122046-44-0Relevant articles and documents
The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis
Gorina, Svetlana S.,Grechkin, Alexander N.,Iljina, Tatiana M.,Mukhtarova, Lucia S.,Smirnova, Elena O.,Toporkova, Yana Y.
, (2020/09/16)
The CYP74 family of cytochromes P450 includes four enzymes of fatty acid hydroperoxide metabolism: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and epoxyalcohol synthase (EAS). The present work is concerned with catalytic specificities of three recombinant DESs, namely, the 9-DES (LeDES, CYP74D1) of tomato (Solanum lycopersicum), 9-DES (NtDES, CYP74D3) of tobacco (Nicotiana tabacum), and 13-DES (LuDES, CYP74B16) of flax (Linum usitatissimum), as well as their alterations upon the site-directed mutagenesis. Both LeDES and NtDES converted 9-hydroperoxides of linoleic and α?linolenic acids to divinyl ethers colneleic and colnelenic acids (respectively) with only minorities of HPL and EAS products. In contrast, LeDES and NtDES showed low efficiency towards the linoleate 13-hydroperoxide, affording only the low yield of epoxyalcohols. LuDES exhibited mainly the DES activity towards α?linolenate 13-hydroperoxide (preferred substrate), and HPL activity towards linoleate 13-hydroperoxide, respectively. In contrast, LuDES converted 9-hydroperoxides primarily to the epoxyalcohols. The F291V and A287G mutations within the I-helix groove region (SRS-4) of LuDES resulted in the loss of DES activity and the acquirement of the epoxyalcohol synthase activity. Thus, the studied enzymes exhibited the versatility of catalysis and its qualitative alterations upon the site-directed mutagenesis.
Epoxyalcohol Synthase RjEAS (CYP74A88) from the Japanese Buttercup (Ranunculus japonicus): Cloning and Characterization of Catalytic Properties
Toporkova,Fatykhova,Gorina,Mukhtarova,Grechkin
, p. 171 - 180 (2019/04/01)
Cytochromes P450 of the CYP74 family play a key role in the lipoxygenase cascade generating oxylipins (products of polyunsaturated fatty acid oxidation). The CYP74 family includes allene oxide synthases, hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases. In this work, we cloned the CYP74A88 gene from the Japanese buttercup (Ranunculus japonicus) and studied the properties of the encoded recombinant protein. The CYP74A88 enzyme specifically converts linoleic acid 9-and 13-hydroperoxides to oxiranyl carbinols 9,10-epoxy-11-hydroxy-12-octadecenoic acid and 11-hydroxy-12,13-epoxy-9-octadecenoic acid, respectively, which was confirmed by GC-MS analysis and kinetic studies. Therefore, the CYP74A88 enzyme is a specific epoxyalcohol synthase.
Replacement of two amino acids of 9R-dioxygenase-allene oxide synthase of Aspergillus niger inverts the chirality of the hydroperoxide and the allene oxide
Sooman, Linda,Wennman, Anneli,Hamberg, Mats,Hoffmann, Inga,Oliw, Ernst H.
, p. 108 - 118 (2016/01/08)
The genome of Aspergillus niger codes for a fusion protein (EHA25900), which can be aligned with ～50% sequence identity to 9S-dioxygenase (DOX)-allene oxide synthase (AOS) of Fusarium oxysporum, homologues of the Fusarium and Colletotrichum complexes and with over 62% sequence identity to homologues of Aspergilli, including (DOX)-9R-AOS of Aspergillus terreus. The aims were to characterize the enzymatic activities of EHA25900 and to identify crucial amino acids for the stereospecificity. Recombinant EHA25900 oxidized 18:2n-6 sequentially to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE) and to a 9R(10)-allene oxide. 9S- and 9R-DOX-AOS catalyze abstraction of the pro-R hydrogen at C-11, but the direction of oxygen insertion differs. A comparison between twelve 9-DOX domains of 9S- and 9R-DOX-AOS revealed conserved amino acid differences, which could contribute to the chirality of products. The Gly616Ile replacement of 9R-DOX-AOS (A. niger) increased the biosynthesis of 9S-HPODE and the 9S(10)-allene oxide, whereas the Phe627Leu replacement led to biosynthesis of 9S-HPODE and the 9S(10)-allene oxide as main products. The double mutant (Gly616Ile, Phe627Leu) formed over 90% of the 9S stereoisomer of HPODE. 9S-HPODE was formed by antarafacial hydrogen abstraction and oxygen insertion, i.e., the original H-abstraction was retained but the product chirality was altered. We conclude that 9R-DOX-AOS can be altered to 9S-DOX-AOS by replacement of two amino acids (Gly616Ile, Phe627Leu) in the DOX domain.
