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 60kDa. 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 Km for pyruvate was calculated to be 1.37mM, while the Kc for thiamin diphosphate (ThDP) and Mg2+ were found to be 0.031μM and 1.27mM, respectively. Negligible absorbance at 450nm 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.
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