Thermostable malate synthase of Streptomyces thermovulgaris.
The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006358:Hot Temperature; D007218:Industrial Microbiology / Q000379:methods*; D008292:Malate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D008292:Malate Synthase