The versatile CYP74 clan enzyme CYP440A19 from the European lancelet Branchiostoma lanceolatum biosynthesizes novel macrolactone, epoxydiene, and related oxylipins

Elena O Smirnova; Natalia V Lantsova; Mats Hamberg; Yana Y Toporkova; Alexander N Grechkin

doi:10.1016/j.bbalip.2024.159507

The versatile CYP74 clan enzyme CYP440A19 from the European lancelet Branchiostoma lanceolatum biosynthesizes novel macrolactone, epoxydiene, and related oxylipins

Biochim Biophys Acta Mol Cell Biol Lipids. 2024 May 11;1869(6):159507. doi: 10.1016/j.bbalip.2024.159507. Online ahead of print.

Authors

Elena O Smirnova¹, Natalia V Lantsova², Mats Hamberg³, Yana Y Toporkova², Alexander N Grechkin⁴

Affiliations

¹ Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, P.O. Box 261, 420111 Kazan, Russia. Electronic address: ye.o.smirnova@gmail.com.
² Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, P.O. Box 261, 420111 Kazan, Russia.
³ Unit of Integrative Metabolomics, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
⁴ Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center of RAS, P.O. Box 261, 420111 Kazan, Russia. Electronic address: grechkin@kibb.knc.ru.

PMID: 38740178
DOI: 10.1016/j.bbalip.2024.159507

Abstract

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.

Keywords: Cytochrome P450; Epoxyalcohols; Epoxydienes; Lancelet Branchiostoma lanceolatum; Macrolactones; Oxylipins.