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Oxygenation reactions catalyzed by the F557V mutant of soybean Lipoxygenase-1: Evidence for two orientations of substrate binding.

Dillon J Hershelman,K. Kahler,Morgan J Price,Iris Lu,Yuhan Fu,P. Plumeri,Fred Karaisz,Natasha F. Bassett,P. M. Findeis,C. Clapp

Published 2019 in Archives of Biochemistry and Biophysics

ABSTRACT

Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD) or 9-hydroperoxy-10Z,12E-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl. Acad. Sci. USA96 (1999) 4192-4197) have identified an important residue, corresponding to phe-557 in soybean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of 13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced 33% 9-HPOD (S:R = 87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12% with the F557V, R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to 13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence that 13(S)-HPOD arises from tail-first binding.

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