Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis.
Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).
Gebhardt
Yvonne Helen
YH
Institut für Pharmazeutische Biologie , Philipps-Universität Marburg, Marburg, Germany.
Witte
Simone
S
Steuber
Holger
H
Matern
Ulrich
U
Martens
Stefan
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019143:Evolution, Molecular; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D028528:Petroselinum / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D040681:Structural Homology, Protein
D006899:Mixed Function Oxygenases; C065998:flavone synthase I; C048797:flavanone 3-dioxygenase