genome sequencing and transcriptome analysis has revealed that engB is expressed as part of the essential gene set . Based on RNAseq analysis, engB demonstrates consistent expression levels during growth on various carbon sources . Unlike some other acetic acid bacteria, G. oxydans maintains higher expression of translational machinery genes, including GTP-binding proteins like EngB, especially under high substrate concentration conditions . This is likely related to the organism's unique incomplete oxidation metabolism, which requires robust translational machinery to support its specialized periplasmic oxidation functions .

• How is EngB typically classified within bacterial essential gene families?

EngB is classified as part of the essential bacterial GTPase family that plays crucial roles in ribosome assembly and cellular growth. In the comprehensive transposon mutagenesis studies of G. oxydans B58, EngB was identified among the 550 non-disrupted genes, suggesting its essential nature . Gene ontology (GO) enrichment analysis has demonstrated that EngB clusters with proteins involved in ribosome biogenesis, translation, and GTP binding . In structural genomics studies, EngB has been categorized as a potential antimicrobial drug target due to three key properties: (1) no close human homolog, (2) involvement in essential metabolic pathways, particularly ribosome assembly, and (3) the presence of a deep binding pocket that could accommodate small molecule inhibitors .

• What is known about the basic structural features of EngB in G. oxydans?

The EngB protein from G. oxydans is a relatively small protein (~20-25 kDa) that contains the characteristic G domains (G1-G5) found in GTPase superfamily members. X-ray crystallography studies of EngB, as reported in the Protein Data Bank entry 4DHE, reveal a structure with a central β-sheet surrounded by α-helices, forming the nucleotide-binding pocket . The protein typically contains Switch I and Switch II regions that undergo conformational changes upon GTP binding and hydrolysis. The GTP-binding site contains conserved motifs including a phosphate-binding loop (P-loop), which interacts with the β- and γ-phosphates of GTP. Comparative structural analysis with other bacterial EngB proteins shows high conservation of the GTP-binding domain but variable regions that likely confer species-specific functions .

Expression and Purification Methodologies

• What are the optimal conditions for expressing recombinant EngB from G. oxydans in E. coli expression systems?

For optimal heterologous expression of recombinant G. oxydans EngB in E. coli, the following approach is recommended based on similar protocols used for other G. oxydans proteins:

Expression conditions:

• Host strain: E. coli BL21(DE3) shows higher yield and solubility for G. oxydans proteins compared to other strains

• Expression vector: pET-based vectors with T7 promoter system provide strong, controllable expression

• Growth temperature: Initial growth at 37°C to OD600 of 0.6-0.8, followed by induction at lower temperature (16-18°C) overnight to enhance protein folding

• Induction conditions: 0.1-0.5 mM IPTG typically provides balanced expression levels without inclusion body formation

• Growth medium: Terrific Broth (TB) with glycerol supplement enhances yield

For G. oxydans EngB specifically, codon optimization may be required as G. oxydans has a relatively high GC content (60.8%) which can lead to ribosomal stalling in E. coli . Supplementation with rare codons through specialized strains like Rosetta(DE3) may improve expression of full-length protein .

• What purification strategy yields the highest purity and activity for recombinant G. oxydans EngB?

Based on purification protocols developed for GTP-binding proteins and other G. oxydans recombinant proteins, a multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA for His-tagged EngB

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM MgCl2 (essential for structural integrity of GTPases)

  • Gradient elution: 20-250 mM imidazole

• Intermediate purification: Ion exchange chromatography

  • Buffer: 20 mM Tris-HCl pH 7.5, 5 mM MgCl2, with NaCl gradient (50-500 mM)

• Polishing step: Size exclusion chromatography

  • Buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT

  • Column: Superdex 75 or 200, depending on oligomeric state

Special considerations for EngB include maintaining 5 mM MgCl2 in all buffers to stabilize nucleotide binding and prevent protein aggregation. For activity studies, avoiding EDTA is critical as it chelates the Mg2+ required for GTPase function .

• How can researchers verify the correct folding and activity of purified recombinant EngB?

Verification of correctly folded and active recombinant EngB should include multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure composition

  • Thermal shift assays to assess protein stability (properly folded EngB typically shows Tm ~55-65°C)

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to verify monomeric state and absence of aggregation

• Functional verification:

  • GTPase activity assay using malachite green phosphate detection system

  • Nucleotide binding assessment via isothermal titration calorimetry (ITC)

  • Differential scanning fluorimetry (DSF) with and without GTP/GDP to confirm nucleotide-dependent thermal stability shifts

• Interaction studies:

  • Pull-down assays to verify interactions with known binding partners (such as ribosomal components)

  • Surface plasmon resonance (SPR) to measure binding kinetics with potential interaction partners

Reference values for GTP hydrolysis by properly folded EngB typically show a kcat in the range of 5-15 min-1 with Km values for GTP around 5-20 μM under standard conditions (37°C, pH 7.5) .

Functional Characterization

• What approaches can be used to determine the physiological role of EngB in G. oxydans?

To determine the physiological role of EngB in G. oxydans, a multi-faceted approach is necessary given its essential nature:

• Conditional depletion strategies:

  • Generate strains with inducible promoter-controlled engB expression

  • Use systems like tetracycline-responsive promoters to titrate expression levels

  • Monitor effects on growth, morphology, and metabolic activity at various depletion levels

• Transcriptomic and proteomic profiling:

  • RNA-seq analysis comparing wild-type and EngB-depleted strains to identify affected pathways

  • Ribosome profiling to assess impact on translation

  • Quantitative proteomics to identify proteins with altered abundance upon EngB depletion

• Localization and interaction studies:

  • Fluorescence microscopy with EngB-GFP fusions to determine subcellular localization

  • Co-immunoprecipitation coupled with mass spectrometry to identify interaction partners

  • Bacterial two-hybrid screening to identify direct protein interactors

• Comparative genomics:

  • Analyze engB conservation and genetic context across related species

  • Assess co-evolution patterns with interacting components

Recent studies with GTPases in bacteria indicate close association with ribosome assembly factors, suggesting microscopy co-localization studies with ribosomal markers would be particularly informative .

• How does EngB contribute to stress response in G. oxydans, particularly under high substrate concentration conditions?

EngB likely plays a significant role in the stress response of G. oxydans under high substrate concentrations through several mechanisms:

• Translation regulation during osmotic stress:

  • In environments with high substrate concentrations (>400 g/L glucose), G. oxydans undergoes significant osmotic stress and requires adaptation of protein synthesis machinery

  • RNA-seq data from high osmotic stress conditions (600 g/L glucose) shows differential expression of translation-related genes, which would involve GTPases like EngB

  • As an essential GTPase involved in ribosome function, EngB likely participates in modulating translation to conserve energy and redirect resources during stress adaptation

• Energy conservation:

  • Under high substrate stress, G. oxydans shifts from membrane-bound dehydrogenase activity to enhanced substrate-level phosphorylation

  • EngB is likely involved in the translational reprogramming required for this metabolic shift

  • ATP levels increase significantly after adaptation to high substrate conditions, suggesting coordinated changes in protein synthesis machinery where EngB functions

• Growth phase-dependent regulation:

  • RNA stability studies show significant changes in the half-lives of mRNAs encoding translational machinery components during different growth phases

  • As G. oxydans adapts to high substrate conditions, the stability and function of translation-associated factors including EngB are differentially regulated

The observed adaptation period of G. oxydans to extremely high substrate concentrations (16-20 hours in 600 g/L glucose) likely reflects the time needed for translational machinery reorganization involving EngB and related factors .

• How does G. oxydans EngB compare with its homologs in other bacterial species in terms of structure and function?

Comparative analysis reveals both conserved and distinctive features of G. oxydans EngB relative to homologs in other bacteria:

Structurally, the G. oxydans EngB maintains the canonical GTPase fold with G1-G5 motifs, but has a distinctive Switch II region that may contribute to its adaptation to acidic environments characteristic of acetic acid bacteria. While the core catalytic function in ribosome assembly appears conserved, the G. oxydans EngB shows distinctive interaction patterns with ribosomal components that likely reflect the organism's unique metabolism and ecological niche .

Structural Analysis and Engineering

• What structural features make EngB a potential antibiotic target, and how might researchers exploit these features?

EngB possesses several structural features that make it an attractive antibiotic target:

• Essential binding pocket characteristics:

  • Deep, well-defined GTP-binding pocket with distinctive architecture compared to human GTPases

  • Conserved P-loop region with bacterial-specific residues that differ from eukaryotic counterparts

  • Presence of a hydrophobic cavity adjacent to the GTP-binding site that is absent in human homologs

• Bacterial-specific structural elements:

  • Unique Switch II region conformation that controls GTP hydrolysis

  • Bacterial-specific interface regions that mediate essential protein-protein interactions

  • Species-specific surface loops that could be targeted for selective inhibition

• Strategic approaches for exploitation:

  • Develop nucleotide analogs that selectively bind bacterial EngB over human GTPases

  • Design allosteric inhibitors targeting the bacterial-specific regions adjacent to the active site

  • Create peptidomimetics that disrupt essential EngB protein-protein interactions

Structural analysis of PDB entry 4DHE from the Seattle Structural Genomics Center for Infectious Disease provides a template for structure-based drug design targeting EngB . Computational approaches like molecular dynamics simulations and virtual screening against this structure could identify lead compounds that selectively inhibit bacterial EngB function without affecting human GTPases.

• What mutations in EngB might enhance G. oxydans tolerance to high substrate concentrations?

Based on the understanding of G. oxydans adaptation to high substrate concentrations and the role of translational machinery, several strategic mutations in EngB could potentially enhance tolerance:

• GTPase activity modifications:

  • Mutations in the G1 motif (P-loop) such as A11V or G15S could modulate GTP hydrolysis rates

  • Switch II region modifications (e.g., T42I or G44A) to alter the conformational dynamics during GTP hydrolysis

  • These modifications would potentially tune translation rates under stress conditions

• Protein stability enhancements:

  • Introduction of stabilizing salt bridges in surface-exposed loops

  • Addition of proline residues in flexible regions to reduce entropy and increase thermostability

  • These changes would maintain EngB function under osmotic stress conditions

• Interaction interface modifications:

  • Mutations at predicted ribosome-binding interfaces to optimize interactions under high osmotic pressure

  • Alterations in C-terminal regions to enhance interactions with stress-response factors

Specific residue targets for mutagenesis could be identified through comparative analysis of EngB sequences from extremophilic bacteria that naturally tolerate high solute concentrations. For example, analysis of G. oxydans strains with enhanced tolerance to high D-sorbitol concentrations (>300 g/L) showed mutations in translational proteins that could serve as templates for EngB engineering .

• How can researchers use cryo-EM to understand the macromolecular interactions of EngB in G. oxydans?

Cryo-electron microscopy (cryo-EM) offers powerful approaches for studying EngB's interactions within the cellular context:

• Technical setup for G. oxydans EngB complex visualization:

  • Sample preparation: Gentle cell lysis using spheroplasting rather than sonication to preserve native complexes

  • Grid optimization: Graphene oxide or ultrathin carbon support films to improve particle distribution

  • Data collection: High-magnification imaging (39,000-62,000×) with total electron dose of 40-60 e-/Ų

  • Processing: Focus on classification approaches to separate heterogeneous ribosome assembly states

• Specific experiments and expected outcomes:

  • Direct visualization of EngB-ribosome intermediates at various assembly stages

  • Identification of conformational changes in EngB upon GTP hydrolysis

  • Mapping of interaction networks between EngB and other assembly factors

• Comparative approaches:

  • Side-by-side analysis of EngB-ribosome complexes from wild-type G. oxydans versus stress-adapted strains

  • Examination of how osmotic stress affects the dynamics of EngB-mediated ribosome assembly

Advanced Research Applications

• How can CRISPR-interference be adapted for studying essential genes like engB in G. oxydans?

CRISPR interference (CRISPRi) can be strategically adapted for studying essential genes like engB in G. oxydans through the following approaches:

• System optimization for G. oxydans:

  • Use of a catalytically dead Cas9 (dCas9) under control of a titratable promoter like P2703 or P0943, which show strong activity in G. oxydans

  • Design of sgRNAs targeting the non-template strand of engB at varying distances from the transcription start site to achieve different levels of repression

  • Implementation of an inducible system using either tetracycline or rhamnose-inducible promoters compatible with G. oxydans

• Experimental design for partial depletion studies:

  • Titration experiments with varying inducer concentrations to achieve 20-80% reduction in EngB levels

  • Time-course studies to identify primary versus secondary effects of EngB depletion

  • Combination with reporter systems to monitor cellular responses to EngB depletion

• Genetic background considerations:

  • Implementation in wild-type versus stress-adapted G. oxydans strains

  • Combination with mutations in stress response pathways to identify genetic interactions

A significant advantage of CRISPRi for studying engB in G. oxydans is the ability to achieve partial knockdown rather than complete knockout, allowing observation of phenotypes that would be lethal in deletion mutants. Based on similar approaches in other bacteria, targeting the region 50-100 bp downstream of the engB transcription start site should achieve 70-90% reduction in expression without completely eliminating the protein .

• What proteomics approaches would be most effective for identifying the protein interaction network of EngB in G. oxydans?

To comprehensively identify the protein interaction network of EngB in G. oxydans, a multi-faceted proteomics strategy is recommended:

• Proximity-dependent labeling approaches:

  • APEX2 or TurboID fusions with EngB for in vivo biotinylation of proximal proteins

  • Optimization of labeling conditions for G. oxydans (30°C, pH 4.5-6.0, 2-5 min labeling window)

  • Stringent controls including catalytically inactive EngB fusions to differentiate specific from non-specific interactions

• Quantitative affinity purification coupled with mass spectrometry (qAP-MS):

  • SILAC or TMT labeling to enable quantitative comparison between specific and control pulldowns

  • Crosslinking prior to cell lysis to capture transient interactions

  • Sequential purification using tandem affinity tags to reduce non-specific binding

• Thermal proximity co-aggregation (TPCA):

  • Heating of intact cells to identify proteins that co-aggregate with EngB

  • Analysis of aggregation profiles across temperature gradients to identify true interactors

  • Particularly useful for membrane-associated complexes that may form during ribosome assembly

• Data integration and network analysis:

  • Combination of multiple datasets using computational approaches such as SAINT or CompPASS

  • Validation of key interactions using targeted approaches like bacterial two-hybrid or FRET

  • Network visualization highlighting interaction dynamics under different growth conditions

Expected outcomes would include identification of:

• Core ribosome assembly factors that directly interact with EngB

• Stress response proteins that associate with EngB under high substrate conditions

• Potential regulatory factors that modulate EngB activity in response to metabolic changes

• How can metabolic flux analysis be integrated with EngB functional studies to understand its role in G. oxydans metabolism?

Integrating metabolic flux analysis with EngB functional studies provides a systems-level understanding of its role in G. oxydans metabolism:

• Experimental design for 13C metabolic flux analysis:

  • Culture G. oxydans strains (wild-type and EngB-depleted) with 13C-labeled glucose or sorbitol

  • Implement parallel labeling strategies (1-13C, U-13C, and positionally labeled substrates)

  • Sample at multiple time points during growth and stress adaptation phases

  • Analyze intracellular metabolites and proteinogenic amino acids for isotope distribution

• Integration with EngB activity modulation:

  • Correlate changes in metabolic fluxes with controlled depletion of EngB using CRISPRi

  • Measure GTP/GDP ratios and energy charge (ATP/ADP/AMP) in parallel with flux measurements

  • Assess ribosome assembly status at corresponding time points

• Mathematical modeling and data integration:

  • Develop constraint-based models incorporating translational machinery parameters

  • Use flux balance analysis (FBA) with constraints on protein synthesis rates

  • Implement dynamic FBA to capture the temporal effects of EngB depletion

Expected outcomes include:

• Quantification of the relationship between EngB activity and central carbon metabolism flux distribution

• Identification of metabolic bottlenecks that arise during EngB depletion

• Understanding how translation machinery optimization could enhance G. oxydans productivity

Based on previous metabolic flux studies in G. oxydans, particular attention should be paid to the pentose phosphate pathway and Entner-Doudoroff pathway, which are the primary routes for cytoplasmic glucose metabolism in this organism .

Methodological Approaches for G. oxydans EngB Research

• What are the most effective strategies for site-directed mutagenesis of EngB in G. oxydans?

For site-directed mutagenesis of EngB in G. oxydans, the following optimized strategies are recommended:

• Two-step allelic exchange procedure:

  • Construct a suicide vector containing the mutated engB gene flanked by 500-1000 bp homology arms

  • Use pK19mobsacB as the backbone, which contains kanamycin resistance and sucrose sensitivity markers

  • First selection on kanamycin plates to identify single-crossover events

  • Counter-selection on sucrose plates to identify double-crossover events

  • Verification by PCR and sequencing

• CRISPR-Cas9 approach for G. oxydans:

  • Design sgRNA targeting near the desired mutation site

  • Include repair template with desired mutation and silent mutations that disrupt the PAM site

  • Use temperature-sensitive plasmids for transient expression of Cas9 and guide RNA

  • Screen colonies using HRMA (High Resolution Melting Analysis) followed by sequencing

• Lambda Red recombineering adapted for G. oxydans:

  • Express phage lambda Red proteins (Gam, Bet, Exo) under control of a G. oxydans-compatible promoter

  • Introduce linear DNA fragments containing the desired mutations and selection markers

  • Use FLP recombinase to remove selection markers if necessary

Special considerations for EngB mutagenesis:

• For essential genes like engB, implement mutations on merodiploid strains containing a second wild-type copy

• Use inducible promoters to control expression of the mutant variant

• Consider temperature-sensitive mutations that function normally at permissive temperatures

For creating libraries of EngB variants, methods such as error-prone PCR or site-saturation mutagenesis coupled with high-throughput screening have been successfully applied in G. oxydans for other proteins and could be adapted for EngB studies .

• How can researchers develop a high-throughput screening system to identify G. oxydans EngB variants with enhanced properties?

Developing a high-throughput screening system for G. oxydans EngB variants requires linking EngB function to an easily detectable phenotype:

• Growth-based selection systems:

  • Engineer G. oxydans strains where growth under selective conditions depends on EngB function

  • Create conditions where wild-type EngB provides minimal growth (e.g., high substrate concentrations)

  • Screen for enhanced growth indicating improved EngB variants

  • Implement in 96-well or 384-well format with automated growth monitoring

• Reporter gene-based screening:

  • Develop a system where EngB function is coupled to expression of a reporter gene

  • Use fluorescent proteins optimized for G. oxydans expression (e.g., superfolder GFP)

  • Design the coupling mechanism through ribosome assembly efficiency markers

  • Implement using fluorescence-activated cell sorting (FACS) for ultra-high-throughput screening

• Enzyme activity-based screening:

  • Create an in vitro assay for EngB GTPase activity adaptable to 384-well format

  • Optimize a colorimetric or fluorescent readout for GTP hydrolysis

  • Implement using cell lysates or purified proteins from variant libraries

• Stress resistance screening:

  • Challenge G. oxydans libraries with increasing concentrations of stressors (high substrate, temperature)

  • Select survivors and characterize EngB sequences

  • Implement as sequential rounds of stress with increasing severity

Practical considerations for G. oxydans EngB screening:

• Maintain selection pressure throughout to prevent false positives

• Include multiple controls on each plate to account for plate-to-plate variation

• Validate hits through secondary screens with different readouts

• Sequence full engB gene from hits to identify all mutations

This approach has been successfully implemented for other G. oxydans proteins, particularly membrane-bound dehydrogenases, and similar principles can be applied to EngB engineering .

• What are the challenges and solutions for studying protein-RNA interactions of EngB in G. oxydans?

Studying protein-RNA interactions of EngB in G. oxydans presents several challenges with corresponding solutions:

• Challenges in cell lysis and RNA preservation:

  • G. oxydans has a complex cell envelope with periplasmic oxidation systems

  • Solution: Optimize gentle lysis procedures using lysozyme-EDTA treatment followed by French press at 15,000 psi

  • Implement immediate RNase inhibition using commercial inhibitors plus high salt (>300 mM KCl)

• Challenges in capturing physiologically relevant interactions:

  • EngB-RNA interactions may be transient or context-dependent

  • Solution: Implement in vivo UV crosslinking (254 nm, 400 mJ/cm²) prior to cell harvest

  • Use formaldehyde crosslinking (1%, 10 min, room temperature) as a complementary approach

  • Perform experiments under different growth conditions to capture context-specific interactions

• Challenges in identifying specific RNA targets:

  • EngB likely interacts with highly structured ribosomal RNAs

  • Solution: Implement CLIP-seq (Crosslinking Immunoprecipitation-Sequencing) with EngB-specific antibodies

  • For recombinant studies, use epitope-tagged EngB expressed at near-native levels

  • Include appropriate controls (untagged strains, non-crosslinked samples)

• Challenges in data analysis:

  • Distinguishing direct from indirect interactions

  • Solution: Implement computational approaches like PureCLIP for peak calling

  • Include structural information to validate interaction sites

  • Perform site-directed mutagenesis to confirm key interaction residues

• Challenges specific to G. oxydans:

  • 23S rRNA fragmentation observed in G. oxydans may affect EngB interactions

  • Solution: Characterize rRNA fragmentation patterns in parallel with EngB binding studies

  • Investigate whether EngB plays a role in rRNA processing

These approaches should enable mapping of EngB binding sites on ribosomal RNA and potentially other RNA targets, providing insight into its role in ribosome assembly and potential regulatory functions .

• How can researchers measure the impact of EngB activity on ribosome assembly and protein synthesis rates in G. oxydans?

Measuring the impact of EngB activity on ribosome assembly and protein synthesis in G. oxydans requires multi-faceted approaches:

• Ribosome assembly profiling:

  • Sucrose gradient ultracentrifugation (10-40% gradients) to separate ribosomal subunits and assembly intermediates

  • Quantification of 30S, 50S, 70S, and pre-ribosomal particles under normal and EngB-depleted conditions

  • RNA-seq of gradient fractions to identify accumulating precursors

  • Northern blotting for specific rRNA precursors to track processing defects

• Translation rate measurements:

  • Ribosome profiling to measure ribosome occupancy genome-wide

  • Pulse-labeling with 35S-methionine to measure global protein synthesis rates

  • Puromycin incorporation assays adapted for G. oxydans to measure translation elongation

  • Luciferase reporter systems for measuring translation of specific mRNAs

• In vivo dynamics:

  • Fluorescently tagged ribosomal proteins to track assembly in living cells

  • FRAP (Fluorescence Recovery After Photobleaching) to measure assembly kinetics

  • Co-localization studies with fluorescently tagged EngB and ribosome markers

• Correlation with cellular energetics:

  • Simultaneous measurement of GTP/GDP ratios using targeted metabolomics

  • ATP measurements to assess energy status during EngB depletion

  • NAD+/NADH ratios to understand redox impact of translation impairment

Expected outcomes and interpretation:

• In EngB-depleted cells, expect accumulation of 50S precursors (based on role in other bacteria)

• Translation rates likely show substrate-specific effects (more severe in high concentration media)

• Assembly defects should precede translation rate decreases, confirming direct vs. indirect effects

• Energy charge may initially increase due to reduced consumption, then decrease as metabolism is compromised

These approaches have been successfully applied to study other bacterial GTPases and can be adapted for G. oxydans considering its unique growth requirements and metabolic properties .

• What computational approaches can predict the functional impact of EngB mutations in G. oxydans?

Multiple computational approaches can predict the functional impact of EngB mutations in G. oxydans:

• Structural modeling and molecular dynamics:

  • Homology modeling based on crystal structure (PDB: 4DHE) for regions with sequence differences

  • Molecular dynamics simulations (100-500 ns) to assess conformational changes caused by mutations

  • Free energy calculations (MM/PBSA or FEP) to predict changes in GTP binding affinity

  • Identification of allosteric pathways using methods like PARS (Protein Allosteric and Regulatory Sites)

• Sequence-based approaches:

  • Conservation analysis across acetic acid bacteria to identify functionally critical residues

  • Coevolution analysis using methods like Direct Coupling Analysis to identify residue networks

  • Machine learning classifiers trained on known GTPase mutations to predict functional effects

  • Evolutionary trace methods to map sequence conservation onto structural elements

• Systems biology modeling:

  • Integration of EngB activity with genome-scale metabolic models of G. oxydans

  • Flux balance analysis incorporating constraints on protein synthesis rates

  • Agent-based models of ribosome assembly incorporating EngB kinetic parameters

  • Prediction of growth phenotypes based on altered translation efficiency

• Specific G. oxydans considerations:

  • Account for the acidic growth environment (pH 4-6) in electrostatic calculations

  • Consider interactions with G. oxydans-specific ribosomal proteins and rRNA sequences

  • Model effects under high substrate concentration environments characteristic of industrial applications

To validate computational predictions, prioritize mutations based on:

• Predicted effect size (large > small)

• Consistency across multiple computational approaches

• Location in functionally important regions (GTP-binding pocket, Switch regions, interaction interfaces)

• Evolutionary conservation (highly conserved > variable)

This integrated approach has successfully predicted functional impacts of mutations in other bacterial GTPases and should provide valuable insights for EngB engineering in G. oxydans .