Journal Article

Interconversion of the Product Specificity of Type I Eubacterial Farnesyl Diphosphate Synthase and Geranylgeranyl Diphosphate Synthase through One Amino Acid Substitution

Takashi Kawasaki, Yoshimitsu Hamano, Tomohisa Kuzuyama, Nobuya Itoh, Haruo Seto and Tohru Dairi

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 133, issue 1, pages 83-91
Published in print January 2003 | ISSN: 0021-924X
Published online January 2003 | e-ISSN: 1756-2651 | DOI: https://dx.doi.org/10.1093/jb/mvg002
Interconversion of the Product Specificity of Type I Eubacterial Farnesyl Diphosphate Synthase and Geranylgeranyl Diphosphate Synthase through One Amino Acid Substitution

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Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) Biosci. Biotechnol. Biochem. 65, 1627–1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the –50th, –8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity.

Keywords: Key words: farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, product specificity, site-directed mutagenesis, Streptomyces.; Abbreviations: DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; GDP, (E)-geranyl diphosphate; FDP, (E,E)-farnesyl diphosphate; FDPSase, farnesyl diphosphate synthase; GGDP, (E,E,E)-geranylgeranyl diphosphate; GGDPSase, geranylgeranyl diphosphate synthase; ORF, open reading frame.

Journal Article.  5938 words.  Illustrated.

Subjects: Biochemistry

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