Recombinant UPF0173 metal-dependent hydrolase STH3160 (STH3160)

What is UPF0173 metal-dependent hydrolase STH3160?

UPF0173 metal-dependent hydrolase STH3160 is a 224-amino acid protein (24.1 kDa) from Symbiobacterium thermophilum (strain T / IAM 14863) that belongs to the UPF0173 family of hydrolases . The "UPF" designation (Uncharacterized Protein Family) indicates it was initially identified through genomic sequencing before detailed functional characterization. The protein likely requires metal ion cofactors for its catalytic activity, as suggested by the "metal-dependent hydrolase" classification, similar to other characterized members of this family found in various thermophilic and mesophilic organisms.

What are optimal conditions for heterologous expression of STH3160?

For recombinant expression of STH3160, researchers should consider its thermophilic origin when designing expression protocols. Recommended approaches include:

• Expression system: E. coli BL21(DE3) with pET-based vectors optimized for thermophilic proteins

• Growth conditions:

  • Initial growth at 37°C until OD600 reaches 0.6-0.8

  • IPTG induction (0.1-0.5 mM)

  • Temperature downshift to 20-25°C during expression phase (12-18 hours)

• Media supplementation:

  • Addition of divalent metal ions (Zn²⁺, Mg²⁺, or Mn²⁺) at 0.1-1.0 mM concentrations

  • 5-10% glycerol to enhance protein stability

This approach balances protein yield with proper folding, particularly important for metal-dependent enzymes from thermophilic sources .

What purification strategies maximize recovery of catalytically active STH3160?

A multi-step purification strategy is recommended to maintain the native conformation and metal co-factor binding essential for STH3160 activity:

• Initial clarification via high-speed centrifugation (20,000-30,000 × g, 30-45 min)

• Heat treatment (optional): Exploiting STH3160's thermostability by heating lysate to 60-65°C for 15-20 min to precipitate E. coli proteins

• IMAC purification using Ni-NTA or Co-NTA columns if working with His-tagged constructs

• Buffer optimization to include:

  • 50 mM Tris-HCl or HEPES (pH 7.5-8.0)

  • 150-300 mM NaCl

  • 1-5 mM of appropriate metal ions

  • Avoiding metal chelators like EDTA

• Size exclusion chromatography as a final polishing step

This approach typically yields enzyme preparations with >95% purity while preserving catalytic activity .

How does the structure of STH3160 compare to other UPF0173 family proteins?

Computational models of UPF0173 family proteins show remarkably high structural conservation despite varying degrees of sequence identity. AlphaFold predictions of related UPF0173 proteins demonstrate exceptionally high confidence scores:

These high confidence scores (>90 is considered "very high" confidence) suggest that the predicted structures closely approximate the actual protein structures . The core structure likely consists of a central β-sheet surrounded by α-helices, with a conserved metal-binding site coordinated by histidine and aspartate residues positioned for optimal catalysis.

What techniques are most effective for investigating STH3160's metal coordination environment?

Investigating the metal coordination environment of STH3160 requires a multi-technique approach:

• Spectroscopic methods:

  • X-ray Absorption Spectroscopy (XAS) to determine metal identity, oxidation state, and coordination geometry

  • Electron Paramagnetic Resonance (EPR) for paramagnetic metal centers

  • UV-Vis spectroscopy to track metal-binding through characteristic absorption bands

• Structural methods:

  • X-ray crystallography with anomalous scattering to precisely locate metal ions

  • Nuclear Magnetic Resonance (NMR) to study metal-protein interactions in solution

• Mutagenesis approaches:

  • Systematic mutation of potential metal-coordinating residues (His, Asp, Glu, Cys)

  • Metal substitution experiments with various divalent metals

  • Activity correlation with metal binding

• Computational methods:

  • Quantum mechanical calculations of the metal center

  • Molecular dynamics simulations of metal-ligand interactions

This integrated approach can provide comprehensive insights into how metal coordination influences STH3160's catalytic mechanism .

What substrates does STH3160 likely hydrolyze, and how should substrate profiling be conducted?

While specific substrates for STH3160 have not been definitively characterized in the available literature, systematic substrate profiling can determine its specificity:

• Initial screening with common hydrolase substrate classes:

  • Para-nitrophenyl esters with varying acyl chain lengths (C2-C16)

  • Thioester substrates

  • Fluorogenic substrates (umbelliferyl derivatives)

  • Glycosidic substrates to rule out glycosidase activity

• Kinetic characterization with promising substrates:

  • Determination of kcat, KM, and kcat/KM values

  • pH-rate profiles to identify catalytically important ionizable groups

  • Temperature-activity profiling (especially relevant for thermophilic enzymes)

• Specialty substrates based on initial results:

  • If short-chain ester preference is observed (similar to enzymes in result ), focus on physiologically relevant short-chain esters

  • If promiscuous activity is observed, test structurally diverse substrates

The enzyme may exhibit substrate preferences similar to other characterized metal-dependent hydrolases, potentially favoring shorter-chain esters as suggested by studies on related enzymes .

How can the catalytic mechanism of STH3160 be elucidated?

Elucidating the catalytic mechanism of STH3160 requires a comprehensive approach combining structural, functional, and computational methods:

• Identification of catalytic residues:

  • Site-directed mutagenesis of predicted catalytic residues followed by activity measurements

  • Chemical modification studies targeting specific amino acid types

  • pH-dependency studies to determine pKa values of catalytic residues

• Reaction intermediate characterization:

  • Pre-steady-state kinetics to capture transient species

  • Cryoenzymology at sub-zero temperatures to slow reaction steps

  • Mass spectrometry to identify reaction intermediates

  • Trapping experiments with mechanism-based inhibitors

• Metal role determination:

  • Metal substitution experiments to assess catalytic competence

  • Spectroscopic monitoring of metal coordination during catalysis

  • Metal-binding affinity measurements correlated with activity

• Computational analysis:

  • QM/MM studies of the reaction pathway

  • Transition state modeling

This multi-faceted approach can establish whether STH3160 follows mechanisms similar to other metal-dependent hydrolases, where a metal-activated water molecule typically serves as the nucleophile in the hydrolytic reaction .

How can conformational dynamics studies enhance understanding of STH3160 function?

Conformational dynamics studies can reveal crucial aspects of STH3160 function that static structural information alone cannot provide:

• Potential conformational changes during catalysis:

  • Studies on other hydrolases like FTT258 have revealed large conformational changes in flexible loops essential for substrate binding and catalysis

  • These changes may create hydrophobic pockets that determine substrate specificity

• Recommended methodologies:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic regions

  • Molecular dynamics simulations at different timescales

  • NMR relaxation measurements for solution dynamics

  • Single-molecule FRET to observe conformational states

  • Temperature-dependent structural studies (particularly relevant for thermophilic enzymes)

• Target regions for analysis:

  • Peripheral loops that may control substrate access

  • Regions surrounding the metal-binding site

  • Domain interfaces if multiple domains are present

Understanding these dynamic aspects is crucial for a complete mechanistic picture and may reveal unexpected regulatory mechanisms or substrate specificity determinants .

What approaches are most effective for developing selective inhibitors of STH3160?

Developing selective inhibitors for STH3160 requires a methodical approach leveraging both structural information and high-throughput screening:

• Structure-based design strategies:

  • Virtual screening of compound libraries against the active site

  • Fragment-based approaches targeting specific binding pockets

  • Metal-binding group incorporation to target the metal center

• Rapid inhibitor development approaches:

  • Click chemistry methods, such as those used to develop 1,2,3-triazole ureas as hydrolase inhibitors

  • This approach enables:

    • Rapid diversification of inhibitor scaffolds

    • Systematic exploration of structure-activity relationships

    • Generation of focused libraries targeting specific binding pockets

• Evaluation methods:

  • Activity-based protein profiling (ABPP) for selectivity assessment

  • Enzyme kinetic studies to determine inhibition mechanisms

  • Structural studies of enzyme-inhibitor complexes

Successful inhibitor development requires focusing on unique structural features of STH3160 compared to other hydrolases to maximize selectivity .

How can STH3160 be used as a model system for studying enzyme adaptation in thermophiles?

STH3160 from the thermophilic bacterium Symbiobacterium thermophilum provides an excellent model system for studying enzyme adaptation:

• Comparative analysis approaches:

  • Sequence and structure comparison with mesophilic homologs to identify thermoadaptation features

  • Analysis of amino acid composition biases typical of thermophiles (increased charged residues, decreased thermolabile residues)

  • Identification of stabilizing structural features (additional salt bridges, hydrophobic packing)

• Experimental investigation methods:

  • Thermal denaturation studies (DSC, CD spectroscopy)

  • Activity and stability measurements across temperature gradients

  • Reciprocal mutations between thermophilic and mesophilic homologs

  • Chimeric enzymes combining domains from differently adapted homologs

• Research applications:

  • Understanding fundamental principles of protein thermostability

  • Developing predictive models for engineering thermostable enzymes

  • Insight into evolutionary adaptation mechanisms

The remarkable structural conservation of UPF0173 family proteins across species from diverse thermal environments makes this an ideal system for such comparative studies .

How should researchers approach collaboration when studying novel enzymes like STH3160?

Effective collaboration strategies for STH3160 research should integrate diverse expertise:

• Interdisciplinary team composition:

  • Biochemists for enzyme characterization

  • Structural biologists for 3D structure determination

  • Computational scientists for modeling and simulation

  • Microbiologists for physiological context

  • Evolutionary biologists for phylogenetic analysis

• Communication frameworks:

  • Regular structured meetings with defined outcomes

  • Shared data repositories with standardized formats

  • Clearly defined project milestones and responsibility assignments

• Overcoming collaboration barriers:

  • Research suggests scientists' perceptions of "who does science" can impact participation and question-asking behavior in scientific forums

  • Implementing inclusive practices such as "Scientist Spotlight" approaches has been shown to positively shift students' ability to relate to scientists

  • Being aware that demographic factors may influence communication patterns in research settings

Effective collaboration requires both technical infrastructure and attention to social dynamics that promote inclusive participation from all team members .

What strategies optimize scientific communication when presenting STH3160 research?

Research on scientific communication offers insights for effectively presenting STH3160 research:

• Publication strategies:

  • Frame research narratives that connect structural details to broader biological significance

  • Highlight methodological innovations alongside results

  • Consider multiple publication formats (research articles, methods papers, reviews) to maximize impact

• Conference presentation considerations:

  • Create presentation structures that accommodate audience members with varying levels of specialized knowledge

  • Use visual representations of protein structure that effectively communicate key findings

  • Prepare for question-answer sessions with attention to inclusive participation

• Evidence-based communication practices:

  • Studies show that scientific communication skills significantly predict research career intention and science identity

  • Developing communication self-efficacy positively impacts science identity

  • Mentoring practices in scientific communication influence trainees' career trajectories