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Purification and Characterization of Listeria monocytogenes HMG‐CoA Reductase
Author(s) -
Theivagt Amy Elizabeth,
Friesen Jon Alan
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a472-b
Subject(s) - reductase , listeria monocytogenes , enzyme , coenzyme a , nad+ kinase , biochemistry , hmg coa reductase , escherichia coli , cofactor , bacteria , chemistry , biology , gene , genetics
Listeria monocytogenes is a gram positive, rod‐shaped bacterium that causes the disease listeriosis, potentially fatal in humans, especially in immunocompromised individuals, pregnant women, and infants. HMG‐CoA Reductase (HMGR) catalyzes the NAD(P)H dependent reduction of HMG‐CoA to mevalonate. This enzyme is critical for cellular cholesterol synthesis in mammals and cell wall isoprenoid synthesis in certain pathogenic bacteria. We have isolated the gene encoding HMGR from Listeria monocytogenes and expressed the recombinant 6x‐His‐tagged form in Escherichia coli . The purified enzyme exhibited dual coenzyme specificity, utilizing both NADPH and NADH in catalysis. The enzyme efficiently catalyzed HMG‐CoA reduction; however, no activity was detected for mevalonate oxidation in vitro. Using NADPH, the enzyme exhibits a V max value of 19.77 μmoles/min/mg and a K’ (Hill constant) value of 2.19 μM for HMG‐CoA and a K’ value of 2.21 μM for NADPH. Using NADH, the enzyme exhibits a V max value of 0.656 μmoles/min/mg and a K’ value of 1.83 μM for HMG‐CoA and a K’ value of 0.97 μM for NADH. The statins mevinolin and mevastatin are weak inhibitors of L. monocytogenes HMGR in either the lactone or acid form as demonstrated by K i values in the micromolar range. Chemical crosslinking using the heterobifunctional crosslinker sulfo‐SMCC indicated the enzyme is multimeric, likely a tetramer.