Point mutation of (+)-germacrene A synthase from Ixeris dentata.
Sesquiterpene cyclases catalyze the conversion of common precursor, farnesyl pyrophosphate, into various terpene backbones. X-ray crystallography of tobacco epi-aristolochene synthase has previously proposed a cyclization mechanism wherein the allylic carbocation intermediate is stabilized by the main chain carbonyl oxygens of three consecutive threonine residues. Alignment of amino acid sequences of plant terpene cyclases shows that the first position of the triad is almost invariably threonine or serine. To probe the carbocation-stabilizing role, the amino acid residues of the 433TSA435 triad in (+)-germacrene A synthase from Ixeris dentata were altered by site-directed mutagenesis. Enzyme kinetic measurements of the mutants and GC/MS analysis of the enzyme reaction products indicate that mutations of the triad decreased enzyme catalysis rather than substrate binding but did not affect its structural rearrangement in the catalytic mechanism. This is the first report that the hydroxyl group of threonine at the first position of the triad is required for the cyclase activity.
Chang
Yung-Jin
YJ
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
Jin
Jianming
J
Nam
Hee-Young
HY
Kim
Soo-Un
SU
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000502:physiology*; D000818:Animals; D019659:Asteraceae / Q000235:genetics*; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D002384:Catalysis; D018360:Crystallography, X-Ray; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D045788:Sesquiterpenes, Germacrane / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D010940:Plant Proteins; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; D019883:Alkyl and Aryl Transferases; C505068:germacrene A synthase, Ixeris dentata