Isolation of cysteine biosynthetic cDNA and characterization of cy-o-acetylserine (thiol) lyase from Leucaena leucocephala

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Yafuso, Jannai Tetsuko
cysteine formation; OAS-TL
thesis / dissertation description
In plants, the final two steps of cysteine formation are catalyzed by O-acetylserine (thiol) lyase (OAS-TL) and serine acetyltransferase (SAT). The purpose of this study was to isolate and characterize an OAS-TL from the tree-legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a putative chloroplastic OAS-TL and cytosolic SAT were isolated through Reverse Transcriptase-PCR using degenerate primers. An additional cDNA for a cytosolic leucaena OAS-TL (cy-OAS-TL) isoform was previously obtained through interspecies suppression subtractive hybridization and was selected for further characterization. SAT and cy-OAS-TL were successfully over-expressed and purified from Escherichia coli. A 40-kDa recombinant cy-OAS-TL was further studied through enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the Km was calculated to be 1.850 ± 0.4146 mM sulfide and the Vmax was 200.6 ± 19.92 μM cysteine min-1. The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate 3-hydroxy-4-pyridone for mimosine formation. The His-tag-cleaved cy-OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as β-substituted alanine synthases.