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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

Evolution of Flavone Synthase I from Parsley Flavanone 3-Hydroxylase by Site-Directed Mutagenesis^1,[W],[OA]

Institut für Pharmazeutische Biologie (Y.H.G., S.W., U.M., S.M.) and Institut für Pharmazeutische Chemie (H.S.), Philipps-Universität Marburg, 35037 Marburg, Germany

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 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 -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).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Stefan Martens (stefan.martens{at}staff.uni-marburg.de).

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.107.098392

^* Corresponding author; e-mail stefan.martens{at}staff.uni-marburg.de; fax 49–6421–2825366.

Received February 21, 2007; accepted May 16, 2007; published May 25, 2007.