Trichodiene synthase. Probing the role of the highly conserved aspartate-rich region by site-directed mutagenesis.
Trichodiene synthase catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon trichodiene. The enzyme normally requires a divalent cation, Mg2+, which can be substituted by Mn2+. Trichodiene synthase from Fusarium sporotrichioides has a highly conserved aspartate rich region, aa 100-104 (DDSKD). Three mutants were constructed by site-directed mutagenesis in which each aspartate residue was individually replaced by glutamate. The mutants were each overexpressed and purified to homogeneity. The importance of Asp100 and Asp101 for catalysis was established by the observation of an increase in Km as well as a reduction in kcat in the corresponding Glu mutants. Replacement of the Asp104 residue with Glu had little effect on either Km or kcat. All three mutants produced anomalous sesquiterpene products in addition to trichodiene when incubated with farnesyl diphosphate. Interestingly, when Mg2+ was replaced by Mn2+ in the incubation buffer, the kcat/Km of both wild type trichodiene synthase and the D104E dropped significantly, while those of the other two mutants were not much affected. The proportion of anomalous products increased significantly when the D100E and D101E mutants were incubated in the presence of Mn2+. These observations all lend weight to the proposal that the aspartate residues mediate substrate binding by chelation of the divalent metal ion. Asp100 and Asp101 appear to play a relatively more important role than Asp104.
Cane
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Department of Chemistry, Brown University, Providence, Rhode Island 02912, USA. David_Cane@brown.edu
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Fitzsimons
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D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019755:Carbon-Carbon Lyases; D002384:Catalysis; D017124:Conserved Sequence; D053138:Cyclohexenes; D004926:Escherichia coli / Q000235:genetics; D005670:Fusarium / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism
D053138:Cyclohexenes; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D001224:Aspartic Acid; D008345:Manganese; C012075:trichodiene; C004808:farnesyl pyrophosphate; D008190:Lyases; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase; D008274:Magnesium