Identification of Functionally Important Residues of Arabidopsis thaliana S-Adenosylmethionine Decarboxylase

The Arabidopsis thaliana S-adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank U63633) was cloned, and the AdoMetDC protein was expressed, purified, and characterized. The Km value for S-adenosylmethionine (AdoMet) is 23.1μM and the K1 value for methylglyoxal bis-(guanylhydrazone) (MGBG) is 0.15 μM. Site-specific muta-genesis was performed on the AdoMetDC to introduce mutations at conserved cysteine (Cys50, Cys83, and Cys230) and lysine81 residues, chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. The AdoMetDC mutants K81A and C83A retained up to 60 and 10% of wild type activity, respectively, demonstrating that lysyl and sulfhydryl groups are required for full catalytic activity. However, changing Cys50 and Cys230 to alanine had minimal effects on the catalytic activity. Changing Lys81 to alanine produced an altered substrate specificity. When lysine was used as a substrate instead of AdoMet, the substrate specificity for lysine increased 6-fold. The Km value for AdoMet is 11-fold higher than that of the wild type, but the Vmax value is more than 60%. Taken together, the results suggest that the lysine81 residue is critical for substrate binding.