Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583

Sang Choon Lee, Jinheung Kim, Im Joung La, Soon Kil Kim, Moon Young Yoon

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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.

Original languageEnglish
Pages (from-to)54-59
Number of pages6
JournalEnzyme and Microbial Technology
Volume52
Issue number1
DOIs
StatePublished - 10 Jan 2013

Keywords

  • Acetolactate synthase
  • Catabolic
  • Enterococcus faecalis
  • Flavin adenine dinucleotide
  • ThDP

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