Abstract
We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacillus stearothermophilus heptaprenyl diphosphate synthase, 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-methoxy-1,4-benzoquinone. These pieces of information led us to identify the ORF-2 gene product. The cell-free homogenate of the transformant of E. coli with an expression 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 concluded that the ORF-1, ORF-2, and ORF-3 genes, designated heps-1, menG, and heps-2, respectively, form another 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.
Highlights
We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacillus stearothermophilus heptaprenyl diphosphate synthase, 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 and ORF-3 (Koike-Takeshita, A., Koyama, T., Obata, S., and Ogura, K. (1995) J
The cell-free homogenate of the transformant of E. coli with an expression vector of ORF-2 catalyzed the incorporation of Sadenosyl-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
Summary
HepPP, heptaprenyl diphosphate; MK, three prenyl diphosphate synthases in Bacillus stearothermophilus. To identify the function of ORF-2 as the 2-heptaprenyl-1,4naphthoquinone methyltransferase gene, we constructed an expression system of ORF-2 and examined the enzymatic activity for catalysis of the terminal step in MK biosynthesis This is the first report on the cloning of the gene encoding the methyltransferase catalyzing the terminal step in MK biosynthesis as well as on the existence of another gene cluster involved in MK biosynthesis. Menaquinone; IPP, isopentenyl diphosphate; FPP, (E,E)-farnesyl diphosphate; PCR, polymerase chain reaction; ORF, open reading frame; SAM, S-adenosyl-L-methionine; 14C-SAM, S-adenosyl-L[methyl-14C]methionine. We examined complementation of the growth of AN70 in M9 medium by the expression vector of o251
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