Conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis.
Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.
Ohnuma
S
S
Department of Biochemistry and Engineering, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-77 Japan.
Nakazawa
T
T
Hemmi
H
H
Hallberg
A M
AM
Koyama
T
T
Ogura
K
K
Nishino
T
T
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002645:Chickens; D017931:DNA Primers / Q000737:chemistry; D051231:Farnesyltranstransferase; D051228:Geranyltranstransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase; D051231:Farnesyltranstransferase