THE ROLE OF ISOCHORISMATE HYDROXYMUTASE GENES ENTC AND MENF IN ENTEROBACTIN AND MENAQUINONE BIOSYNTHESIS IN ESCHERICHIA-COLI

Klebsiella pneumoniae 62-1, a triple mutant impaired in aromatic amino acid biosynthesis (Phe(-), Tyr(-), Trp(-)), excretes chorismic acid i nto the culture broth. When transformed with plasmids harbouring Esche richia coli genes entC or menF the mutant excretes a mixture of both c horismic and isochorismic acid indicating that not only entC but also menF encodes an isochorismate hydroxymutase (isochorismate synthase, E C 5.4.99.6) enzyme. These enzymes catalyze the first step in enterobac tin or menaquinone biosynthesis, respectively. Although both gene prod ucts (EntC and MenF) catalyze the same reaction, they play distinct ro les in the biosynthesis of menaquinone (MK8) and enterobactin. An E. c oli mutant (PBB7) with an intact menF but a disrupted entC produced me naquinone (MK8) but no enterobactin, whereas a mutant (PBB9) with an i ntact entC but a disrupted menF produced enterobactin and only a trace of menaquinone (MK8). When both menF and entC were disrupted (mutant PBB8) neither menaquinone (MK8) nor enterobactin was detectable. Our p revious assumption that entC is responsible for both menaquinone and e nterobactin biosynthesis is inconsistent with these mutant studies and has to be revised. The presence in the promoter region of menF of a p utative cAMP receptor protein binding site indicates that menF is regu lated differently from entC. The menF gene was overexpressed as a fusi on gene and its product (6 x His-tagged MenF) isolated. The enzyme cat alyzed the formation of isochorismic from chorismic acid and as oppose d to a previous publication also the reverse reaction. The enzyme was characterized and its kinetic data determined. (C) 1998 Elsevier Scien ce B.V. All rights reserved.