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J Bacteriol, March 1998, p. 1425-1430, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Conserved Histidine Is Essential for Glycerolipid Acyltransferase Catalysis

Richard J. Heath1 and Charles O. Rock1,2,*

Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105,1 and Department of Biochemistry, University of Tennessee, Memphis, Tennessee 381632

Received 5 September 1997/Accepted 23 December 1997

Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration. We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli. Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution. The invariant aspartic acid residue in the HX4D pattern was also important. The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.

* Corresponding author. Mailing address: Department of Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-3491. Fax: (901) 525-8025. E-mail: charles.rock{at}stjude.org.




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