title: Determination of a functional lysine residue of a plant cysteine synthase by site-directed mutagenesis, and the molecular evolutionary implications.
abstract: 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 Lys 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, cysK and 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 beta-carbon of amino acids.
authors.affiliation: Faculty of Pharmaceutical Sciences, Chiba University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Saito
authors.forename: M
authors.initials: M
authors.lastname: Kurosawa
authors.forename: I
authors.initials: I
authors.lastname: Murakoshi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D003547:Cysteine Synthase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000235:genetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000378:metabolism*; D010957:Plasmids; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D011732:Pyridoxal Phosphate; D003547:Cysteine Synthase; D008239:Lysine; D005998:Glycine