Journal of Biochemistry Advance Access originally published online on December 5, 2006
Journal of Biochemistry 2007 141(1):37-45; doi:10.1093/jb/mvm004
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© 2006 The Japanese Biochemical Society.
Functional Analysis of Eubacterial ent-Copalyl Diphosphate Synthase and Pimara-9(11),15-Diene Synthase with Unique Primary Sequences
1Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan; and 2Faculty of Applied Bio-science, Tokyo University of Agriculture, Setagaya-ku, 156-8502, Japan
*To whom correspondence should be addressed at: Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan. Tel: +81-766-56-7500, Fax: +81-766-56-2498, E-mail: dairi{at}pu-toyama.ac.jp
Received October 19, 2006; Accepted November 13, 2006
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Abstract |
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We have previously cloned a DNA fragment that contained four ORFs and was confirmed to participate in viguiepinol {3-hydroxypimara-9(11),15-diene} biosynthesis by a heterologous expression experiment, from Streptomyces sp. strain KO-3988. Of the four ORFs, ORF2 and ORF4 were confirmed to encode an ent-CDP synthase and a GGDP synthase, respectively, by experiments using recombinant enzymes. In this study, ORF3, that did not show similarities with any other known proteins was expressed in Escherichia coli and used for functional analysis. The purified ORF3 product clearly converted ent-CDP into PMD. Since ORF2 and ORF3 are the first examples of enzymes with these biosynthetic functions from prokaryotes, enzymatic properties of both enzymes were investigated. ORF2 is likely to be a dimer and requires a divalent cation such as Mg2+ and Zn2+ for its activity. The optimum pH and temperature were 5.5 and 35°C. The Km value was calculated to be 13.7 ± 1.0 µM for GGDP and the kcat value was 3.3 x 10–2/sec. ORF3 is likely to be a monomer and also requires a divalent cation. The optimum pH and temperature were 7.0 and 30°C. The Km value for ent-CDP was estimated to be 2.6 ± 0.2 µM and the kcat value was 1.4 x 10–3/sec.
Key Words: ent-copalyl diphosphate synthase, diterpene, isoprenoid cyclase, pimara-9(11),15-diene synthase, Streptomyces
Abbreviations: CDP, copalyl diphosphate; FDP, farnesyl diphosphate; GGDP, geranylgeranyl diphosphate; MBP, maltose binding protein; ORF, open reading frame; PMD, pimara-9(11),15-diene; TDP, terpentedienol diphosphate
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