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 La ctobacillus casei folylpoly-gamma-glutamate synthetase (FPGS) protein (residues 49 to 52), which is highly conserved when compared to the Es cherichia coil and human FPGS proteins. The amino acid sequence of thi s region, GKGS/T, is similar to the consensus sequence for the A regio n of a nucleotide binding site, a motif which encodes a phosphate-bind ing loop. Mutation G49A or K50R, with substitution to amino acids of s imilar size and charge, resulted in decreases in V-max/K-m of 40- to o ver 100-fold, depending on the variable substrate. Alteration of G51 t o S or T resulted in a large increase in the K-m for glutamate. The K- m for ATP was not affected more than 4-fold by any of the mutations. O ur studies indicate that the conserved region is essential for FPGS fu nction, since many of the mutations resulting in functionally conserva tive substitutions produced inactive enzymes. However, the mutations a ffected binding of all three substrates, so there is no direct evidenc e for involvement of the region in ATP binding. (C) 1994 Academic Pres s, Inc.