Identification of a Novel Gene Cluster Participating in Menaquinone (Vitamin K2) Biosynthesis

Abstract

We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacil- lus stearothermophilus heptaprenyl diphosphate syn- thase, which catalyzes the synthesis of the prenyl side chain of menaquinone-7 of this bacterium. Sequence analyses revealed the presence of three open reading frames (ORFs), designated as ORF-1, ORF-2, and ORF-3, and the structural genes of the heptaprenyl diphosphate synthase were proved to consist of ORF-1 (heps-1) and ORF-3 (heps-2) (Koike-Takeshita, A., Koyama, T., Obata, S., and Ogura, K. (1995) J. Biol. Chem. 270, 18396–18400). The predicted amino acid sequence of ORF-2 (234 amino acids) contains a methyltransferase consensus sequence and shows a 22% identity with UbiG of Escherichia coli, which catalyzes S-adenosyl-L-methionine-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-me- thoxy-1,4-benzoquinone. These pieces of information led us to identify the ORF-2 gene product. The cell-free ho- mogenate of the transformant of E. coli with an expres- sion vector of ORF-2 catalyzed the incorporation of S- adenosyl-L-methionine into menaquinone-8, indicating that ORF-2 encodes 2-heptaprenyl-1,4-naphthoquinone methyltransferase, which participates in the terminal step of the menaquinone biosynthesis. Thus it is con- cluded that the ORF-1, ORF-2, and ORF-3 genes, desig- nated heps-1, menG, and heps-2, respectively, form an- other cluster involved in menaquinone biosynthesis in addition to the cluster of menB, menC, menD, and menE already identified in the Bacillus subtilis and E. coli chromosomes.