Received February 21, 2007
Accepted May 16, 2007
Evolution of Flavone Synthase I from Parsley Flavanone 3
-Hydroxylase by Site-Directed Mutagenesis
Yvonne Helen Gebhardt , Simone Witte , Holger Steuber , Ulrich Matern , and Stefan Martens *
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstr. 17A, 35037 Marburg, Germany; Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35037 Marburg, Germany
* Corresponding author; email: stefan.martens{at}staff.uni-marburg.de.
Flavanone 3
-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 few Apiaceae species while FHTs are more abundant. Domain swapping experiments joining the N-terminus of parsley 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 vs. FNS I genes. Homology modelling 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 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 ‘-face’-configurated 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).
