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J. Biol. Chem., Vol. 282, Issue 2, 1257-1264, January 12, 2007

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The C-terminal Domain of the Escherichia coli WaaJ Glycosyltransferase Is Important for Catalytic Activity and Membrane Association^*

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

The waaJ gene encodes an -1,2-glucosyltransferase involved in the synthesis of the outer core region of the lipopolysaccha-ride of some Escherichia coli and Salmonella isolates. WaaJ belongs to glycosyltransferase CAZy family 8, characterized by the GT-A fold, a DXD motif, and by retention of configuration at the anomeric carbon of the donor sugar. Detailed kinetic and structural information for bacterial family 8 glycosyltransferases has resulted from studies of Neisseria meningitidis LgtC. As many as 28 amino acids could be deleted from the C terminus of LgtC without affecting its in vitro catalytic behavior. This C-terminal domain has a high ratio of positively charged and hydrophobic residues, a feature conserved in WaaJ and some other family 8 representatives. Unexpectedly, deletion of as few as five residues from the C terminus of WaaJ resulted in substantially reduced in vivo activity. With deletions of 15 residues or less, activity was only detected when levels of expression were elevated. No in vivo activity was detected after the removal of 20 amino acids, regardless of expression levels. Longer deletions (20 residues and greater) compromised the ability of WaaJ to associate with the membrane. However, the reduced in vivo activity in enzymes lacking 5–12 C-terminal residues also reflected a dramatic drop in catalytic activity in vitro (a 294-fold decrease in the apparent k_cat/K_m,_LPS). Deletions removing 20 or more residues resulted in a protein showing no detectable in vitro activity. Therefore, the C-terminal domain of WaaJ plays a critical role in enzyme function.

Received for publication, August 24, 2006 , and in revised form, October 17, 2006.

^* This work was supported by funds (to C. W.) from the Natural Sciences and Engineering Research Council of Canada. 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 Table I.

¹ Present address: Infection, Immunity, Injury and Repair Program, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

² To whom correspondence should be addressed. Tel.: 519-824-4120 (Ext. 53361); Fax: 519-837-1802; E-mail: cwhitfie{at}uoguelph.ca.

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