Two conserved histidine residues are critical to the function of the TagF-like family of enzymes.
The TagF protein from Bacillus subtilis 168 is the poly(glycerol phosphate) polymerase responsible for the synthesis of wall teichoic acid and is the prototype member of a poorly understood family of similar teichoic acid synthetic enzymes. Here we describe in vitro and in vivo characterization of TagF, which localizes the active site to the carboxyl terminus of the protein and identifies residues that are critical for catalysis. We also establish the first mechanistic link among TagF and similar proteins by demonstrating that the identified residues are also critical in the function of TagB, a homologous enzyme implicated as the glycerophosphotransferase responsible for priming poly(glycerol phosphate) synthesis. We investigated the dependence of TagF activity on pH and showed that deprotonation of a residue with a pK(a) near neutral is critical for proper function. Alteration of histidine residues 474 and 612 by site-directed mutagenesis abolished TagF activity in vitro (5000-fold reduction in k(cat)/K(m)) while variants in four other conserved acidic residues showed minimal loss of activity. Complementation using H474A and H612A mutant alleles failed to suppress a lethal temperature-sensitive tagF defect in vivo despite confirmation of robust expression by Western blot. When corresponding mutations were made to the homologous tagB gene, these alleles were unable to suppress a tagB temperature-sensitive lethal phenotype. These results extend the mechanistic observations for TagF across a wider family of enzymes and provide the first biochemical evidence for the relatedness of these two enzymes.
Schertzer
Jeffrey W
JW
Antimicrobial Research Centre and Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
Bhavsar
Amit P
AP
Brown
Eric D
ED
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D015153:Blotting, Western; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D005816:Genetic Complementation Test; D005994:Glycerophosphates / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D017855:Transferases (Other Substituted Phosphate Groups) / Q000502:physiology*
D005994:Glycerophosphates; D011994:Recombinant Proteins; D006639:Histidine; D017855:Transferases (Other Substituted Phosphate Groups); C072175:CDP glycerol glycerophosphotransferase