title: Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding.
abstract: Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, Ont., Canada M5S 1A8.
authors.forename: Yi
authors.initials: Y
authors.lastname: Sheng
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Cross
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Smith
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Bognar
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D018698:Glutamic Acid / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010453:Peptide Synthases / Q000378:metabolism*; D011622:Pterins / Q000378:metabolism*
chemical_list: D011622:Pterins; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase