Determination of a functional lysine residue of a plant cysteine synthase by site‐directed mutagenesis, and the molecular evolutionary implications

FEBS Letters

Kurosawa, Makoto¹  Murakoshi, Isamu¹  Saito, Kazuki¹ 

[1]Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263, Japan

关键词: Cysteine synthase;    Genetic complementation;    Pyridoxal phosphate;    Spinacia oleracea;    CSase;    cysteine synthase;    PLP;    pyridoxal phosphate;   

DOI  :  10.1016/0014-5793(93)80976-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Comparison of seven deduced amino acid sequences of cysteine synthase (O-acetyl-l-serine (thiol)-lyase, EC 4.2.99.8) from plants and bacteria disclosed the presence of 12 conserved Lys residues, which can be candidates for a functional binding site for pyridoxal phosphate cofactor. These 12 conserved Eys residues in a cDNA clone encoding spinach cysteine synthase A were replaced with Gly by oligonucleotide-directed in vitro mutagenesis. These Lys → Gly mutated cDNAs were transferred into Escherichia coli NK3, a cysteine auxotroph lacking both cysteine synthase loci, cysKand cysM. One mutant replaced at Lys-49 could not complement the cysteine requirement of NK3, whereas other mutants and wild-type clone could. No enzymatic activity of cysteine synthase A was detected either in the cell-free extracts of E coli NK3 transformed with the Lys-49 mutant. These results indicated that Lys-49 is a functional residue for the catalytic activity of cysteine synthase. This Lys residue is conserved in other evolutionarily related amino acid-metabolizing enzymes catalyzing reactions involving the β-carbon of amino acids.

【 授权许可】

Unknown