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J. Biol. Chem., Vol. 280, Issue 44, 36683-36690, November 4, 2005

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Two Conserved Histidine Residues Are Critical to the Function of the TagF-like Family of Enzymes^*

From the Antimicrobial Research Centre and Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

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 (Bhavsar, A. P., Truant, R., and Brown, E. D. (Sept. 2, 2005) J. Biol. Chem. 36691–36700). 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.

Received for publication, July 1, 2005 , and in revised form, August 15, 2005.

^* This work was supported by Canadian Institutes of Health Research Grant MOP-15496, a Canada Research Chair in Microbial Biochemistry (to E. D. B.), and Canadian Institutes of Health Research Canada Graduate Scholarships (to J. W. S. and A. P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables IS and IIS.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22392); Fax: 905-522-9033; E-mail: ebrown{at}mcmaster.ca.

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				J. W. Schertzer and E. D. Brown Use of CDP-Glycerol as an Alternate Acceptor for the Teichoic Acid Polymerase Reveals that Membrane Association Regulates Polymer Length J. Bacteriol., November 1, 2008; 190(21): 6940 - 6947. [Abstract] [Full Text] [PDF]

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