Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli.
The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1). The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.
Daruwala
R
R
Department of Biological Sciences, Northern Illinois University, DeKalb 60115, USA.
Bhattacharyya
D K
DK
Kwon
O
O
Meganathan
R
R
D002413:Cations, Divalent; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases; D007535:Isomerases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008670:Metals; D008970:Molecular Weight; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D014812:Vitamin K / Q000096:biosynthesis*
D002413:Cations, Divalent; D008670:Metals; D014812:Vitamin K; D008623:Mercaptoethanol; D007535:Isomerases; D019751:Intramolecular Transferases; C058486:isochorismate synthase; D005033:Ethylmaleimide