The CYP74B34 Enzyme of Carrot (Daucus carota) with Double Hydroperoxyde Lyase/Epoxyalcohol Synthase Activity: Identification and Biochemical Properties

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Abstract

The lipoxygenase cascade of plants is a source of oxidized fatty acid derivatives, oxylipins, which play an important role in regulatory processes, as well as in the formation of responses to stress factors. One of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPL, synonym “hemiacetal synthase”) of the CYP74B subfamily. This work described the discovery and cloning of the CYP74B34 gene of the carrot (Daucus carota), as well as a description of the biochemical properties of the corresponding recombinant enzyme. The CYP74B34 enzyme was active towards 9- and 13- hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic acids (9-HPOT and 13-HPOT, respectively). CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldoacids (HPL products). The transformation of 13-HPOD led to the formation of aldoacids (as main products) and epoxyalcohols (as minor products). Epoxyalcohols are products of the epoxyalcohol synthase (EAS) activity. At the same time, 9-HPOD conversion resulted in the formation of the epoxyalcohols as main products and aldoacid as the minor one. Thus, the CYP74B34 enzyme is the first enzyme with double HPL/EAS activity described in carrot. The presence of corresponding catalytic activities was confirmed by the results of analyses of oxylipin profiles of roots of young seedlings and mature plants. In addition, the work describes the results of substitution of amino acid residues in one of the catalytically essential sites.

About the authors

Y. Y. Toporkova

Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”

Author for correspondence.
Email: kibmail@kibb.knc.ru

Kazan Institute of Biochemistry and Biophysics

Russian Federation, 420111 Kazan

S. S. Gorina

Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”

Email: kibmail@kibb.knc.ru

Kazan Institute of Biochemistry and Biophysics

Russian Federation, 420111 Kazan

T. M. Iljina

Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”

Email: kibmail@kibb.knc.ru

Kazan Institute of Biochemistry and Biophysics

Russian Federation, 420111 Kazan

N. V. Lantsova

Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”

Email: kibmail@kibb.knc.ru

Kazan Institute of Biochemistry and Biophysics

Russian Federation, 420111 Kazan

A. N. Grechkin

Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”

Email: kibmail@kibb.knc.ru

Kazan Institute of Biochemistry and Biophysics

Russian Federation, 420111 Kazan

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