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Determination of a functional lysine residue of a plant cysteine synthase by site‐directed mutagenesis, and the molecular evolutionary implications
Author(s) -
Saito Kazuki,
Kurosawa Makoto,
Murakoshi Isamu
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)80976-2
Subject(s) - cysteine , biochemistry , site directed mutagenesis , serine , amino acid , enzyme , atp synthase , biology , peptide sequence , mutant , mutagenesis , chemistry , gene
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, 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 β‐carbon of amino acids.