4.2.3.128 H235P site-directed mutagenesis, the mutation converts Cop4 into a much more selective enzyme that produces (-)-germacrene D as the major cyclization product with 50% of total sesquiterpenes products. The mutant makes beta-ylangene, which is a diastereomer of beta-copaene and not synthesized by wild-type Cop4 713839 4.2.3.128 K233I site-directed mutagenesis, mutation of K233, interacting with the second Asp92 in the DDXXD motif of Cop4, does not significantly change the overall product promiscuity of Cop4, though beta-cubebene 4 does become the major product 713839 4.2.3.128 additional information directed mutations of the H-alpha1 loop have a marked effect on the product profile Cop4, loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. H-alpha1 loop swap between Cop4 and Cop6 shifts Cop4 to a germacrene D synthase 713839 4.2.3.128 N238L site-directed mutagenesis, the mutant shows a altered product profile compared to the wild-type enzyme with a slight reduction in beta-cubebene synthesis. The mutant does no longer show production of cubebol and has reduced (-)-germacrene D synthesis activity compared to the wild-type enzyme, synthesis of beta-cubebene, beta-copaene, delta-cadinene, and alpha-cubebene 713839 4.2.3.128 N239L site-directed mutagenesis, no production of beta-cubebene, the mutation converts Cop4 into a much more selective enzyme that produces (-)-germacrene D as the major cyclization product with 50% of total sesquiterpenes products. The mutant makes beta-ylangene, which is a diastereomer of beta-copaene and not synthesized by wild-type Cop4 713839 4.2.3.128 T236L site-directed mutagenesis, the mutant shows a altered product profile compared to the wild-type enzyme with an increase in beta-cubebene synthesis. The mutant does no longer show production of cubebol and (-)-germacrene D compared to the wild-type enzyme 713839