Structural roles of cysteine 50 and cysteine 230 residues in Arabidopsis thaliana S-adenosylmethionine decarboxylase.
The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine Cys(50), Cys(83), and Cys(230), and lys(81) residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the Cys(50) and Cys(230) mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the Cys(50) and Cys(230) mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the Cys(50) and Cys(230) residues are structurally important.
Park
Sung-Joon
SJ
Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea.
Cho
Young-Dong
YD
D012437:Adenosylmethionine Decarboxylase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D018076:DNA, Complementary; D018744:DNA, Plant; D012437:Adenosylmethionine Decarboxylase; D008239:Lysine; D003545:Cysteine