Abstract
Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations
were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a kcat/KM of 0.62 mM−1 s−1 , which is nearly identical to that of the
parent Cop2, which had a kcat /KM of 0.58 mM−1 s−1
were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a kcat/KM of 0.62 mM−1 s−1 , which is nearly identical to that of the
parent Cop2, which had a kcat /KM of 0.58 mM−1 s−1
Originalsprache | englisch |
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Seiten (von - bis) | 4013-4020 |
Fachzeitschrift | Organic & Biomolecular Chemistry |
Jahrgang | 12 |
DOIs | |
Publikationsstatus | Veröffentlicht - 2014 |
Extern publiziert | Ja |
Fields of Expertise
- Human- & Biotechnology