Recombinant Staphylococcus aureus UPF0173 metal-dependent hydrolase SAR1785 (SAR1785)

What is SAR1785 and what is its known function in Staphylococcus aureus?

SAR1785 is a UPF0173 family metal-dependent hydrolase from Staphylococcus aureus strain MRSA252, a clinically important methicillin-resistant strain. The protein has 229 amino acids and belongs to a class of enzymes that catalyze hydrolysis reactions using metal cofactors . While the specific physiological function of SAR1785 remains under investigation, it is part of S. aureus's extensive repertoire of hydrolytic enzymes that may contribute to nutrient acquisition, bacterial survival, and host immune evasion .

Metal-dependent hydrolases in pathogenic bacteria often play crucial roles in virulence by degrading host molecules or modifying bacterial surface components. As S. aureus produces numerous secreted enzymes that function in immune evasion and tissue degradation, SAR1785 might participate in these processes, although its specific substrates and exact mechanistic role require further characterization .

What are the optimal storage conditions for maintaining SAR1785 activity?

According to product specifications, the shelf life of recombinant SAR1785 depends on several factors including storage state, buffer composition, temperature, and the inherent stability of the protein itself. Generally:

• Liquid form: 6 months stability at -20°C/-80°C

• Lyophilized form: 12 months stability at -20°C/-80°C

• Working aliquots can be stored at 4°C for up to one week

Repeated freezing and thawing is not recommended as it can compromise protein integrity. For optimal results, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol (final concentration) added before aliquoting for long-term storage .

What expression systems are optimal for high-yield production of active SAR1785?

The commercially available recombinant SAR1785 is produced in yeast expression systems , which suggests this host is suitable for obtaining properly folded, active protein. For researchers planning to express this protein, several considerations should be addressed:

• Expression host selection:

  • Yeast systems (Pichia pastoris or Saccharomyces cerevisiae) provide eukaryotic post-translational processing capability

  • E. coli systems offer high yield but may require optimization for proper folding

  • Insect cell systems may be considered for complex proteins requiring specific folding conditions

• Codon optimization:
  Research indicates that accessibility of translation initiation sites significantly impacts expression success. For recombinant protein production, modifying the first nine codons of mRNAs with synonymous substitutions can significantly improve expression levels. Tools like TIsigner can be employed to optimize codon usage for the target expression system .

• Vector design:

  • Include an appropriate affinity tag (His-tag, GST, etc.) for purification

  • Consider inducible promoters for controlled expression

  • Include appropriate signal sequences if secretion is desired

• Growth conditions:
  Optimized growth temperature, induction timing, and media composition should be empirically determined, as stochastic simulation models show that higher translation initiation site accessibility leads to higher protein production but potentially slower cell growth .

How can the metal dependency of SAR1785 be experimentally determined?

To characterize the metal dependency of SAR1785, a systematic approach combining multiple techniques is recommended:

• Metal chelation studies:

  • Treat the purified enzyme with chelating agents (EDTA, EGTA) and measure residual activity

  • Perform rescue experiments by adding back individual metal ions (Zn²⁺, Mn²⁺, Fe²⁺, Cu²⁺, etc.) to identify which restore activity

• Atomic absorption spectroscopy or ICP-MS:

  • Quantitatively determine the metal content of the purified enzyme

  • Compare metal content in active versus inactive preparations

• Site-directed mutagenesis:

  • Identify putative metal-binding residues based on sequence alignment with related hydrolases

  • Mutate these residues and assess the impact on metal binding and catalytic activity

• Structural studies:

  • X-ray crystallography with and without bound metals can reveal the coordination geometry

  • Similar to studies with S-adenosylhomocysteine hydrolase, structural analysis can reveal domain organization and active site architecture

• Enzymatic assays with different buffers:

  • Test activity in buffers containing different metal ions

  • Monitor enzyme kinetics as a function of metal ion concentration

A comparison table documenting enzyme activity with different metals can provide clear evidence of preferential metal cofactor requirements:

What assays can be used to measure the hydrolase activity of SAR1785?

Without definitively knowing the natural substrate of SAR1785, researchers should employ a panel of assays to characterize its hydrolytic activity:

• Generic hydrolase screening:

  • p-Nitrophenyl ester hydrolysis assays with various chain lengths

  • Fluorogenic substrate libraries to identify preferred substrates

  • Coupled enzyme assays that detect released products

• Specific candidate substrate testing:
  Based on knowledge of related hydrolases and S. aureus biology, test activity against:

  • Peptide bonds (if potentially a peptidase)

  • Ester linkages in lipids

  • Phosphodiester bonds

  • ADP-ribose modifications (if related to ADP-ribosylhydrolases)

• Activity-based protein profiling:

  • Use chemical probes that react specifically with active hydrolases

  • Can identify active site residues and confirm hydrolase classification

• In silico substrate prediction:

  • Structure-based modeling to predict substrate binding and catalysis

  • Molecular docking of candidate substrates

• Differential scanning fluorimetry:

  • Measure thermal stability shifts upon binding of potential substrates or inhibitors

  • Can provide indirect evidence of substrate specificity

What is known about the catalytic mechanism of metal-dependent hydrolases similar to SAR1785?

While the specific mechanism of SAR1785 has not been fully elucidated, insights can be drawn from related metal-dependent hydrolases:

• Catalytic site architecture:
  Metal-dependent hydrolases typically feature a metal ion coordinated by conserved residues (often histidine, aspartate, glutamate) that positions and activates a water molecule for nucleophilic attack on the substrate .

• Proposed catalytic steps:
  The general mechanism likely involves:

  • Substrate binding in proximity to the metal center

  • Polarization of a water molecule by the metal ion

  • Nucleophilic attack by the activated water on the substrate

  • Formation of a tetrahedral intermediate

  • Collapse of the intermediate and product release

• Structural dynamics:
  Similar to S-adenosylhomocysteine hydrolase, SAR1785 may undergo conformational changes during catalysis. For example, a comparison of human S-adenosylhomocysteine hydrolase with and without inhibitor suggested a 17-degree rigid body movement of the catalytic domain upon substrate binding .

• Proton transfer pathways:
  The mechanism likely involves proton transfer networks, with conserved residues acting as general bases or acids. For example, in the catalytic mechanism of tyrosine phenol-lyase, specific residues like Lys-257 act as the base abstracting protons, while others stabilize reaction intermediates .

How can site-directed mutagenesis be used to probe the active site of SAR1785?

A systematic mutagenesis approach can reveal critical residues involved in catalysis:

• Selection of target residues:

  • Conserved residues identified through sequence alignment with related hydrolases

  • Predicted metal-binding residues (His, Asp, Glu)

  • Residues likely involved in substrate binding or catalysis

  • Residues forming the hydrophobic pocket if present

• Types of mutations to consider:

  • Conservative mutations (e.g., Asp to Glu) to test spatial requirements

  • Non-conservative mutations (e.g., His to Ala) to remove functional groups

  • Introduction of bulky side chains to probe steric constraints

  • Charge reversal to test electrostatic contributions

• Analysis of mutant proteins:

  • Determine expression levels and solubility

  • Assess structural integrity through circular dichroism or thermal shift assays

  • Measure metal binding capacity

  • Determine kinetic parameters (kcat, Km) for comparison with wild-type

• Integration with structural data:

  • Map mutations onto structural models

  • Correlate functional effects with structural features

• Example mutagenesis plan:

What is the potential role of SAR1785 in S. aureus pathogenicity and virulence?

S. aureus employs numerous virulence factors including toxins, enzymes, and immune evasion proteins to establish infection and counter host defenses . While the specific role of SAR1785 in pathogenicity is not explicitly documented in the search results, several hypotheses can be formulated based on knowledge of hydrolases in bacterial pathogenesis:

• Potential functions in pathogenesis:

  • Degradation of host antimicrobial peptides

  • Modification of bacterial cell surface to evade immune recognition

  • Processing of bacterial virulence factors

  • Nutrient acquisition during infection

  • Contribution to biofilm formation or regulation

• Comparative analysis with known virulence factors:
  S. aureus produces numerous exoenzymes that contribute to virulence, including proteases (aureolysin, V8 protease, staphopains), nucleases, lipases, and hyaluronidase . SAR1785 may have complementary or redundant functions with these enzymes.

• Immune evasion mechanisms:
  S. aureus has evolved sophisticated mechanisms to evade the host immune response, including inhibition of neutrophil chemotaxis, resistance to antimicrobial peptides, and complement evasion . Metal-dependent hydrolases could potentially contribute to these processes through enzymatic modification of host defense molecules.

• Integration with regulatory networks:
  Virulence factor expression in S. aureus is controlled by complex regulatory networks including the accessory gene regulator (agr) system and staphylococcal accessory regulator (sarA) . Understanding how SAR1785 expression is regulated within these networks could provide insights into its role during infection.

How can animal models be used to study the role of SAR1785 in S. aureus infections?

To investigate the contribution of SAR1785 to S. aureus pathogenesis, several animal model approaches can be employed:

• Gene knockout studies:

  • Generate SAR1785 deletion mutants in relevant S. aureus strains

  • Compare virulence of wild-type and mutant strains in infection models

  • Perform complementation studies to confirm phenotypes are due to SAR1785 deletion

• Infection models:

  • Systemic infection models (intravenous challenge)

  • Skin and soft tissue infection models

  • Pneumonia models

  • Catheter-associated biofilm models

  • Arthritis models similar to those used for evaluating fatty acid-sensitive mutants

• Host response analysis:

  • Assess inflammatory markers and cytokine profiles

  • Evaluate neutrophil recruitment and function

  • Measure bacterial burden in tissues

  • Histopathological examination of infected tissues

• In vivo expression studies:

  • Use reporter constructs to monitor SAR1785 expression during infection

  • Identify conditions that induce or repress expression

• Combination with other virulence factor mutations:

  • Create double or triple mutants to assess functional redundancy

  • Evaluate cumulative effects on virulence

How does SAR1785 compare structurally and functionally to other UPF0173 family hydrolases?

A comprehensive comparative analysis would include:

• Sequence alignment and phylogenetic analysis:

  • Identify conserved domains and catalytic residues

  • Determine evolutionary relationships within the UPF0173 family

  • Compare SAR1785 from MRSA252 with orthologs from other S. aureus strains and related species

• Structural comparison:

  • If crystal structures are available, compare folding patterns, active site architecture, and substrate binding pockets

  • In the absence of SAR1785 crystal structure, homology modeling based on related structures can provide insights

  • Analysis of potential conformational changes during catalysis similar to those observed in S-adenosylhomocysteine hydrolase

• Functional comparison:

  • Compare substrate specificity profiles

  • Analyze metal preferences and catalytic parameters

  • Evaluate expression patterns and regulation

• Evolutionary considerations:

  • Assess conservation across bacterial species

  • Identify potential horizontal gene transfer events

  • Evaluate selective pressures on different domains

What are the challenges in crystallizing SAR1785 for structural studies and how can they be addressed?

Protein crystallization for structural determination presents several challenges:

• Common challenges and solutions:

• Specific considerations for metal-dependent hydrolases:

  • Metal binding can induce conformational changes, affecting crystallization

  • Try crystallization with and without bound metals

  • Consider co-crystallization with substrate analogs or inhibitors to capture mechanistically relevant states

• Alternative approaches:

  • Cryo-electron microscopy (cryo-EM) for structure determination without crystals

  • NMR spectroscopy for solution structure and dynamics studies

  • Small-angle X-ray scattering (SAXS) for low-resolution envelope determination

• Case study from related proteins:
  The structure of S-adenosylhomocysteine hydrolase was solved using a combination of crystallographic direct methods and multiwavelength anomalous diffraction data . Similar approaches could be applicable to SAR1785.

How can genomic and proteomic approaches be used to identify natural substrates of SAR1785?

Identifying the natural substrates of SAR1785 requires integrative approaches:

• Comparative genomics:

  • Analyze gene neighborhood and operon structure

  • Identify co-evolving genes that may encode substrates or pathway components

  • Compare presence/absence patterns across bacterial strains with different phenotypes

• Transcriptomic correlation:

  • Identify genes co-expressed with SAR1785 under various conditions

  • Analyze expression patterns during infection or stress conditions

  • Perform RNA-seq on wild-type versus SAR1785 knockout strains

• Metabolomics approaches:

  • Compare metabolite profiles between wild-type and SAR1785 mutant strains

  • Look for accumulated precursors or depleted products

  • Use labeled substrates to trace metabolic pathways

• Proteomic strategies:

  • Activity-based protein profiling with hydrolase-specific probes

  • Affinity purification using catalytically inactive SAR1785 to capture substrates

  • Differential proteomics comparing wild-type and mutant strains

• Structural prediction and docking:

  • In silico screening of potential substrates based on binding pocket analysis

  • Molecular docking simulations to predict binding affinities

  • Virtual screening of metabolite libraries

What is the potential of SAR1785 as a drug target against MRSA infections?

Evaluating SAR1785 as a therapeutic target requires consideration of several factors:

• Target validation criteria:

  • Essentiality: Determine if SAR1785 is essential for S. aureus growth or virulence

  • Conservation: Assess conservation across clinical isolates to ensure broad-spectrum activity

  • Uniqueness: Evaluate structural or functional differences from human homologs to minimize off-target effects

  • Accessibility: Consider cellular location and accessibility to inhibitors

• Inhibitor development approaches:

  • Structure-based design if crystal structure is available

  • High-throughput screening of compound libraries

  • Fragment-based drug discovery

  • Natural product screening

• Potential advantages as a target:

  • If metal-dependent, metal chelation could be exploited for inhibition

  • Enzyme active sites often provide well-defined binding pockets for inhibitors

  • If involved in virulence rather than growth, inhibitors might reduce selective pressure for resistance

• Challenges and considerations:

  • Redundancy: S. aureus has multiple virulence factors and immune evasion mechanisms that might compensate for SAR1785 inhibition

  • Delivery: Ensuring inhibitors reach their target, particularly for intracellular infections

  • Resistance development: Assessing potential mechanisms of resistance

How can high-throughput screening be optimized to identify inhibitors of SAR1785?

Designing an effective high-throughput screening campaign requires careful consideration of assay design and compound selection:

• Assay development:

  • Primary assay: Develop a robust enzymatic assay with appropriate signal-to-noise ratio

  • Counter-screen: Include assays to identify false positives (e.g., compounds that interfere with detection method)

  • Secondary assays: Confirm hits with orthogonal assay formats

  • Tertiary assays: Test activity in cellular contexts (bacterial growth, infection models)

• Compound library selection:

  • Diversity-oriented libraries for broad chemical space exploration

  • Focused libraries targeting metal-dependent hydrolases

  • Natural product libraries that may include evolved inhibitors

  • Fragment libraries for identifying starting points for optimization

• Screening strategy:

  • Consider quantitative high-throughput screening (qHTS) with dose-response curves

  • Implement automated liquid handling and data analysis

  • Include appropriate controls for assay quality assessment (Z' factor)

  • Use clustering and machine learning for hit prioritization

• Hit validation and optimization pipeline:

  • Confirm structure and purity of hits

  • Determine mechanism of inhibition

  • Assess specificity against related enzymes

  • Evaluate physicochemical properties and optimize for drug-like characteristics

  • Test activity against multiple S. aureus strains