Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli

Abstract

The first committed step in the biosynthesis of menaquinone (vitamin K 2 ) 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°C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg . In the presence of 30 mM ␤-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.

Isochorismate Synthase (PchA), the First and Rate-limiting Enzyme in Salicylate Biosynthesis of Pseudomonas aeruginosa

“…Kinetic Properties of PchA-Like the entC and menF isoenzymes having ICS activity in Escherichia coli (30,31), the PchA enzyme of P. aeruginosa strictly depended on the presence of Mg 2ϩ . The PchA enzyme, pretreated with 1 mM EDTA and incubated in an incubation mixture without Mg 2ϩ , was inactive (Յ 1.1% activity).…”

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

“…Indeed, we found that overexpression of AtICS1 under control of its native promoter resulted in increased stressinduced SA accumulation suggesting that the amount of AtICS1 and not available chorismate limits induced SA synthesis. 5 The affinity of AtICS1 for chorismate also suggests the possibility that it can compete with a subset of chorismate-utilizing enzymes involved in "constitutive" aromatic amino acid and folate production. For example, AtICS1 could successfully compete for chorismate with all three characterized A. thaliana chorismate mutases (37, 38), whereas the recently characterized Arabidopsis aminodeoxychorismate synthase exhibits Ͼ10-fold higher affinity for chorismate than does AtICS1 (78).…”

“…For these ics1 transgenics, AtICS1-V5 expressed under control of the native AtICS1 promoter restored pathogen-induced SA biosynthesis, and induction of SA-dependent PR1 expression. 5 The size, shape, chlorophyll autofluorescence, and 4Ј,6-diamidino-2-phenylindole-staining of double-stranded DNA of the plastid shown in Fig. 3 are hallmarks of mesophyll chloroplasts.…”

“…In bacteria, ICS enzymes have been associated with either the synthesis of low molecular weight Fe 3ϩ chelators (siderophores) or menaquinones that function as electron acceptors. For example, Escherichia coli contains two ICS genes located in distinct operons encoding proteins with distinct biochemical properties: EntC is expressed under iron-limited conditions and associated with siderophore biosynthesis, and MenF is required for menaquinone biosynthesis and expressed under anaerobic conditions where it plays an essential role in anaerobic electron transport (2)(3)(4)(5). The initial steps in bacterial siderophore production from IC involve the synthesis of salicylic acid (2-hydroxybenzoic acid) or 2,3-dihydroxybenzoic acid; these compounds are then incorporated into siderophores such as pyochelin (Pseudomonas aeruginosa (6)) or enterobactin (E. coli (7)), respectively.…”

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

“…Genes encoding ICS have been cloned from bacteria such as Escherichia coli (Ozenberger et al, 1989), Pseudomonas aeruginosa (Serino et al, 1995), Aeromonas hydrophila (Barghouthi et al, 1991), Flavobacterium K 3-15 (Schaaf et al, 1993), Hemophilus influenzae (Fleischmann et al, 1995), and Bacillus subtilis (Rowland and Taber, 1996). Both E. coli and B. subtilis have two distinct ICS genes; one is involved in siderophore biosynthesis and the other is involved in menaquinone production (Daruwala et al, 1996(Daruwala et al, , 1997Mü ller et al, 1996;Rowland and Taber, 1996). The biochemical properties of the two ICS enzymes from E. coli are different (Daruwala et al, 1997;Liu et al, 1990).…”