OPEN ACCESS ARTICLE

This Article Free via Open Access: OA OA Full Text Full Text (PDF) Supplemental Data OA All Versions of this Article: 144/3/1442    most recent pp.107.098392v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Gebhardt, Y. H. Articles by Martens, S. Search for Related Content PubMed PubMed Citation Articles by Gebhardt, Y. H. Articles by Martens, S. Agricola Articles by Gebhardt, Y. H. Articles by Martens, S.

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.

^[OA] Open Access articles can be viewed online without a subscription.

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.