title: Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583.
abstract: The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60 kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent K(m) for pyruvate was calculated to be 1.37 mM, while the K(c) for thiamin diphosphate (ThDP) and Mg(2+) were found to be 0.031 μM and 1.27 mM, respectively. Negligible absorbance at 450 nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol.
authors.affiliation: Department of Chemistry and Research Institute of Natural Sciences, Hanyang University, Seoul 133-791, South Korea.
authors.forename: Sang-Choon
authors.initials: SC
authors.lastname: Lee
authors.forename: Jinheung
authors.initials: J
authors.lastname: Kim
authors.forename: Im-Joung
authors.initials: IJ
authors.lastname: La
authors.forename: Soon-Kil
authors.initials: SK
authors.lastname: Kim
authors.forename: Moon-Young
authors.initials: MY
authors.lastname: Yoon
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000096:Acetone / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology; D013696:Temperature; D013835:Thiamine Pyrophosphate / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012997:Solvents; D000096:Acetone; D005182:Flavin-Adenine Dinucleotide; D000095:Acetolactate Synthase; D013835:Thiamine Pyrophosphate; D004121:Dimethyl Sulfoxide