本工作报道了欧洲柳叶刀(Branchiostomalanceolatum)的新CYP74氏族基因的检测和克隆以及重组蛋白CYP440A19的生化特征。CYP440A19对亚油酸和α-亚麻酸的13-氢过氧化物具有环氧醇合酶(EAS)活性,转化为环氧乙烷基甲醇,即,(11S,12R,13S)-11-羟基-12,13-环氧衍生物。9-氢过氧化物的转化产生不同的产物。亚油酸9(S)-过氧化氢(9-HPOD)主要转化为9,14-二醇(10E,12E)-9,14-二羟基-10,12-十八碳二烯酸和大内酯9(S),10(R)-环氧基-11(E)-十八碳烯-13(S)-内酯。此外,形成了(8Z)-松香酸。在己烷-水的双相系统中将酶与9-HPOD进行短暂孵育,从而可以分离出短寿命的9,10-环氧二烯(9S,10R,11E,13E)-9,10-环氧-11,13-十八碳二烯酸。环氧醇的结构和立体化学,大内酯,(8Z)-松酸(Me),和9,10-环氧二烯(Me)通过1H-NMR确认,1H-1H-COSY,1H-13C-HSQC,和1H-13C-HMBC光谱。大环内酯和顺式-9,10-环氧二烯是新产品。α-亚麻酸的9-氢过氧化物主要转化为大内酯9(S),10(R)-环氧-11(E),15(Z)-十八二烯-13(S)-内酯和少数二乙烯基醚,特别是(8Z)-亚油酸。酶催化的多功能性,以及CYP74和其他参与羟脂素生物合成的酶的多样性,证明了刺血针中脂加氧酶途径的复杂性。
The present work reports the detection and cloning of a new CYP74 clan gene of the European lancelet (Branchiostoma lanceolatum) and the biochemical characterization of the recombinant protein CYP440A19. CYP440A19 possessed epoxyalcohol synthase (EAS) activity towards the 13-hydroperoxides of linoleic and α-linolenic acids, which were converted into oxiranylcarbinols, i.e., (11S,12R,13S)-11-hydroxy-12,13-epoxy derivatives. The conversion of 9-hydroperoxides produced distinct products. Linoleic acid 9(S)-hydroperoxide (9-HPOD) was mainly converted into 9,14-diol (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid and macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In addition, (8Z)-colneleic acid was formed. Brief incubations of the enzyme with 9-HPOD in a biphasic system of hexane-water enabled the isolation of the short-lived 9,10-epoxydiene (9S,10R,11E,13E)-9,10-epoxy-11,13-octadecadienoic acid. The structure and stereochemistry of the epoxyalcohols, macrolactone, (8Z)-colneleic acid (Me), and 9,10-epoxydiene (Me) were confirmed by 1H-NMR, 1H-1H-COSY, 1H-13C-HSQC, and 1H-13C-HMBC spectroscopy. Macrolactone and cis-9,10-epoxydiene are novel products. The 9-hydroperoxide of α-linolenic acid was mainly converted into macrolactone 9(S),10(R)-epoxy-11(E),15(Z)-octadecadiene-13(S)-olide and a minority of divinyl ethers, particularly (8Z)-colnelenic acid. The versatility of enzyme catalysis, as well as the diversity of CYP74s and other enzymes involved in oxylipin biosynthesis, demonstrates the complexity of the lipoxygenase pathway in lancelets.