Mutagenesis of the Lactobacillus casei folylpolyglutamate synthetase gene at essential residues resembling an ATP binding site.
Site-directed mutagenesis studies were performed on a region of the Lactobacillus casei folylpoly-gamma-glutamate synthetase (FPGS) protein (residues 49 to 52), which is highly conserved when compared to the Escherichia coli and human FPGS proteins. The amino acid sequence of this region, GKGS/T, is similar to the consensus sequence for the A region of a nucleotide binding site, a motif which encodes a phosphate-binding loop. Mutation G49A or K50R, with substitution to amino acids of similar size and charge, resulted in decreases in Vmax/Km of 40- to over 100-fold, depending on the variable substrate. Alteration of G51 to S or T resulted in a large increase in the Km for glutamate. The Km for ATP was not affected more than 4-fold by any of the mutations. Our studies indicate that the conserved region is essential for FPGS function, since many of the mutations resulting in functionally conservative substitutions produced inactive enzymes. However, the mutations affected binding of all three substrates, so there is no direct evidence for involvement of the region in ATP binding.
Department of Microbiology, University of Toronto, Ontario, Canada.
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Bognar
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D006801:Humans; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase