Biosynthesis of New Epoxy Fatty Acids from C18 Polyunsaturated Fatty
Acids by Linoleate 9-Lipoxygenase from Sphingopyxis
macrogoltabida
Abstract
Epoxy fatty acids (EFAs), which exist in the human body, are signaling
molecules that maintain homeostasis. They are involved in
anti-inflammation and are precursors of dihydroxy fatty acids. EFAs
derived from the C20 polyunsaturated fatty acid (PUFA) arachidonic acid
by lipoxygenases, such as
5,6-epoxy-7E,9E,11Z,14Z-eicosatetraenoic
acid and
8,9-epoxy-5Z,10E,12E,14Z-eicosatetraenoic
acid, are found in humans and mice, respectively. However, EFAs derived
from C18 PUFAs by lipoxygenases have not been identified to date. In
this study, the putative lipoxygenase gene of Spingopyxis
macrogoltabida was cloned and expressed in Escherichia coli. The
activity and catalytic efficiency
(kcat/Km) of the
recombinant enzyme were the highest for linoleic acid among the C18
PUFAs, including also α-linolenic acid and γ-linolenic acid. The product
obtained from the conversion of linoleic acid by the putative
lipoxygenase was identified as
9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE) by
high-performance liquid chromatography using 9-HODE and
13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE)
standards. These results indicate that the enzyme is a linoleate
9-lipoxygenase. The enzyme converted linoleic acid, α-linolenic acid,
and γ-linolenic acid into 9-HODE,
13-hydroxy-9Z,11E,15Z-octadecatrienoic acid, and
9-hydroxy-6Z,10E,12Z-octadecatrienoic acid,
respectively. Moreover, the enzyme also converted the three C18 PUFAs
into 9,10-epoxy-11E,13E-octadecadienoic acid,
12,13-epoxy-8E,10E,15Z-octadecatrienoic acid, and
9,10-epoxy-6Z,11E,13E-octadecatrienoic acid,
respectively, which were identified as new EFAs by liquid
chromatography-mass spectrometry/mass spectrometry. To our knowledge,
this is the first report on the biosynthesis of EFAs from C18 PUFAs via
a lipoxygenase.