Biochemical characterization of the decaprenyl diphosphate synthase of Rhodobacter sphaeroides for coenzyme Q10 production.
Coenzyme Q(10) (CoQ(10)), like other CoQs of various organisms, plays indispensable roles not only in energy generation but also in several other processes required for cells' survival. In this study, a gene encoding for a decaprenyl diphosphate synthase (Rsdds) was cloned from Rhodobacter sphaeroides in Escherichia coli. The in vivo catalytic activity and product specificity of Rsdds were compared with those of a counterpart enzyme from Agrobacterium tumefaciens (Atdds) in E. coli as a heterologous host. In contrast with Atdds, Rsdds showed lower catalytic activity but higher product specificity for CoQ(10) production, as indicated by the amount of CoQ(9) formation. The higher product specificity of Rsdds was also confirmed by utilizing both Rsdds and Atdds for in vitro synthesis of polyprenyl diphosphates. Thin layer chromatography indicated that the Rsdds enzyme resulted in relatively much less solanesyl diphosphate formation. The purified Rsdds catalyzed the addition of isopentenyl diphosphate to dimethyl allyl diphosphate, geranyl diphosphate, omega,E,E-farnesyl diphosphate (FPP), and omega,E,E,E-geranylgeranyl diphosphate as priming substrates. The kinetic parameters of V (max) (pmol/min), K (M) (microM), k (cat) (1/min), and k (cat) /K (M) of the enzyme using FPP as the most appropriate substrate were determined to be 264.6, 13.1, 8.8, and 0.67, respectively.
Zahiri
Hossein Shahbani
HS
Department of Microbiology, Gyeongsang National University, Gazwa-dong 900, Jinju 660-701, South Korea.
Noghabi
Kambiz Akbari
KA
Shin
Yong Chul
YC
D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D003067:Coenzymes; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D045782:Hemiterpenes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D012717:Sesquiterpenes / Q000378:metabolism; D013379:Substrate Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis
D003067:Coenzymes; D004269:DNA, Bacterial; D011756:Diphosphates; D004224:Diterpenes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C511282:geranyl diphosphate; D014451:Ubiquinone; C004809:isopentenyl pyrophosphate; C063778:solanesyl pyrophosphate; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C039821:decaprenyl pyrophosphate synthetase; C024989:coenzyme Q10; C030571:ubiquinone 9