Functional Analysis of Eubacterial ent-Copalyl Diphosphate Synthase and Pimara-9(11),15-Diene Synthase with Unique Primary Sequences

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Abstract
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 Mg 2+ and Zn 2+ for its activity. The optimum pH and temperature were 5.5 and 35°C. The K m value was calculated to be 13.7 ± 1.0 μM for GGDP and the k cat value was 3.3 × 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 K m value for ent -CDP was estimated to be 2.6 ± 0.2 μM and the k cat value was 1.4 × 10 −3 /sec.

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