An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase.
In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). 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 cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in 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±414 μM 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 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.
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Rd., Honolulu, HI, 96822, USA.
Jannai T
JT
Yafuso
Vishal Singh
VS
Negi
Jon-Paul
JP
Bingham
Dulal
D
Borthakur
D003545:Cysteine / Q000096:biosynthesis*; D003547:Cysteine Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology*; D008898:Mimosine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D008898:Mimosine; D003547:Cysteine Synthase; D003545:Cysteine