Abstract
The genes encoding two dissociable components essential for Bacillus stearothermophilus heptaprenyl diphosphate synthase (all-trans-hexparenyl-diphosphate:isopentenyl-diphosphate hexaprenyl-trans-transferase, EC 2.5.1.30) were cloned, and their nucleotide sequences were determined. Sequence analyses revealed the presence of three open reading frames within 2,350 base pairs, designated as ORF-1, ORF-2, and ORF-3 in order of nucleotide sequence, which encode proteins of 220, 234, and 323 amino acids, respectively. Deletion experiments have shown that expression of the enzymatic activity requires the presence of ORF-1 and ORF-3, but ORF-2 is not essential. As a result, this enzyme was proved genetically to consist of two different protein compounds with molecular masses of 25 kDa (Component I) and 36 kDa (Component II), encoded by two of the three tandem genes. The protein encoded by ORF-1 has no similarity to any protein so far registered. However, the protein encoded by ORF-3 shows a 32% similarity to the farnesyl diphosphate synthase of the same bacterium and has seven highly conserved regions that have been shown typical in prenyltransferases (Koyama, T., Obata, S., Osabe, M., Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K. (1993) J. Biochem. (Tokyo) 113, 355-363).
Highlights
From the tBio Research Laboratory, Toyota Motor Corporation, Toyota-cho 1, Toyota, Aichi 471-71 and the §Institute for Chemical Reaction Science, Tohoku University, Aoba-ku, Sendai, Miyagi 980, Japan
The protein encoded by ORF-3 shows a 32% similarity to the farnesyl diphosphate synthase of the same bacterium and has seven highly conserved regions that have been shown typical in prenyltransferases (Koyama, T., Obata, S., Osabe, M., Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K. (1993) J
Product Analysis of the Reaction Catalyzed by Prenyltransferase Expressed in E. coli-Mter the enzymatic reaction at 55°C, the radioactive prenyl diphosphate in the reaction mixture was hydrolyzed to the corresponding alcohol with potato acid phosphate according to our method reported previously [21]
Summary
Vol 270, No 31, Issue of August 4, pp. 18396~18400, Printed in U.S.A. Molecular Cloning and Nucleotide Sequences of the Genes for Two Essential Proteins Constituting a Novel Enzyme System for Heptaprenyl Diphosphate Synthesis*. Deletion experiments have shown that expression of the enzymatic activity requires the presence of ORF-l and ORF-3, but ORF-2 is not essential As a result, this enzyme was proved genetically to consist of two different protein components with molecular masses of 25 kDa (Component I) and 36 kDa (Component II), encoded by two of the three tandem genes. In order to shed light on the significance of such an unusual two-component system for prenyl diphosphate synthesis and the role of each component in functional expression, we cloned and sequenced the genes encoding the HepPP synthase of Bacillus stearothermophilus. This is the first report of the genes encoding two essential proteins involved in prenyl diphosphate synthesis
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