title: Separation of enzymatic functions and variation of spin state of rice allene oxide synthase-1 by mutation of Phe-92 and Pro-430.
abstract: Rice allene oxide synthase-1 mutants carrying F92L, P430A or F92L/P430A amino acid substitution mutations were constructed, recombinant mutant and wild type proteins were purified and their substrate preference, UV-vis spectra and heme iron spin state were characterized. The results show that the hydroperoxide lyase activities of F92L and F92L/P430A mutants prefer 13-hydroperoxy substrate to other hydroperoxydienoic acids or hydroperoxytrienoic acids. The Soret maximum was completely red-shifted in P430A and F92L/P430A mutants, but it was partially shifted in the F92L mutant. ESR spectral data showed that wild type, F92L and P430A mutants occupied high and low spin states, while the F92L/P430A mutant occupied only low spin state. The extent of the red shift of the Soret maximum increased as the population of low spin heme iron increased, suggesting that the spectral shift reflects the high to low transition of heme iron spin state in rice allene oxide synthase-1. Relative to wild type allene oxide synthase-1, the hydroperoxide lyase activities of F92L and F92L/P430A are less sensitive to inhibition by imidazole with (13S or 9S)-hydroperoxydienoic acid as substrate and more sensitive than wild type with (13S)-hydroperoxytrienoic acid as substrate. Our results suggest that hydroperoxydienoic acid is the preferred substrate for the hydroperoxide lyase activity and (13S)-hydroperoxytrienoic acid is the preferred substrate for allene oxide synthase activity of allene oxide synthase-1.
authors.affiliation: Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea.
authors.forename: Sereyvath
authors.initials: S
authors.lastname: Yoeun
authors.affiliation: Zavoisky Physical - Technical Institute, Sibirsky tract, 10/7, Kazan 420029, Russia.
authors.forename: Andrey
authors.initials: A
authors.lastname: Sukhanov
authors.affiliation: Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea. Electronic address: oshan@chonnam.ac.kr.
authors.forename: Oksoo
authors.initials: O
authors.lastname: Han
mesh_terms: D004305:Dose-Response Relationship, Drug; D007093:Imidazoles / Q000494:pharmacology; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation; D012275:Oryza / Q000201:enzymology*; D010649:Phenylalanine / Q000235:genetics*; D011392:Proline / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D007093:Imidazoles; D010649:Phenylalanine; D011392:Proline; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase
keywords: Allene oxide synthase; Cytochrome P450; Hydroperoxide lyase; Oxylipin; Site-directed mutation