5.5.1.13 A710C no change in reaction product profile compared to wild-type 714971 5.5.1.13 A710C site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS 714971 5.5.1.13 A710F mutant converts geranylgeranyl diphosphate to ent-kaurene as the sole product 714971 5.5.1.13 A710F site-directed mutagenesis, the mutant protein shows the same enzymic activity as wild-type Jungermannia subulata JsCPS/KS 714971 5.5.1.13 A710G no change in reaction product profile compared to wild-type 714971 5.5.1.13 A710G site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS 714971 5.5.1.13 A710L the substitution changes the ratio of ent-kaurene and 16alpha-hydroxy-entkaurane produced. The production of ent-kaurene is increased to the same level as that of 16alpha-hydroxyent-kaurane 714971 5.5.1.13 A710M mutant converts geranylgeranyl diphosphate to ent-kaurene as the sole product 714971 5.5.1.13 A710M site-directed mutagenesis, the mutant protein shows the same enzymic activity as wild-type Jungermannia subulata JsCPS/KS 714971 5.5.1.13 A710N no change in reaction product profile compared to wild-type 714971 5.5.1.13 A710N site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS 714971 5.5.1.13 A710S no change in reaction product profile compared to wild-type 714971 5.5.1.13 A710S site-directed mutagenesis, the mutant shows unaltered activity and reaction product profile compared to the wild-type Jungermannia subulata JsCPS/KS 714971 5.5.1.13 C717A site-directed mutagenesis, the mutant protein shows the same enzymic activity as wild-type Jungermannia subulata JsCPS/KS 714971 5.5.1.13 D320A the mutation leads to complete enzyme activity 492252 5.5.1.13 D503A the mutant exhibits a 7fold reduction in kcat 729301 5.5.1.13 D656A the mutation causes a small reduction in activity 492252 5.5.1.13 E211A the mutation results in a nearly 500fold reduction in kcat 729301 5.5.1.13 additional information construction of chimeric proteins of Physcomitrella patens PpCPS/KS with Jungermannia subulata, the chimeric cyclases Pp131-566 /Js574-886 and Pp131-504 /Js512-886 and produce only ent-kaurene. Chimeric cyclases, Pp131-622 /Js630-886 and Pp131-714 /Js722-886, lose all enzymic activity 714971 5.5.1.13 additional information Jungermannia subulata JsCPS/KS peptide fragments are replaced by the corresponding Physcomitrella patens PpCPS/KS region. A PCR-amplified Jungermannia subulata JsCPS/KS DNA fragment corresponding to amino acids 574-746 is replaced by Physcomitrella patens PpCPS/KS amino acids 566-740. Four chimeric cyclases, Physcomitrella patens Pp566/Js574-746/Pp740, Pp566/Js574-721/Pp715, Pp627/Js635-721/Pp715, and Pp666/Js674-721/Pp715, have enzymic activity and produce only ent-kaurene from geranylgeranyl diphosphate, like Jungermannia subulata JsCPS/KS. The chimeric cyclase Pp566/Js574-634/Pp628 shows the same activity as wild-type PpCPS/KS, converting geranylgeranyl diphosphate to both ent-kaurene and 16alpha-hydroxy-ent-kaurane. Overview mutant chimeric constructs and enzymatic activity 714971 5.5.1.13 additional information sequence contains a DVDD motif responsible for copalyl diphosphate synthase activity and a DDYFD motif responsible for ent-kaurene synthase activity. Mutation of DVDD motif to AVAD leads to loss of function, mutation of DDYFD motif to AAYFD results in accumulation of ent-copalyl diphosphate 679843 5.5.1.13 N425A the mutant exhibits a 13fold reduction in kcat 729301 5.5.1.13 R340A the mutant exhibits an 850fold reduction in kcat 729301 5.5.1.13 T421A the mutant exhibits a 163fold reduction in kcat (KM is increased 2fold) 729301 5.5.1.13 T421S the mutant exhibits a 3fold reduction in kcat 729301