Characterization of O‐acetylserine (thiol) lyase from Leucaena leucocephala

Abstract

In plants, the final step of cysteine formation involves the addition of sulfide to O‐acetylserine (OAS) and the release of acetate, catlyzed by O‐acetylserine (thiol) lyase (OASTL). The purpose of this study was to isolate and characterize recombinant OASTL from Leucaena leucocephala (leucaena), a protein‐rich legume used as a fodder. Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also produced by OASTL. In order to understand the function of this enzyme, we first studied cysteine synthesis. The cDNA for a cytosolic OASTL was obtained through interspecies subtractive hybridization and Rapid Amplification of cDNA Ends. The sequence was codon optimized and expressed in Escherichia coli under the control of a T7 promoter. A 37‐kDa protein was isolated and used in in vitro enzyme activity assays with Na2S and OAS as substrates where cysteine was detected and quantified using High Performance Liquid Chromatography. The Km was 1.850±0.4146 mM Na2S and the Vmax was 200.6±19.92 μM cysteine minute−1. Central to the mimosine and cysteine biosynthetic pathways is OASTL. Characterization of this enzyme will be essential in the development of a toxin‐free leucaena for its use as a widespread forage crop. This research was funded by NSF Award No. CBET 08–27057.