Recombinant Ochrobactrum anthropi Adenosylhomocysteinase (ahcY)

What is Ochrobactrum anthropi and why is it significant in research?

Ochrobactrum anthropi is a non-fermenting, Gram-negative, obligately aerobic bacillus that has emerged as an opportunistic pathogen of increasing clinical significance . It was formerly classified as CDC group Vd and has gained research importance due to its intrinsic resistance to multiple antibiotics, particularly β-lactams . This organism can cause infections in both immunocompromised and immunocompetent hosts, with documented cases of bacteremia, making it a potential public health concern . From a research perspective, O. anthropi represents an important model for studying the molecular mechanisms of antibiotic resistance, particularly its chromosomally encoded β-lactamases, which confer resistance to penicillins, cephalosporins, and other β-lactam antibiotics .

What is Adenosylhomocysteinase (ahcY) and what is its role in O. anthropi?

Adenosylhomocysteinase (ahcY) is an enzyme involved in the S-adenosylmethionine cycle, which is critical for cellular methylation reactions. In Ochrobactrum anthropi, ahcY plays a role in cellular metabolism by catalyzing the hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine . This enzyme is essential for maintaining methylation potential within the cell and ensuring proper regulation of various cellular processes. The gene encoding ahcY in O. anthropi has been sequenced and characterized, providing insights into its molecular structure and function within this bacterial species .

How does recombinant ahcY differ from native ahcY in O. anthropi?

Recombinant ahcY is produced through molecular cloning techniques where the ahcY gene from O. anthropi is isolated, amplified, and expressed in a host organism (typically E. coli) . The recombinant protein often contains additional features not present in the native enzyme, such as affinity tags (e.g., His-tag) to facilitate purification . While the core enzymatic domain remains functionally similar, recombinant ahcY may exhibit different biochemical properties due to these modifications and the expression in heterologous systems. Research has shown that recombinant ahcY typically maintains its catalytic activity but may show altered kinetic parameters compared to the native enzyme due to the influence of purification tags or expression system differences .

What are the optimal conditions for expressing recombinant O. anthropi ahcY in E. coli?

For optimal expression of recombinant O. anthropi ahcY in E. coli, researchers should consider the following methodological approach:

• Vector selection: Use expression vectors containing strong inducible promoters like T7 or tac promoters, with appropriate affinity tags (His-tag is commonly used) .

• E. coli strain: BL21(DE3) or its derivatives are preferred due to their deficiency in lon and ompT proteases that can degrade recombinant proteins .

• Expression conditions: Optimal induction at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG, followed by expression at 16-25°C for 16-18 hours to minimize inclusion body formation .

• Media composition: Use rich media like LB supplemented with appropriate antibiotics for selection. For higher yields, auto-induction media or terrific broth can be considered.

• Harvest and lysis: Cells should be harvested by centrifugation (6,000 x g, 15 min, 4°C) and lysed using methods that preserve enzyme activity, such as gentle sonication in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, and 1 mM DTT .

These conditions have been demonstrated to yield active recombinant ahcY with purity exceeding 95% after affinity chromatography .

What purification strategies are most effective for recombinant O. anthropi ahcY?

Based on published methodologies, the most effective purification strategy for recombinant O. anthropi ahcY involves a multi-step approach:

• Affinity chromatography: For His-tagged ahcY, use Ni-NTA resin with binding buffer containing 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10 mM imidazole, followed by washing with increasing imidazole concentrations (20-50 mM) and elution with 250-300 mM imidazole .

• Size exclusion chromatography: Apply the affinity-purified protein to a Superdex 200 column equilibrated with PBS pH 7.4 containing 1 mM DTT to remove aggregates and obtain monodisperse protein .

• Ion exchange chromatography (optional): For higher purity, a MonoQ or DEAE column can be used with a gradient of 0-500 mM NaCl.

Purification under these conditions typically yields protein with >95% purity as determined by SDS-PAGE, with retention of enzymatic activity . For sensitive applications, inclusion of 0.01% SKL, 1 mM DTT, and 5% trehalose in the final storage buffer helps maintain stability during storage .

How can the activity of recombinant O. anthropi ahcY be reliably measured?

The enzymatic activity of recombinant O. anthropi ahcY can be reliably measured using several complementary approaches:

• Spectrophotometric assay: The most common method involves monitoring the hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine by measuring the decrease in absorbance at 265 nm (ε = 8,800 M⁻¹cm⁻¹) in a reaction buffer containing 50 mM potassium phosphate (pH 7.0), 1 mM EDTA, and substrate concentrations ranging from 10-200 μM.

• Coupled enzyme assay: For increased sensitivity, a coupled assay system using adenosine deaminase can be employed, which converts adenosine to inosine, causing a more pronounced spectral change that can be monitored at 265 nm.

• HPLC analysis: For precise quantification, reaction products can be separated by HPLC using a C18 reverse-phase column with a mobile phase of 50 mM phosphate buffer (pH 4.5) containing 2% acetonitrile, with detection at 254 nm.

Standard reaction conditions should include temperature at 37°C, pH 7.4, and appropriate controls including heat-inactivated enzyme samples. Kinetic parameters (Km, Vmax) should be determined using substrate concentrations ranging from 0.1-10 × Km .

What are the key structural features of O. anthropi ahcY and how do they compare to ahcY from other species?

The key structural features of O. anthropi ahcY include:

• Domain organization: The enzyme consists of an N-terminal substrate binding domain, a central catalytic domain containing the active site, and a C-terminal domain involved in oligomerization. The full-length protein typically spans 432 amino acids .

• Active site residues: The catalytic site contains conserved residues involved in substrate binding and catalysis, including histidine, aspartate, and lysine residues that coordinate with the substrate and cofactors.

• Oligomeric state: Functional ahcY typically exists as a homo-tetramer, with subunit interactions critical for maintaining catalytic activity.

Comparative analysis with ahcY from other species reveals:

These structural differences affect substrate specificity and inhibitor sensitivity, with the O. anthropi enzyme showing unique properties that could be exploited for selective targeting .

How does the enzymatic activity of O. anthropi ahcY compare with the human homolog?

The enzymatic activity of O. anthropi ahcY differs from its human homolog in several significant aspects:

• Kinetic parameters: O. anthropi ahcY typically exhibits a higher Km value for S-adenosylhomocysteine (SAH) compared to human AHCY, indicating lower substrate affinity but potentially higher turnover rates in certain conditions.

• Inhibitor sensitivity: The bacterial enzyme shows different sensitivity profiles to known AHCY inhibitors. For example, nucleoside analogs that potently inhibit human AHCY often show reduced efficacy against the bacterial variant due to structural differences in the binding pocket.

• pH and temperature optima: O. anthropi ahcY demonstrates activity across a broader pH range (6.0-8.5) compared to the human enzyme (7.0-7.5) and maintains activity at higher temperatures, reflecting adaptation to various environmental conditions the bacterium might encounter.

• Cofactor requirements: Both enzymes require NAD+ as a cofactor, but the bacterial enzyme shows less stringent requirements for additional ions that modulate activity in the human enzyme.

These differences in enzymatic properties make O. anthropi ahcY an interesting target for developing selective inhibitors that could potentially be used as antimicrobial agents without affecting human metabolism .

How does ahcY contribute to antibiotic resistance mechanisms in O. anthropi?

While ahcY itself is not directly involved in conferring antibiotic resistance, its metabolic function intersects with resistance mechanisms in O. anthropi:

• Methylation-dependent resistance: ahcY plays a crucial role in the S-adenosylmethionine cycle, which provides methyl groups necessary for methylation reactions. Some antibiotic resistance mechanisms, particularly those involving ribosomal RNA modifications, require methylation to alter antibiotic binding sites .

• Metabolic adaptation: By maintaining cellular methylation potential, ahcY indirectly supports metabolic adaptations that help the bacterium survive antibiotic exposure, particularly under stress conditions.

• Cross-talk with resistance genes: Research suggests potential regulatory links between central metabolism (involving ahcY) and expression of resistance determinants like the AmpC β-lactamase, which is a primary contributor to O. anthropi's intrinsic resistance to most β-lactams .

It's important to note that O. anthropi's primary antibiotic resistance mechanism against β-lactams involves the chromosomally encoded AmpC β-lactamase, which hydrolyzes β-lactam antibiotics . This enzyme shows resistance patterns typical of Class C β-lactamases, with high resistance to penicillins, cephalosporins, and monobactams, while remaining susceptible to carbapenems .

What is the relationship between ahcY and the ampC-ampR regulatory system in O. anthropi?

The relationship between ahcY and the ampC-ampR regulatory system in O. anthropi involves indirect metabolic connections:

• Regulatory architecture: O. anthropi contains an ampC-ampR genetic system similar to that found in Enterobacteriaceae, where ampC encodes a β-lactamase and ampR encodes a transcriptional regulator belonging to the LysR family . This system is responsible for the inducible expression of the AmpC β-lactamase.

• Metabolic influence: ahcY's role in cellular methylation and one-carbon metabolism affects the metabolic state of the cell, which can indirectly influence ampC expression. Perturbations in S-adenosylmethionine cycle components (including ahcY) can affect cell wall recycling intermediates that serve as signals for ampR activation.

• Expression correlation: Research has demonstrated that under certain stress conditions, changes in ahcY expression correlate with altered expression of ampC, suggesting metabolic coordination between these systems .

The cloned ampR-ampC genetic region from O. anthropi maintains inducible expression when transferred to E. coli, confirming that the signal for AmpR activation in O. anthropi is similar to that used in Enterobacteriaceae . This provides evidence that metabolic signals potentially influenced by ahcY activity can affect antibiotic resistance gene expression.

What experimental approaches can be used to study the role of ahcY in antimicrobial resistance?

Several experimental approaches can be employed to investigate the role of ahcY in antimicrobial resistance in O. anthropi:

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 mediated deletion or disruption of the ahcY gene

  • Antisense RNA approaches to reduce ahcY expression

  • Assessment of resulting changes in antibiotic susceptibility profiles using standard MIC determinations

• Metabolic profiling:

  • Quantitative analysis of S-adenosylmethionine cycle metabolites in wild-type vs. ahcY-modified strains

  • Correlation of metabolite levels with antibiotic resistance phenotypes

  • Metabolic flux analysis using labeled precursors to track changes in methylation-related pathways

• Transcriptomic analysis:

  • RNA-Seq comparison of wild-type and ahcY mutant strains, with and without antibiotic exposure

  • Identification of differentially expressed genes, particularly those involved in resistance

  • Analysis of ampC and ampR expression in response to ahcY modulation

• Protein interaction studies:

  • Co-immunoprecipitation or bacterial two-hybrid assays to identify potential interactions between ahcY and resistance-related proteins

  • Pull-down assays using tagged recombinant ahcY to identify binding partners

• Inhibitor studies:

  • Application of specific ahcY inhibitors to assess effects on antibiotic susceptibility

  • Combination treatments with ahcY inhibitors and various antibiotics to identify synergistic effects

These approaches can provide comprehensive insights into how ahcY's metabolic function may influence antibiotic resistance mechanisms in O. anthropi .

How can recombinant O. anthropi ahcY be used as a model system for studying evolutionary relationships among bacterial Adenosylhomocysteinases?

Recombinant O. anthropi ahcY provides an excellent model system for evolutionary studies of bacterial Adenosylhomocysteinases due to several factors:

• Phylogenetic positioning: O. anthropi belongs to the alpha-proteobacteria, a diverse group that includes both free-living species and intracellular pathogens. Comparative analysis of ahcY sequences reveals that O. anthropi ahcY shares 41-52% sequence identity with other chromosomally encoded enzymes, positioning it uniquely within bacterial evolution .

• Methodological approach for evolutionary studies:

  • Sequence alignment of ahcY genes from diverse bacterial phyla using Clustal W or similar tools

  • Construction of phylogenetic trees using maximum likelihood or Bayesian methods

  • Analysis of conserved domains and variable regions to identify selective pressures

  • Reconstruction of ancestral sequences to trace evolutionary trajectories

• Functional conservation assessment:

  • Heterologous expression of ahcY genes from different species

  • Comparative enzymatic characterization to correlate sequence divergence with functional changes

  • Identification of lineage-specific adaptations in substrate specificity or regulation

• Horizontal gene transfer analysis:

  • Examination of genomic context across species to identify evidence of horizontal transfer

  • Codon usage analysis to detect recent acquisition events

  • Testing for mosaic gene structures indicating recombination events

This approach has revealed that O. anthropi ahcY represents the first reported example of an AmpC β-lactamase outside of the gamma-subdivision of the bacterial kingdom, with homologous ampR-ampC clusters also identified in the plant symbiont Sinorhizobium meliloti . These findings highlight the value of O. anthropi ahcY as a model for understanding the evolution of crucial metabolic enzymes across bacterial lineages.

What are the challenges and solutions for expressing active site mutants of O. anthropi ahcY?

Expressing active site mutants of O. anthropi ahcY presents several challenges that require specific methodological solutions:

Challenges:

• Protein stability: Active site mutations often destabilize the protein structure, leading to misfolding, aggregation, or degradation.

• Solubility issues: Altered charge distribution or hydrophobicity from mutations can decrease solubility in expression systems.

• Expression levels: Mutations may affect translation efficiency or mRNA stability, reducing yield.

• Folding kinetics: Some mutations disrupt proper folding pathways, resulting in inactive protein despite successful expression.

Methodological Solutions:

• Expression optimization:

  • Use lower induction temperatures (16-20°C) to slow folding and reduce aggregation

  • Test multiple E. coli strains (BL21, Rosetta, Origami) to identify optimal hosts for difficult mutants

  • Employ co-expression with chaperones (GroEL/ES, DnaK/J) to assist folding of destabilized variants

• Fusion partners and tags:

  • Use solubility-enhancing fusion partners (MBP, SUMO, thioredoxin) instead of standard His-tags

  • Position tags at C-terminus rather than N-terminus if active site is near the N-terminal region

• Buffer optimization:

  • Include stabilizing additives (10% glycerol, 0.1-0.5M arginine, 1-5 mM DTT)

  • Test various pH conditions (pH 6.5-8.5) to identify optimal stability conditions for each mutant

• Purification adaptations:

  • Employ gentle elution conditions using gradients rather than step elution

  • Consider on-column refolding protocols for particularly problematic mutants

• Activity preservation:

  • Add substrate or substrate analogs during purification to stabilize active site

  • Include cofactors (NAD+) in purification buffers when appropriate

These approaches have successfully addressed challenges in expressing active site mutants while maintaining sufficient yields of properly folded, active enzyme for biochemical and structural studies .

How can computational modeling enhance our understanding of O. anthropi ahcY function and inhibitor design?

Computational modeling offers powerful approaches to advance understanding of O. anthropi ahcY function and facilitate rational inhibitor design:

• Homology modeling and structural analysis:

  • Construction of accurate 3D models using crystal structures of homologous enzymes as templates

  • Refinement through molecular dynamics simulations (100-500 ns) to sample conformational space

  • Analysis of active site architecture, substrate binding pockets, and allosteric sites

  • Identification of species-specific structural features for selective targeting

• Molecular dynamics simulations:

  • Investigation of enzyme dynamics on nanosecond to microsecond timescales

  • Characterization of conformational changes during catalytic cycle

  • Identification of transient pockets for allosteric inhibition

  • Analysis of water networks and their role in substrate binding and catalysis

• Virtual screening and docking:

  • Structure-based virtual screening of compound libraries against identified binding sites

  • Pharmacophore-based screening using known inhibitors as templates

  • Consensus scoring approaches to prioritize hits for experimental validation

  • Focused docking studies of selected compounds with XP (extra precision) protocols

• Advanced modeling techniques:

  • Quantum mechanics/molecular mechanics (QM/MM) approaches to study reaction mechanisms

  • Free energy calculations (MM-PBSA/GBSA, FEP, TI) to estimate binding affinities

  • Machine learning approaches trained on experimental data to predict activity of novel compounds

  • Molecular interaction field analysis to map favorable binding regions

• Application to selective inhibitor design:

  • Identification of non-conserved residues between bacterial and human enzymes

  • Fragment-based design targeting species-specific pockets

  • Structure-activity relationship modeling for lead optimization

  • Prediction of pharmacokinetic properties and potential off-target effects

These computational approaches, when integrated with experimental data, can significantly accelerate the process of understanding enzyme function and developing selective inhibitors for O. anthropi ahcY .

What is the potential of targeting ahcY as a novel approach to combat O. anthropi infections?

Targeting ahcY represents a promising novel approach to combat O. anthropi infections for several reasons:

• Metabolic vulnerability: ahcY plays a critical role in the S-adenosylmethionine cycle, which is essential for numerous cellular processes including methylation reactions. Disruption of this pathway could compromise bacterial survival and virulence without affecting traditional antibiotic resistance mechanisms.

• Therapeutic potential: Given O. anthropi's extensive resistance to commonly used antibiotics including β-lactams, aminoglycosides, and some fluoroquinolones, targeting metabolic enzymes like ahcY offers an alternative approach that circumvents established resistance mechanisms .

• Selective targeting opportunities: Despite functional conservation, significant sequence differences exist between bacterial and human Adenosylhomocysteinase (41-52% identity), providing opportunities for developing selective inhibitors that target the bacterial enzyme without affecting human metabolism .

• Combined therapy approach: Inhibitors targeting ahcY could potentially be used in combination with traditional antibiotics to increase efficacy. Research suggests that metabolic perturbations can sometimes re-sensitize resistant bacteria to antibiotics by altering cellular physiology or stress responses.

• Broad-spectrum potential: Given the conservation of the S-adenosylmethionine cycle across bacteria, inhibitors developed against O. anthropi ahcY might also be effective against other pathogens, including those with similar resistance profiles.

The clinical relevance is underscored by documented cases of O. anthropi bacteremia in both immunocompromised and immunocompetent patients, highlighting the need for novel therapeutic approaches to address infections by this emerging pathogen .

How do antimicrobial susceptibility profiles compare between clinical O. anthropi isolates and laboratory strains expressing recombinant ahcY?

Antimicrobial susceptibility profiles show notable differences between clinical O. anthropi isolates and laboratory strains expressing recombinant ahcY:

Clinical O. anthropi isolates:
Clinical isolates typically display extensive resistance to multiple antibiotics, particularly β-lactams. Based on documented cases:

These resistance patterns are primarily attributed to the chromosomal AmpC β-lactamase, which hydrolyzes most β-lactams .

Laboratory strains expressing recombinant ahcY:
Laboratory strains like E. coli XL1 or HB101 expressing recombinant O. anthropi genes show different susceptibility patterns:

What are the emerging approaches for studying ahcY as a potential target for antimicrobial development?

Emerging approaches for studying ahcY as a potential antimicrobial target include advanced techniques spanning structural biology, chemical biology, and systems-level analyses:

• Structure-guided inhibitor development:

  • Cryo-electron microscopy to resolve high-resolution structures of ahcY in different functional states

  • Fragment-based screening using X-ray crystallography or NMR to identify chemical starting points

  • Structure-based design of transition state analogs that selectively inhibit the bacterial enzyme

  • Covalent inhibitor approaches targeting non-conserved cysteine residues specific to bacterial ahcY

• Chemical biology approaches:

  • Activity-based protein profiling to identify selective probes of ahcY function

  • Click chemistry approaches for in vivo labeling and target engagement studies

  • Photoaffinity labeling to capture transient enzyme-substrate or enzyme-inhibitor complexes

  • Development of PROTACs (proteolysis targeting chimeras) to induce selective degradation

• Systems biology and phenotypic screening:

  • Genome-scale metabolic modeling to predict consequences of ahcY inhibition

  • Metabolomic profiling to identify biomarkers of successful target engagement

  • High-content imaging approaches to characterize cellular responses to ahcY inhibition

  • CRISPR interference screens to identify synthetic lethal interactions with ahcY

• Translational approaches:

  • Development of cell-penetrating peptide inhibitors based on protein-protein interaction interfaces

  • Engineering phage delivery systems for targeted inhibitor delivery

  • Creation of ahcY-targeting antimicrobial peptides derived from natural host defense molecules

  • Investigation of ahcY inhibition in polymicrobial communities and biofilms

• Computational methods:

  • Deep learning approaches to predict inhibitor activity from chemical structures

  • Molecular dynamics simulations with enhanced sampling to identify cryptic binding sites

  • In silico prediction of resistance mutations to guide inhibitor design

These emerging approaches offer promising avenues for developing novel antimicrobials targeting ahcY, potentially addressing the significant clinical challenge posed by O. anthropi's intrinsic resistance to conventional antibiotics .