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 cysteme synthase ( O-acetyl-L-serine (thtol)-lyase, EC 4.2.99.8) from plants and bacterta disclosed the presence of I2 conserved Lys residues. which can be candtdates for a functional bindmg site for pyrtdoxal phosphate cofactor. These 12 conserved Lys restdues in a cDNA clone encoding spmach cysteine synthase A were replaced with Gly by oligonucleotlde-directed in vttro mutagenests.These Lys -+ Gly mutated cDNAs were transferred mto ~s~~~~~~c~~ff coit NK3, a cysteme auxotroph lacking both cysteme synthase loci, qsK and cysM. One mutant replaced at Lys-49 could not complement the cysteme requirement of NK3, whereas other mutants and wild-type clone could. No enzymatic acttvity of cysteme synthase A was detected either in the cell-free extracts of E co11 NK3 transformed with the Lys-49 mutant These results mdtcated that Lys-49 IS a functional residue for the catalytic activity of cysteme synthase. Thts Lys residue is conserved in other evoluttonarily related amino actd-metabohzmg enzymes catalyzing reactions involvmg the p-carbon of ammo acids.

Characterization of Transsulfuration and Cysteine Biosynthetic Pathways in the Protozoan Hemoflagellate, Trypanosoma cruzi

“…Thus, trypanosomal CBS is one of the shortest CBS enzymes reported to date. The consensus sequence for the putative pyridoxal phosphate-binding domain and three of four lysine residues shown to be important for the catalytic activity of spinach CS-A by site-directed mutagenesis (35) are very well conserved in TcCBS.…”

Characterization of an Arabidopsis thaliana cDNA encoding an S‐adenosylmethionine‐sensitive threonine synthase Threonine synthase from higher plants

“…2 shows, there are two lysine residues (indicated by asterisks) that are conserved between the six sequences. Amino acid comparisons [7,31] between evolutionarily related PLP-dependent enzymes suggest that Lys-107 in E. coli threonine synthase is the PLP binding site. Consequently, Lys-202 in A. thaliana threonine synthase which is aligned with Lys-107 in E. coli threonine synthase may be linked to PLP.…”

Characterization of Two Genes Encoding Putative Cysteine Synthase Required for Cysteine Biosynthesis inSchizosaccharomyces pombe

“…The deduced amino acid sequence of cys1a þ and cys1b þ had 66% and 44% identity with that of A. nidulans cysB, respectively. Both of them had two lysine residues which bind the pyridoxal 5 0 -phosphate cofactor, 18) which are highly conserved among cysteine synthases of plants and bacteria (Fig. 2-i).…”