A conserved histidine is essential for glycerolipid acyltransferase catalysis - PubMed (original) (raw)

A conserved histidine is essential for glycerolipid acyltransferase catalysis

R J Heath et al. J Bacteriol. 1998 Mar.

Abstract

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.

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Figures

FIG. 1

Acyltransferase domain sequence similarity. Sequences in the GenBank database from bacteria, yeast, plants, nematodes, and mammals with homology to the E. coli PlsB G3P acyltransferase (G3PAT), the E. coli PlsC lysophosphatidic acid acyltransferase (LPAAT), and 2-acyl-GPE acyltransferase (LPEAT) were aligned by using the PILEUP program in the Genetics Computer Group software package. Sequences are aligned according to overall similarity. •, invariant His and Asp; Φ, conserved hydrophobic residue. Genus names not specified in the text: A., Arabidopsis; S., Salmonella; M., Mus; H., Haemophilus; N., Neisseria; L., Limnanthes; Myc., Mycobacterium.

FIG. 2

Effects of substitutions at His306 and Asp311 on the G3P acyltransferase activity of PlsB. Membranes were prepared from strain SJ22 (plsB) harboring either a control plasmid (pACYC177) or a plasmid expressing either a wild-type or mutant plsB gene and assayed for G3P acyltransferase activity as described in Materials and Methods. (A) Effect of the PlsB[H306A] mutation, examined by assaying membranes from strain SJ22 (plsB) harboring pRJ22 (wild-type PlsB) (•), pRJ50 (PlsB[H306A]) (○) and pACYC177 (×); (B) G3P acyltransferase activity, measured in membranes prepared from strain 8 (chromosomal wild type for plsB) (•) or from strain SJ22 (plsB) harboring either pRJ52 (PlsB[D311E]) (○), pRJ51 (PlsB[D311A]) (▪), or pACYC177 (×).

FIG. 3

G3P _Km_s for the PlsB[H306A] and PlsB[D311A] mutants. Membranes were prepared from strain SJ22 (plsB) harboring either a control plasmid (pACYC177) or a plasmid expressing either the PlsB[H306A] (pRJ50) or PlsB[D311E] (pRJ52) mutant and assayed for G3P acyltransferase activity as a function of G3P concentrations as described in Materials and Methods. (A) Direct plot of activity versus G3P concentration for PlsB[H306A] (•) and PlsB[D311E] (○) mutants; (B) double-reciprocal analysis of the data for the PlsB[D311E] mutant. No activity was detected in the PlsB[H306A] mutant, and the apparent Km for G3P in the PlsB[D311E] mutant was 200 μM.

FIG. 4

Expression levels of wild-type and mutant epitope-tagged PlsB proteins. Flag-tagged PlsB wild-type and mutant expression vectors were transformed into strain SJ22 (plsB). Membranes were prepared, 10 μg of membrane protein was loaded into each lane, and the presence of the flag-tagged PlsB proteins was determined by immunoblotting as described in Materials and Methods. Expression of the PlsB protein was detected in membranes from cells transformed with plasmids expressing PlsB[D311E], PlsB[H306A], and PlsB. Protein expression was not detected with PlsB[D311A] and control membranes (pRJ22).

FIG. 5

The Aas[H36A] mutant lacks acyltransferase activity but retains acyl-ACP synthetase activity. Wild-type Aas (A) and Aas[H36A] (B), present in membranes prepared from LCH26 (aas) containing the appropriate plasmid, were assayed for both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities as described in Materials and Methods.

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A conserved histidine is essential for glycerolipid acyltransferase catalysis - PubMed (original) (raw)