Recombinant Squalus acanthias L-lactate dehydrogenase A chain (ldha)

What structural features govern the catalytic activity of Squalus acanthias L-LDHA?

The catalytic activity of Squalus acanthias L-LDHA is defined by its conserved active site architecture, which includes critical residues such as His195, Arg171, and Asp168. These residues coordinate substrate binding and proton transfer during the interconversion of pyruvate and lactate . Structural refinements at 2.0 Å resolution revealed a sulfate ion bound near His195, forming hydrogen bonds with Asn140 and Asp168 . This sulfate occupies the putative pyruvate-binding site, mimicking the substrate’s carboxyl group. Researchers analyzing catalytic mechanisms should prioritize mutagenesis of these residues and employ competitive inhibition assays using sulfate analogs.

Key Structural Parameters from Refined Crystallographic Data

How can researchers resolve discrepancies in crystallographic data for L-LDHA?

Discrepancies in L-LDHA structural data often arise from pH-dependent ligand binding or sequence misassignments. For example, the original sequence (WNALKE, residues 207–211) in Squalus acanthias L-LDHA was revised to NVASIK after electron density analysis . To address such issues:

• Validate sequence alignment using de novo sequencing or cryo-EM density maps.

• Compare multiple datasets collected under varying conditions (e.g., pH 6.0 vs. 7.8) to assess ligand occupancy .

• Utilize restrained least-squares refinement to minimize model bias, especially for flexible loops (residues 97–123) .

What methodological approaches are optimal for expressing recombinant L-LDHA in bacterial systems?

The heterologous expression of Squalus acanthias L-LDHA in Escherichia coli requires codon optimization and stringent purification protocols. Key steps include:

• Vector design: Use pET or pGEX systems with inducible promoters (e.g., T7 or lacUV5).

• Codon optimization: Replace rare codons in the LDHA gene to match E. coli tRNA abundance.

• Affinity purification: Employ His-tag or GST-tag systems, followed by size-exclusion chromatography to remove aggregates .

• Activity validation: Perform coupled assays with NADH and pyruvate, monitoring absorbance at 340 nm .

How do post-translational modifications (PTMs) regulate L-LDHA activity in experimental models?

Lysine acetylation is a critical PTM that inhibits L-LDHA activity by disrupting substrate binding. In Streptococcus mutans, acetylation by ActA reduces lactic acid production by 40–60% . To study PTMs:

• Overexpress acetyltransferases (e.g., ActA) in host systems and monitor acetylation via anti-acetyllysine immunoblotting .

• Map acetylation sites using LC-MS/MS after tryptic digestion. Ten acetylation sites were identified in S. mutans LDH, seven of which are conserved in vertebrates .

• Correlate PTMs with activity: Use enzymatic assays under varying acetylation states (e.g., ±Ac-CoA) .

Impact of Acetylation on L-LDHA Activity

What advanced techniques are used to study L-LDHA’s role in hypoxia adaptation?

Comparative studies in hypoxia-tolerant organisms, such as Macrobrachium nipponense, reveal LDH’s regulation by hypoxia-inducible factor 1 (HIF-1). Methodologies include:

• Promoter analysis: Clone the LDHA promoter upstream of luciferase and quantify hypoxia-induced expression .

• RNA interference: Knock down HIF-1α/β subunits and measure LDHA mRNA/protein levels via qRT-PCR and Western blotting .

• Enzymatic profiling: Compare lactate production in normoxic vs. hypoxic conditions using spectrophotometric assays .

How can researchers address contradictions in LDHA kinetic data across studies?

Discrepancies in kinetic parameters (e.g., K_m for pyruvate) often stem from assay conditions. Standardize protocols by:

• Buffering systems: Use 50 mM Tris-HCl (pH 7.5) to avoid pH fluctuations during assays .

• Cofactor concentrations: Maintain NADH at 0.2–0.4 mM to prevent substrate inhibition .

• Temperature control: Conduct assays at 25°C for comparability with crystallographic data .

What strategies improve the stability of recombinant L-LDHA during storage?

Protein stability is enhanced by:

• Cryoprotectants: Add 20% glycerol and store at −80°C .

• Ligand stabilization: Incubate with 10 mM oxamate or sulfate to stabilize the active site .

• Avoid freeze-thaw cycles: Aliquot purified protein into single-use volumes.

How does conformational flexibility impact L-LDHA substrate specificity?

The “flexible loop” (residues 97–123) undergoes substrate-induced closure, positioning Arg105 and Gln102 for hydrogen bonding with pyruvate . To study flexibility:

• Perform molecular dynamics simulations using crystallographic coordinates (PDB: 6LDH) .

• Use fluorescence quenching to monitor loop movement upon substrate binding.