Recombinant Acinetobacter baumannii Serine hydroxymethyltransferase (glyA)

What is Serine hydroxymethyltransferase (glyA) and what is its role in A. baumannii metabolism?

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. In A. baumannii, this enzyme serves as a critical component of one-carbon metabolism, providing essential one-carbon units for biosynthetic pathways including purine, thymidylate, and methionine synthesis.

The enzyme plays multiple metabolic roles in A. baumannii:

• Facilitates interconversion between serine and glycine, two important amino acids

• Contributes to folate metabolism, impacting nucleotide synthesis and methylation reactions

• Supports bacterial growth by generating precursors for essential cellular components

• May contribute to metabolic adaptations during infection processes

Given that A. baumannii is an opportunistic pathogen associated with hospital-acquired infections including pneumonia, meningitis, bacteremia, and wound infections, the metabolic pathways supported by glyA likely play important roles in bacterial survival within host environments .

What is the structural organization of A. baumannii glyA and how does it compare to homologs from other species?

The crystal structure of glycine hydroxymethyltransferase from Acinetobacter baumannii has been determined and is available in protein structure databases. According to the SWISS-MODEL Repository data, the enzyme consists of approximately 417 amino acids and typically functions as a homodimer, with each monomer containing a PLP cofactor binding site .

Structural analysis reveals:

• A typical SHMT fold consisting of a large domain containing the PLP binding site and a smaller domain involved in substrate binding

• The protein shares approximately 72.29% sequence identity with the structure template 1dfo.1.A, which corresponds to E. coli SHMT

• Key catalytic residues involved in PLP binding and substrate interaction are conserved across bacterial species

The oligomeric state of the functional enzyme appears to be monomeric or dimeric based on available structural data, which differs from some bacterial SHMTs that function as tetramers .

What expression systems are most effective for producing recombinant A. baumannii glyA?

For recombinant production of A. baumannii serine hydroxymethyltransferase, several expression systems have been employed with varying advantages:

E. coli Expression System:

• Most commonly used and generally most effective for bacterial proteins like A. baumannii glyA

• Provides high yield and cost-effectiveness with relatively short production timelines

• Typical strains include BL21(DE3) or Rosetta for accommodating rare codons

• Compatible with pET-series vectors, especially pET28a with His-tag for simplified purification

• Challenges may include protein solubility and proper folding

Alternative Expression Systems:

• Yeast, baculovirus, and mammalian cell systems are also viable options as indicated by commercial sources

• These systems offer improved protein folding and post-translational modifications but with lower yields and higher costs

• Generally unnecessary for A. baumannii glyA unless specific experimental requirements exist

Standard purification protocols typically achieve ≥85% purity as determined by SDS-PAGE , which is sufficient for most biochemical and structural studies. The choice of expression system should be guided by the specific research application, with E. coli being the most practical choice for most academic research scenarios.

What enzymatic assays are most effective for characterizing recombinant A. baumannii glyA activity?

Several complementary approaches can accurately measure the enzymatic activity of recombinant A. baumannii glyA:

Spectrophotometric Assays:

• Coupled Dehydrogenase Assay:

  • Principle: Couples formation of 5,10-methylenetetrahydrofolate to reduction of NADP+ using methylenetetrahydrofolate dehydrogenase

  • Detection: Increased absorbance at 340 nm due to NADPH formation

  • Components: Serine, tetrahydrofolate, NADP+, coupling enzyme, and A. baumannii glyA

  • Conditions: 25-37°C, pH 7.5-8.0, with PLP as cofactor

• Formaldehyde Detection Assay:

  • Principle: When glycine is used as substrate, formaldehyde is formed

  • Detection: Colorimetric detection using Nash reagent (absorbance at 412 nm)

  • Advantages: Simpler setup without coupling enzymes

Radiometric Methods:

• Utilization of 14C-labeled serine or glycine

• Detection of labeled products via scintillation counting

• Provides high sensitivity but requires radioactive material handling

Chromatographic Analysis:

• HPLC or LC-MS/MS quantification of reaction products

• Enables direct measurement of substrate consumption and product formation

• Useful for complex kinetic studies and inhibitor screening

A comprehensive characterization should include determination of:

• Km values for serine, glycine, and tetrahydrofolate

• kcat and catalytic efficiency (kcat/Km)

• pH and temperature optima

• Cofactor (PLP) binding affinity

• Inhibition parameters (Ki) for potential inhibitors

These values represent typical ranges for bacterial SHMTs; specific parameters for A. baumannii glyA should be experimentally determined.

What are the optimal purification strategies for maintaining A. baumannii glyA stability and activity?

Purification of recombinant A. baumannii glyA requires careful consideration of enzyme stability and activity. A comprehensive purification protocol would include:

Buffer Optimization:

• Maintaining pH 7.0-8.0 (typically 50 mM Tris-HCl or phosphate buffer)

• Including glycerol (10-20%) to enhance stability

• Adding reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation

• Including pyridoxal-5'-phosphate (PLP, 50-100 μM) as the essential cofactor

• Considering stabilizing agents like trehalose or sucrose (5-10%)

Chromatography Strategy:

• Affinity Chromatography: For His-tagged protein, use Ni-NTA with imidazole gradient elution

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250-300 mM imidazole

• Ion Exchange Chromatography: For further purification

  • Typically anion exchange (Q-Sepharose) with NaCl gradient

• Size Exclusion Chromatography: Final polishing step

  • Buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT, 50 μM PLP

Activity Preservation Considerations:

• Minimize freeze-thaw cycles (aliquot before freezing)

• Store with glycerol (25-50%) at -80°C

• Consider storage with excess PLP to maintain cofactor saturation

• Monitor activity at each purification step

• Process samples quickly and keep on ice when possible

This purification approach should yield enzyme preparations with ≥85% purity as assessed by SDS-PAGE , suitable for biochemical and structural studies.

How can structural biology techniques be applied to study A. baumannii glyA interactions with substrates and inhibitors?

Structural biology approaches provide crucial insights into enzyme-substrate and enzyme-inhibitor interactions. For A. baumannii glyA, researchers can employ:

X-ray Crystallography:

• Protein Preparation: Highly purified (>95%) A. baumannii glyA

• Crystallization Strategy:

  • Commercial crystallization screens at 10-15 mg/mL protein concentration

  • Vapor diffusion methods (sitting or hanging drop)

  • Crystal optimization through additive screening

• Ligand Studies:

  • Co-crystallization with substrates (serine, glycine, tetrahydrofolate)

  • Co-crystallization or soaking with potential inhibitors

  • Substrate analogs to trap reaction intermediates

• Data Collection and Analysis:

  • High-resolution data collection at synchrotron facilities

  • Structure determination via molecular replacement using the existing crystal structure

  • Careful analysis of active site architecture and ligand binding modes

The available crystal structure of glycine hydroxymethyltransferase from A. baumannii provides an excellent starting point for these studies .

Complementary Techniques:

• Molecular Dynamics Simulations: To study protein flexibility and transient interactions

• Hydrogen-Deuterium Exchange Mass Spectrometry: For conformational dynamics analysis

• NMR Spectroscopy: For studying weaker interactions and protein dynamics

• Thermal Shift Assays: To screen potential ligands and assess stability

These approaches can identify:

• Key residues involved in substrate binding and catalysis

• Conformational changes during the catalytic cycle

• Potential allosteric sites for inhibitor design

• Species-specific features for selective inhibitor development

What genetic manipulation approaches are most effective for studying glyA function in A. baumannii?

Genetic manipulation of A. baumannii presents unique challenges due to its intrinsic antibiotic resistance and genetic recalcitrance. Effective approaches include:

Gene Knockout Strategies:

• Homologous Recombination:

  • Double crossover with antibiotic resistance cassettes

  • Non-antibiotic selection markers like tellurite resistance

  • Sucrose counter-selection using sacB for marker removal

• CRISPR-Cas9 Systems:

  • Adapted CRISPR-Cas9 for A. baumannii genome editing

  • Specific guide RNAs targeting glyA

  • Delivery via electroporation of ribonucleoprotein complexes

  • Particularly useful for clinical isolates with multiple resistance markers

Conditional Gene Expression:

• Inducible Systems:

  • Tetracycline-responsive promoters

  • Temperature-sensitive alleles

  • Particularly important if glyA proves essential under laboratory conditions

• Gene Knockdown Approaches:

  • Antisense RNA expression

  • CRISPRi (CRISPR interference) using catalytically inactive Cas9

  • Enables partial repression rather than complete knockout

Complementation Strategies:

• Expression of wild-type glyA from plasmid in mutant strain

• Site-specific integration for single-copy complementation

• Essential for confirming phenotypes are specifically due to glyA disruption

Transformation Optimization:

• Electroporation with glycine-treated cells

• Growth phase optimization (early to mid-log phase)

• Buffer optimization (10% glycerol with sucrose)

• High voltage (1.8-2.5 kV) with 1 mm gap cuvettes

When studying potentially essential genes like glyA, a stepwise approach is recommended:

• First attempt conditional knockdown to assess viability

• Provide metabolic bypass (glycine supplementation) if needed

• Create complementation strain before attempting complete knockout

• Test phenotypes under various conditions including infection-relevant environments

These genetic approaches provide powerful tools for dissecting glyA's role in A. baumannii physiology and pathogenesis.

How can inhibitors targeting A. baumannii glyA be rationally designed as potential antimicrobial compounds?

Rational design of inhibitors targeting A. baumannii glyA presents a promising approach for antimicrobial development. Key considerations include:

Structure-Based Design Approaches:

• Utilize the crystal structure of A. baumannii glycine hydroxymethyltransferase

• Focus on the active site containing PLP (pyridoxal phosphate) cofactor

• Identify unique structural features compared to mammalian SHMT isozymes

• Employ molecular docking and virtual screening to identify lead compounds

Target Sites Within the Enzyme:

• PLP Binding Site:

  • Design compounds that form covalent adducts with PLP

  • Target the Schiff base linkage between PLP and catalytic lysine

  • Exploit differences in cofactor binding compared to human enzymes

• Substrate Binding Pockets:

  • Design competitive inhibitors mimicking serine, glycine, or tetrahydrofolate

  • Develop transition-state analogs that mimic reaction intermediates

  • Create bisubstrate analogs linking features of multiple substrates

• Allosteric Sites:

  • Identify potential allosteric sites using computational approaches

  • Design non-competitive inhibitors that affect enzyme conformation

Types of Potential Inhibitors:

• Antifolates: Modified folate derivatives

• Amino acid analogs: Modified serine or glycine structures

• PLP-directed inhibitors: Compounds that interact with the cofactor

• Fragment-based design: Building complex inhibitors from simple binding fragments

Selectivity Considerations:

• Compare A. baumannii glyA with human SHMT isozymes

• Target regions with low sequence conservation

• Exploit differences in active site architecture

• Design compounds with bacterial-specific membrane permeability

Evaluation Pipeline:

• In silico screening and molecular modeling

• Biochemical assays with purified enzyme

• Cellular assays in A. baumannii

• Specificity testing against human enzymes

• Assessment of resistance development potential

This rational design approach can potentially yield selective inhibitors of A. baumannii glyA as novel antimicrobial agents.

What role does glyA play in A. baumannii's metabolic adaptations during infection?

A. baumannii demonstrates remarkable metabolic flexibility during infection, with glyA likely playing several important roles in this adaptation:

Nutritional Adaptation:

• A. baumannii colonizes diverse host environments with varying nutrient availability

• glyA enables interconversion between serine and glycine, allowing utilization of either amino acid

• One-carbon units generated feed into multiple biosynthetic pathways, providing metabolic flexibility

• This adaptability likely contributes to A. baumannii's success in colonizing different host niches including skin tissues, where neolactotetraosylceramide has been identified as a binding target

Stress Response:

• Infection sites expose bacteria to oxidative stress from host immune responses

• glyA contributes to biosynthesis of glutathione precursors (glycine) important for oxidative stress defense

• One-carbon metabolism supports nucleotide synthesis for DNA repair after oxidative damage

• Folate pathway metabolites may influence gene expression under stress conditions

Biofilm Formation:

• A. baumannii forms biofilms on medical devices and host tissues

• Biofilm formation requires specific metabolic adaptations

• Serine metabolism via glyA may contribute to exopolysaccharide production

• One-carbon units support nucleotide synthesis during biofilm development

Experimental Approaches to Study Metabolic Adaptations:

• Transcriptomic Analysis:

  • RNA-Seq under infection-relevant conditions

  • Comparison of glyA expression across stress conditions

• Metabolomic Profiling:

  • Quantification of one-carbon metabolites during infection

  • Isotope labeling to track metabolic flux

• In Vivo Studies:

  • Infection models comparing wild-type and glyA-deficient strains

  • Tissue-specific bacterial burden analysis

Understanding glyA's role in metabolic adaptation could reveal vulnerability points for therapeutic intervention against this challenging pathogen.