Biochemical Characterization, Kinetic Analysis and Molecular Modeling of Recombinant Vegetative Lipoxygenases from Soybean

Abstract

Plants in general have multiple lipoxygenase isoforms that exhibit distinct biochemical attributes and discrete cellular and sub-cellular localization patterns. This suggests that plant lipoxygenases are multifunctional and contribute to several biochemical and physiological processes during plant growth and development. Soybeans have at least eight isoforms and the three that are localized to the seed (L-1; L-2; L-3) have been characterized extensively and L-1 serves as a model. The vegetative organs, however, have five additional isoforms that are not well characterized at present. To better understand the cellular function of these vegetative lipoxygenases, kinetic parameters and pH profiles for the recombinant VLXs were determined for three free fatty acids above and below the corresponding critical micelle concentrations, including free fatty acids and the triglyceride trilinolein. The kinetic data using trilinolein as a substrate from two isoforms, VLX-C & VLX-D, supports a potential role for VLX-C & -D in the metabolism of lipids in soybean leaves as suggested by the significantly higher kcat/km and turnover of lipid substrates for these two isoforms. In addition, structural studies indicate clear differences exist between VLX-A, -B & -E and VLX-C & -D in the exposed loops of the ?-barrel, O2 cavity and entry site of the subcavity IIa. Therefore, based on distinct patterns of activity and structure, the existence of functional subgroups among the five VLX isoforms was confirmed.