Recombinant Putative 8-amino-7-oxononanoate synthase/2-amino-3-ketobutyrate coenzyme A ligase (STH1872)

What is the enzymatic function of STH1872 and in which metabolic pathways does it participate?

STH1872 from Symbiobacterium thermophilum functions as a glycine C-acetyltransferase (EC 2.3.1.29), also known as 8-amino-7-oxononanoate synthase or 2-amino-3-ketobutyrate coenzyme A ligase. The enzyme primarily participates in glycine, serine, and threonine metabolism, as well as broader metabolic pathways . Methodologically, researchers can confirm its function by conducting enzyme assays measuring the conversion of substrates to products using spectrophotometric techniques. Specifically, the enzyme catalyzes the conversion of 2-amino-3-ketobutyrate (from threonine dehydrogenation) to glycine and acetyl-CoA with the participation of CoA as a cofactor .

What is the oligomeric structure of STH1872 and how does this relate to its function?

Based on structural homology with related enzymes like 2-amino-3-ketobutyrate CoA ligase (KBL), STH1872 likely functions as a homodimer, with two active sites located at the dimer interface . Both monomers contribute side chains to each active/substrate binding site, making the dimeric structure essential for catalytic activity. To methodologically investigate the oligomeric state, researchers should employ size exclusion chromatography, analytical ultracentrifugation, or native PAGE. The functional significance of dimerization can be assessed by creating mutations at the dimer interface and measuring subsequent effects on enzyme activity.

What cofactors are essential for STH1872 activity and how should they be incorporated in experimental designs?

STH1872, like other enzymes in its family, is a pyridoxal phosphate (PLP)-dependent enzyme that also requires CoA as a cofactor for its activity . When designing experiments, researchers should ensure that both PLP and CoA are included in reaction mixtures at appropriate concentrations. Typically, PLP should be added at 0.1-0.5 mM and CoA at 0.2-1 mM concentrations. Additionally, experimental buffers should maintain pH conditions (usually 7.5-8.0) that preserve the Schiff base intermediate formation between PLP and substrate. To verify cofactor binding, spectroscopic methods can detect the characteristic absorption of the PLP-enzyme complex at approximately 420 nm.

What expression systems are most suitable for recombinant production of STH1872?

For recombinant expression of STH1872, E. coli is the most commonly used heterologous host due to its simplicity and high yield. For thermophilic proteins like STH1872 from Symbiobacterium thermophilum, E. coli BL21(DE3) or Rosetta strains are particularly suitable. Methodologically, researchers should optimize expression by testing various conditions: induction at OD600 of 0.6-0.8, IPTG concentrations of 0.1-1.0 mM, and post-induction temperatures of 18-30°C. For challenging expressions, consider specialized strains like C41(DE3) or Arctic Express. Alternative expression systems such as insect cells (like Sf9) might be beneficial if E. coli expression results in inclusion bodies or inactive protein.

What purification strategy yields the highest purity and activity for STH1872?

A multi-step purification approach is recommended for STH1872 to achieve both high purity and activity. Begin with affinity chromatography using an N-terminal or C-terminal His-tag, followed by ion-exchange chromatography, and finally size exclusion chromatography. Methodologically, all buffers should contain a reducing agent (typically 1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation of cysteine residues. Additionally, including PLP (0.1 mM) in purification buffers helps maintain the enzyme's structural integrity. After each purification step, assess both protein purity by SDS-PAGE and enzymatic activity to ensure that the purification process maintains functional protein. For long-term storage, add 10-20% glycerol and store at -80°C in small aliquots to avoid freeze-thaw cycles.

How can researchers assess whether recombinant STH1872 is properly folded and functional?

Multiple complementary approaches should be employed to evaluate proper folding and functionality of recombinant STH1872. Circular dichroism (CD) spectroscopy can assess secondary structure content, while thermal shift assays provide information about protein stability and proper folding. For functional assessment, enzymatic activity assays measuring the conversion of 2-amino-3-ketobutyrate to glycine and acetyl-CoA should be performed. Additionally, spectroscopic analysis can confirm PLP binding by detecting characteristic absorption peaks. Methodologically, researchers should compare their recombinant enzyme parameters with published values for related enzymes or wild-type extracts. Proper folding is often indicated by a single peak in size exclusion chromatography, corresponding to the dimeric form of the enzyme.

What are the optimal conditions for measuring STH1872 enzymatic activity?

For optimal measurement of STH1872 activity, researchers should conduct assays at temperatures reflective of Symbiobacterium thermophilum's thermophilic nature, typically around 50-60°C. The reaction buffer should maintain pH 7.5-8.0 (commonly using HEPES or Tris buffer at 50-100 mM). Methodologically, ensure the presence of both essential cofactors: PLP at 0.1-0.5 mM and CoA at 0.2-1 mM. The activity can be measured using either a coupled enzyme assay or by directly detecting product formation through spectrophotometric, HPLC, or mass spectrometry methods. When developing new assays, validate them by confirming linearity with respect to both time and enzyme concentration. Additionally, include appropriate controls such as heat-inactivated enzyme and reactions without substrate to account for background activities.

How does substrate specificity of STH1872 compare with related enzymes from other organisms?

STH1872 shares functional similarity with 2-amino-3-ketobutyrate CoA ligase (KBL) from E. coli and other organisms, but likely exhibits distinct substrate specificity profiles based on structural differences in the active site . Methodologically, researchers should perform comprehensive substrate screening using structurally related compounds at standardized concentrations (typically 0.1-10 mM depending on solubility). For each potential substrate, determine kinetic parameters (Km, kcat, and kcat/Km) to quantify specificity differences. These experiments should be performed under identical conditions for accurate cross-comparison. Structural alignment of the active sites from different homologs can provide insights into the amino acid residues responsible for observed specificity differences, which can be further validated through site-directed mutagenesis experiments.

How can researchers accurately determine kinetic parameters for STH1872 and its variants?

To accurately determine kinetic parameters for STH1872 and its variants, researchers should employ steady-state kinetic analyses with varying substrate concentrations (typically ranging from 0.2× to 5× the Km value). Methodologically, initial velocity measurements must be taken within the linear range of the reaction, usually within the first 10% of substrate consumption. For bi-substrate reactions, one substrate should be held at saturating concentrations while varying the other. Data should be fitted to appropriate kinetic models (Michaelis-Menten, ping-pong, or ordered bi-bi) using non-linear regression software. For accurate comparisons between wild-type and variants, all parameters should be determined under identical conditions. The table below illustrates how kinetic parameters might be presented, similar to the format used for enzyme variants in related studies:

What crystallization methods are most effective for obtaining STH1872 crystals suitable for X-ray diffraction?

For successful crystallization of STH1872, researchers should initially employ sparse matrix screening to identify promising crystallization conditions. Based on structural homology with related enzymes, vapor diffusion methods (hanging or sitting drop) typically yield the best results. Methodologically, purified protein should be concentrated to 10-15 mg/ml in a buffer containing PLP to ensure homogeneity. Co-crystallization with substrates, product analogs, or inhibitors often improves crystal quality by stabilizing the protein conformation. Crystal optimization techniques including microseeding, additive screening, and varying drop ratios should be systematically explored. For thermophilic proteins like STH1872, crystallization at elevated temperatures (20-30°C) may yield better results than the standard 4°C incubation. Crystals should be cryoprotected using glycerol, PEG, or sucrose before flash-freezing in liquid nitrogen for X-ray diffraction experiments.

How can researchers identify critical residues for substrate binding and catalysis in STH1872?

Identifying critical residues for substrate binding and catalysis requires a multi-faceted approach combining structural and functional analyses. Methodologically, begin with sequence alignment of STH1872 with homologous proteins of known structure/function to identify conserved residues. Based on homology with 2-amino-3-ketobutyrate CoA ligase, residues equivalent to Ser142, Tyr155, His221, and Glu282 might be particularly important, as these form hydrogen bonds with substrates in related enzymes . Create a series of single-point mutations targeting these residues, along with other conserved amino acids in the predicted active site. Characterize each variant using enzyme kinetics, determining changes in Km, kcat, and substrate specificity. Structural validation through X-ray crystallography or hydrogen-deuterium exchange mass spectrometry can further confirm the role of specific residues. The combined data should allow mapping of the substrate binding pocket and catalytic machinery of STH1872.

What computational approaches are valuable for predicting substrate binding modes in STH1872?

Computational approaches provide powerful tools for predicting substrate binding modes when experimental structures with bound substrates are unavailable. Methodologically, researchers should first generate a high-quality homology model of STH1872 using related structures such as E. coli 2-amino-3-ketobutyrate CoA ligase as templates, if a crystal structure is not available. Molecular docking simulations using software like AutoDock, GOLD, or Glide can then predict substrate binding poses, ranking them based on calculated binding energies. These predictions should be refined using molecular dynamics simulations (50-100 ns) to account for protein flexibility and solvent effects. Quantum mechanics/molecular mechanics (QM/MM) calculations can further elucidate the electronic structure of the active site and reaction mechanism. Results from computational studies should be validated experimentally through site-directed mutagenesis of predicted key residues followed by kinetic analyses to confirm their functional significance.

How can STH1872 be engineered for altered substrate specificity or enhanced catalytic efficiency?

Engineering STH1872 for altered properties requires systematic application of both rational design and directed evolution approaches. For rational design, researchers should first identify residues involved in substrate binding through structural analysis or homology modeling. Based on studies of related enzymes, amino acid positions equivalent to Ser142, Tyr155, His221, and Glu282 might be primary targets for mutagenesis . Methodologically, create focused libraries targeting these residues using site-directed mutagenesis or saturation mutagenesis techniques. For broader exploration, employ random mutagenesis methods like error-prone PCR or DNA shuffling to generate diverse variant libraries. Develop high-throughput screening assays specific to the desired property (altered specificity or enhanced activity) to efficiently evaluate variants. Beneficial mutations can then be combined through iterative rounds of screening or computational prediction. Successful engineering efforts should validate improved variants through comprehensive kinetic characterization and, ideally, structural analysis to understand the molecular basis of the enhanced properties.

What are the most effective methods for studying the reaction mechanism of STH1872?

Investigating the reaction mechanism of STH1872 requires integration of multiple biophysical and biochemical approaches. Pre-steady-state kinetics using stopped-flow or rapid-quench techniques can identify reaction intermediates and determine rate-limiting steps. Spectroscopic methods including UV-visible, fluorescence, and circular dichroism can monitor changes in the PLP cofactor during catalysis. Methodologically, researchers should employ isotope effects by comparing reaction rates with deuterated substrates versus normal substrates to identify bond-breaking steps in the rate-limiting transition state. Trapping and characterizing reaction intermediates through chemical quenching or low-temperature studies provides direct evidence of the reaction path. Additionally, X-ray crystallography of enzyme-substrate complexes captured at different reaction stages through the use of substrate analogs or cryocrystallography offers structural insights into the catalytic mechanism. Computational approaches like QM/MM can complement experimental data by calculating energy profiles for proposed mechanisms.

How can STH1872 be integrated into synthetic biology applications for metabolic engineering?

Integrating STH1872 into synthetic biology frameworks requires careful consideration of metabolic context and enzyme properties. Methodologically, researchers should first characterize the enzyme's behavior in heterologous expression systems, including activity, stability, and potential toxicity effects. For pathway design, computational metabolic modeling tools can predict flux distributions and identify potential bottlenecks when incorporating STH1872. When implementing the system, use tunable promoters and optimized ribosome binding sites to control expression levels and balance them with other pathway enzymes. For thermophilic enzymes like STH1872, consider compartmentalization strategies or host organisms capable of growth at elevated temperatures to maximize activity. Evaluation of the engineered system should include metabolite profiling using LC-MS or GC-MS, in vivo enzyme activity measurements, and quantification of pathway end products. Iterative optimization through feedback from these analyses will lead to more efficient synthetic pathways incorporating STH1872.

How can researchers address the issue of poor solubility or inclusion body formation when expressing STH1872?

Poor solubility and inclusion body formation are common challenges when expressing thermophilic proteins like STH1872 in mesophilic hosts. Methodologically, researchers should first optimize expression conditions by reducing growth temperature (16-20°C), lowering inducer concentration (0.1-0.2 mM IPTG), and using enriched media like Terrific Broth. Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ) often improves folding efficiency. If inclusion bodies persist, solubilization strategies include adding solubility tags (SUMO, MBP, or GST) or employing in vitro refolding protocols. For refolding, gradually remove denaturant through dialysis or dilution while maintaining reducing conditions and including PLP to facilitate correct folding. Alternative expression hosts like Brevibacillus choshinensis or cell-free protein synthesis systems may yield soluble protein when E. coli systems fail. Each approach should be evaluated by SDS-PAGE analysis of soluble versus insoluble fractions and enzymatic activity measurements.

What strategies can overcome substrate instability issues during STH1872 enzymatic assays?

Substrate instability, particularly with 2-amino-3-ketobutyrate, presents significant challenges for accurate enzymatic characterization. Methodologically, researchers should prepare fresh substrate solutions immediately before assays and keep them on ice. Consider synthesizing more stable substrate analogs that retain binding ability but demonstrate improved stability. Alternatively, develop coupled enzyme assays where the unstable substrate is generated in situ by an upstream enzyme like threonine dehydrogenase. When direct measurement is necessary, minimize the time between substrate preparation and assay by automating the process using rapid-mixing devices. Lower the assay temperature if compatible with enzyme activity to reduce substrate degradation rates. Quantify spontaneous substrate degradation rates under assay conditions and incorporate these values into kinetic calculations. For accurate measurements, validate results using multiple independent methods such as spectrophotometric assays, HPLC analysis, and mass spectrometry to ensure consistency.

How can inconsistent enzyme activity measurements for STH1872 be resolved?

Inconsistent activity measurements often stem from multiple sources including enzyme heterogeneity, assay variability, and cofactor issues. Methodologically, researchers should first assess protein homogeneity through analytical size exclusion chromatography and native PAGE to detect potential oligomeric state variations. Verify PLP incorporation by measuring the absorbance ratio A280/A420, with higher A420 indicating greater PLP saturation. Prepare larger batches of standardized buffers and reagents to reduce preparation variability, and use internal standards to normalize between experimental runs. For thermophilic enzymes, temperature control during assays is critical—use heated plate readers or water-jacketed spectrophotometers to maintain consistent temperatures. When comparing activities between studies, standardize reporting using specific activity (μmol/min/mg) rather than relative activities. Develop a standard operating procedure for enzyme storage, handling, and assaying to ensure reproducibility across different researchers and laboratories. Finally, statistical analysis of multiple independent experiments should be performed to establish confidence intervals for activity measurements.