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Thematic Review Series: Glycerolipids. Acyltransferases in bacterial glycerophospholipid synthesis

2008; Elsevier BV; Volume: 49; Issue: 9 Linguagem: Inglês

10.1194/jlr.r800005-jlr200

1539-7262

Yongmei Zhang, Charles O. Rock,

Lipid metabolism and biosynthesis

Phospholipid biosynthesis is a vital facet of bacterial physiology that begins with the synthesis of the fatty acids by a soluble type II fatty acid synthase. The bacterial glycerol-phosphate acyltransferases utilize the completed fatty acid chains to form the first membrane phospholipid and thus play a critical role in the regulation of membrane biogenesis. The first bacterial acyltransferase described was PlsB, a glycerol-phosphate acyltransferase. PlsB is a key regulatory point that coordinates membrane phospholipid formation with cell growth and macromolecular synthesis. Phosphatidic acid is then produced by PlsC, a 1-acylglycerol-phosphate acyltransferase. These two acyltransferases use thioesters of either CoA or acyl carrier protein (ACP) as the acyl donors and have homologs that perform the same reactions in higher organisms. However, the most prevalent glycerol-phosphate acyltransferase in the bacterial world is PlsY, which uses a recently discovered acyl-phosphate fatty acid intermediate as an acyl donor. This unique activated fatty acid is formed from the acyl-ACP end products of the fatty acid biosynthetic pathway by PlsX, an acyl-ACP:phosphate transacylase. Phospholipid biosynthesis is a vital facet of bacterial physiology that begins with the synthesis of the fatty acids by a soluble type II fatty acid synthase. The bacterial glycerol-phosphate acyltransferases utilize the completed fatty acid chains to form the first membrane phospholipid and thus play a critical role in the regulation of membrane biogenesis. The first bacterial acyltransferase described was PlsB, a glycerol-phosphate acyltransferase. PlsB is a key regulatory point that coordinates membrane phospholipid formation with cell growth and macromolecular synthesis. Phosphatidic acid is then produced by PlsC, a 1-acylglycerol-phosphate acyltransferase. These two acyltransferases use thioesters of either CoA or acyl carrier protein (ACP) as the acyl donors and have homologs that perform the same reactions in higher organisms. However, the most prevalent glycerol-phosphate acyltransferase in the bacterial world is PlsY, which uses a recently discovered acyl-phosphate fatty acid intermediate as an acyl donor. This unique activated fatty acid is formed from the acyl-ACP end products of the fatty acid biosynthetic pathway by PlsX, an acyl-ACP:phosphate transacylase. Bacterial phospholipid synthesis is a vital facet of bacterial physiology, and the phospholipid head group structures found in the bacterial world come in a truly bewildering variety (1Cronan Jr., J.E. Bacterial membrane lipids: where do we stand?.Annu. Rev. Microbiol. 2003; 57: 203-224Crossref PubMed Scopus (265) Google Scholar). Phosphatidic acid is a universal intermediate in the biosynthesis of these membrane glycerophospholipids in eubacteria, and this review focuses on the two acyltransferase steps that are common reactions in all glycerophospholipid biosynthesis in bacteria, the glycerol-phosphate and 1-acylglycerol-phosphate (LPA) acyltransferases. These enzymes sit at the interface between the soluble type II fatty acid biosynthetic pathway and the creation of a phospholipid molecule that drives membrane expansion. This pivotal position makes the glycerol-phosphate acyltransferases key regulators of both fatty acid and phospholipid synthesis and has spurred considerable research into the function, selectivity, and regulation of the acyltransferase systems. This review will cover the two acyltransferase systems involved in bacterial glycerophospholipid synthesis, the origin and utilization of acyl donors by these pathways, and their roles in regulating membrane biogenesis. The most important acyl donor in bacterial glycerolipid synthesis is acyl-acyl carrier protein (ACP). ACP is a 9 kDa protein that is the acyl group carrier in type II fatty acid synthesis and shuttles the intermediates attached to the sulfhydryl group at the terminus of its 4′-phosphopantetheine prosthetic group between the pathway enzymes (2Zhang Y-M. Marrakchi H. White S.W. Rock C.O. The application of computational methods to explore the diversity and structure of bacterial fatty acid synthase.J. Lipid Res. 2003; 44: 1-10Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 3Rock C.O. Jackowski S. Forty years of fatty acid biosynthesis.Biochem. Biophys. Res. Commun. 2002; 292: 1155-1166Crossref PubMed Scopus (169) Google Scholar). These acyl donors are the end products of the bacterial dissociated type II fatty acid synthesis pathway, and in most bacteria, type II fatty acid synthesis is the sole source of fatty acids for membrane phospholipid synthesis. An acyl-ACP intermediate has two possible fates. It can reenter the fatty acid elongation cycle and be extended by two carbons by the action of the elongation-condensing enzymes of fatty acid synthesis, or the acyl-ACP can be used by the acyltransferase system. The fate of a particular acyl-ACP chain length is determined by the competition between the elongation-condensing enzyme and the PlsB glycerol-phosphate acyltransferase for this intermediate based on their opposing substrate specificities. The 16–18 carbon acyl-ACPs are poorer substrates for the elongation-condensing enzymes than their shorter chain precursors (4Cronan Jr., J.E. Rock C.O. Biosynthesis of membrane lipids.in: Neidhardt F.C. Curtis R. Gross C.A. In Escherichia coliSalmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 612-636Google Scholar), whereas 16 and 18 carbon acyl-ACPs are the preferred substrates for the acyltransferases (5Rock C.O. Goelz S.E. Cronan Jr., J.E. Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol-3-phosphate.J. Biol. Chem. 1981; 256: 736-742Abstract Full Text PDF PubMed Google Scholar), thus accounting for the preponderance of 16–18 carbon fatty acids in bacterial membrane phospholipids. The importance of the relative activities of the competing enzymes, as well as their substrate specificities, are revealed by in vivo experiments in Escherichia coli showing that the inactivation of PlsB results in the accumulation of abnormally long-chain acyl-ACPs due to the continued elongation by the condensing enzymes of fatty acid synthesis (6Cronan Jr., J.E. Weisberg L.J. Allen R.G. Regulation of membrane lipid synthesis in Escherichia coli. Accumulation of free fatty acids of abnormal length during inhibition of phospholipid synthesis.J. Biol. Chem. 1975; 250: 5835-5840Abstract Full Text PDF PubMed Google Scholar), whereas overproduction of the elongation-condensing enzyme FabB leads to a higher proportion of 18 carbon fatty acids in the membrane (7Garwin J.L. Klages A.L. Cronan Jr., J.E. β-Ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis.J. Biol. Chem. 1980; 255: 3263-3265Abstract Full Text PDF PubMed Google Scholar). Long-chain acyl-ACP is not commercially available but is efficiently synthesized from ACP using the acyl-ACP synthetase reaction (8Rock C.O. Garwin J.L. Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein.J. Biol. Chem. 1979; 254: 7123-7128Abstract Full Text PDF PubMed Google Scholar). The idea that there are physical interactions between the enzymes of type II fatty acid synthesis enzymes, between the membrane-associated acyltransferases, or between the type II enzymes and the acyltransferases is intriguing, but to date there is no experimental support for this hypothesis. A surprising recent finding was that the most widely distributed bacterial glycerol-phosphate acyltransferase system (PlsY) uses a novel acyl donor, acyl-phosphate (acyl-PO4), produced by the PlsX enzyme (Fig. 1). Acyl-PO4 is a mixed anhydride of phosphoric acid and a fatty acid that was first synthesized in 1945 by Lehninger (9Lehninger A.L. Synthesis and properties of the acyl phosphates of some higher fatty acids.J. Biol. Chem. 1945; 162: 333-342Abstract Full Text PDF Google Scholar), who prepared these fatty acid derivatives to determine whether they had a role in fatty acid metabolism in mammalian cell extracts. Acyl-PO4 exhibits a higher degree of instability in water than their thioester counterparts (9Lehninger A.L. Synthesis and properties of the acyl phosphates of some higher fatty acids.J. Biol. Chem. 1945; 162: 333-342Abstract Full Text PDF Google Scholar). However, long-chain acyl-PO4 has a half-life of 12 h at pH 7.4 and 37°C (compared with 3 h for acetyl-PO4) (9Lehninger A.L. Synthesis and properties of the acyl phosphates of some higher fatty acids.J. Biol. Chem. 1945; 162: 333-342Abstract Full Text PDF Google Scholar) and is clearly sufficiently stable to play its role as an ephemeral metabolic intermediate. PlsX generates the acyl-PO4 intermediates from the acyl-ACP end products of fatty acid synthesis in a reaction analogous to phosphotransacetylase (10Lu Y-J. Zhang Y-M. Grimes K.D. Qi J. Lee R.E. Rock C.O. Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens.Mol. Cell. 2006; 23: 765-772Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The plsX gene was first recognized as a second site mutation required for the glycerol-phosphate auxotrophic phenotype of plsB mutants (11Larson T.J. Ludtke D.N. Bell R.M. sn-Glycerol-3-phosphate auxotrophy of plsB strains of Escherichia coli: evidence that a second mutation, plsX, is required.J. Bacteriol. 1984; 160: 711-717Crossref PubMed Google Scholar). These data suggested that plsX either encoded a second acyltransferase or was involved in glycerol-phosphate metabolism. However, only the Km defective acyltransferase activity was detected in membranes prepared from cells possessing a wild-type plsX and a mutant plsB gene, suggesting that PlsX is not an alternative glycerol-phosphate acyltransferase. The plsX gene is most often found associated with genes encoding enzymes of bacterial fatty acid synthesis, reflecting the connection between PlsX and fatty acid biosynthesis. For example, in E. coli, a fab gene cluster consists of plsX-fabH-fabD-fabG-acpP-fabF (12Oh W. Larson T.J. Physical location of genes in the rne(ams)-rpmF-plsX-fab region of the Escherichia coli K-12 chromosome.J. Bacteriol. 1992; 174: 7873-7874Crossref PubMed Google Scholar). The PlsX reaction is readily reversible. PlsX is a soluble protein and its crystal structure is known (Protein Data Bank accession numbers 1vi1 and 1u7n), although there is no information available on the role of specific residues in substrate binding or catalysis. However, the PlsX protein appears to associate with the Bacillus subtilis cell membrane in vivo (13Paoletti L. Lu Y-J. Schujman G.E. de Mendoza D. Rock C.O. Coupling of fatty acid and phospholipid synthesis in Bacillus subtilis.J. Bacteriol. 2007; 189: 5816-5824Crossref PubMed Scopus (70) Google Scholar), although the molecular determinants of PlsX-membrane interactions have not been explored. Bioinformatic database searches do not identify any PlsX homologs in mammalian systems. Some bacterial acyltransferases can use acyl-CoA as an alternative acyl donor in addition to acyl-ACP. In bacteria, acyl-CoAs are derived from exogenous fatty acids that are converted to their acyl-CoA derivatives via an acyl-CoA synthetase (FadD) following their entry into the cell (14Black P.N. DiRusso C.C. Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.Microbiol. Mol. Biol. Rev. 2003; 67: 454-472Crossref PubMed Scopus (181) Google Scholar). In both Gram-negative (15DiRusso C.C. Black P.N. Weimar J.D. Molecular inroads into the regulation and metabolism of fatty acids, lessons from bacteria.Prog. Lipid Res. 1999; 38: 129-197Crossref PubMed Scopus (113) Google Scholar) and Gram-positive (16Matsuoka H. Hirooka K. Fujita Y. Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation.J. Biol. Chem. 2007; 282: 5180-5194Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) bacteria, the FadD enzymes are associated with an inducible β-oxidation system that allows exogenous fatty acids to be used as a carbon source for growth. Thus, tagging the fatty acid with either CoA or ACP thioesters serves as a molecular marker to distinguish acyl chains destined for degradation from those intended for phospholipid biosynthesis, respectively. Enzymes that transfer long-chain acyl chains between acyl-CoA and acyl-ACP are not present in the bacterial models examined to date, meaning that bacteria may not generally permit exogenous fatty acids to enter the fatty acid biosynthetic pathway and do not have a mechanism to divert newly synthesized products of de novo fatty acid synthesis for degradation. In E. coli, both PlsB and PlsC use acyl-CoA thioesters as readily as acyl-ACPs, and this dual substrate specificity is extrapolated to be the pattern of substrate selectivity in the γ-proteobacteria. The advantage of acyl-CoA utilization by the acyltransferases in these organisms is that exogenous fatty acids can be used for membrane phospholipid formation in lieu of the energy-expensive fatty acid biosynthesis pathway (4Cronan Jr., J.E. Rock C.O. Biosynthesis of membrane lipids.in: Neidhardt F.C. Curtis R. Gross C.A. In Escherichia coliSalmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 612-636Google Scholar, 17Polacco M.L. Cronan Jr., J.E. Mechanism of the apparent regulation of Escherichia coli unsaturated fatty acid synthesis by exogenous oleic acid.J. Biol. Chem. 1977; 252: 5488-5490Abstract Full Text PDF PubMed Google Scholar). In contrast, the PlsC proteins from Streptococcus pneumoniae (10Lu Y-J. Zhang Y-M. Grimes K.D. Qi J. Lee R.E. Rock C.O. Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens.Mol. Cell. 2006; 23: 765-772Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and B. subtilis (13Paoletti L. Lu Y-J. Schujman G.E. de Mendoza D. Rock C.O. Coupling of fatty acid and phospholipid synthesis in Bacillus subtilis.J. Bacteriol. 2007; 189: 5816-5824Crossref PubMed Scopus (70) Google Scholar) do not accept acyl-CoA as an acyl donor, indicating that Gram-positive bacteria have acyltransferases that cannot utilize acyl-CoA. In the case of S. pneumoniae, this property correlates with the absence of a β-oxidation system and recognizable acyl-CoA synthetases in the genome. On the other hand, B. subtilis has two acyl-CoA synthetases and a β-oxidation pathway (16Matsuoka H. Hirooka K. Fujita Y. Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation.J. Biol. Chem. 2007; 282: 5180-5194Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), but nonetheless, the PlsC from this bacteria does not accept acyl-CoA as substrate (13Paoletti L. Lu Y-J. Schujman G.E. de Mendoza D. Rock C.O. Coupling of fatty acid and phospholipid synthesis in Bacillus subtilis.J. Bacteriol. 2007; 189: 5816-5824Crossref PubMed Scopus (70) Google Scholar). Thus, the major route for exogenous fatty acid/acyl-CoA utilization in bacteria is β-oxidation, and the ability of acyltransferases to interchangeably utilize either acyl-ACP or acyl-CoA may potentially be restricted to γ-proteobacteria. More examples of acyl-ACP-specific and dual-specificity acyltransferases need to be definitively characterized in order for refined bioinformatic tools to be developed that can predict the type of acyltransferase based on sequence information alone. Both acyl-ACP and acyl-CoA have important roles in metabolic regulation in addition to their function as acyltransferase substrates. Long-chain acyl-CoA is a ligand for the FadR transcriptional regulator in E. coli that represses the expression of β-oxidation genes (15DiRusso C.C. Black P.N. Weimar J.D. Molecular inroads into the regulation and metabolism of fatty acids, lessons from bacteria.Prog. Lipid Res. 1999; 38: 129-197Crossref PubMed Scopus (113) Google Scholar) and activates fabA (18Cronan Jr., J.E. Subrahmanyam S. FadR, transcriptional co-ordination of metabolic expediency.Mol. Microbiol. 1998; 29: 937-943Crossref PubMed Scopus (102) Google Scholar) and fabB(19Campbell J.W. Cronan Jr., J.E. Escherichia coli FadR positively regulates transcription of the fabB fatty acid biosynthetic gene.J. Bacteriol. 2001; 183: 5982-5990Crossref PubMed Scopus (99) Google Scholar) gene expression in unsaturated fatty acid synthesis. The DesT transcription factor regulates the expression of genes required for oxidative acyl-CoA desaturation in Pseudomonas aeruginosa (20Zhu K. Choi K-H. Schweizer H.P. Rock C.O. Zhang Y-M. Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa.Mol. Microbiol. 2006; 60: 260-273Crossref PubMed Scopus (94) Google Scholar), and its DNA binding is regulated by the composition of the acyl-CoA pool. Unsaturated acyl-CoA binding results in a tighter association of DesT with DNA repressing desaturase transcription, whereas saturated acyl-CoA triggers the release of DesT from DNA and the induction of desaturase expression (21Zhang Y-M. Zhu K. Frank M.W. Rock C.O. A Pseudomonas aeruginosa transcription factor that senses fatty acid structure.Mol. Microbiol. 2007; 66: 622-632Crossref PubMed Scopus (46) Google Scholar). Acyl-ACP is an allosteric regulator of the initiating steps in fatty acid synthesis, acetyl-CoA carboxylase (22Davis M.S. Cronan Jr., J.E. Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein.J. Bacteriol. 2001; 183: 1499-1503Crossref PubMed Scopus (129) Google Scholar) and β-ketoacyl-ACP synthase III (23Heath R.J. Rock C.O. Inhibition of β-ketoacyl-acyl carrier protein synthase III (FabH) by acyl-acyl carrier protein in Escherichia coli.J. Biol. Chem. 1996; 271: 10996-11000Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Modulation of the long-chain acyl-ACP concentration underlies the coordinated regulation of fatty acid, phospholipid, and macromolecular biosynthesis by the glycerol-phosphate acyltransferases (see below). These regulatory properties of the acyl-ACP and acyl-CoA donors for phospholipid synthesis in controlling bacterial lipid metabolism suggest that acyl-PO4 may also have a regulatory role in addition to its function as a biosynthetic intermediate. A regulatory function for acyl-PO4 may explain the retention of PlsX in bacteria that possess PlsB, and metabolic labeling experiments point to PlsX as a control point for the coordination of fatty acid synthesis, membrane phospholipid formation, and macromolecular synthesis in B. subtilis (13Paoletti L. Lu Y-J. Schujman G.E. de Mendoza D. Rock C.O. Coupling of fatty acid and phospholipid synthesis in Bacillus subtilis.J. Bacteriol. 2007; 189: 5816-5824Crossref PubMed Scopus (70) Google Scholar), a Gram-positive bacterium that lacks PlsB. However, the role, if any, for acyl-PO4 in metabolic regulation remains speculative. PlsB was the first glycerol-phosphate acyltransferase characterized in bacteria, and it participates in the pathway for phosphatidic acid formation outlined in Fig. 2. A novel mutagenesis strategy allowed the Bell laboratory to isolate glycerol-phosphate auxotrophs that possessed a Km defect in a membrane-associated glycerol-phosphate acyltransferase (24Bell R.M. Mutants of Escherichia coli defective in membrane phospholipid synthesis: macromolecular synthesis in an sn-glycerol 3-phosphate acyltransferase Km mutant.J. Bacteriol. 1974; 117: 1065-1076Crossref PubMed Google Scholar, 25Bell R.M. Mutants of Escherichia coli defective in membrane phospholipid synthesis: properties of wild type and Km defective sn-glycerol-3-phosphate acyltransfersae activities.J. Biol. Chem. 1975; 250: 7147-7152Abstract Full Text PDF PubMed Google Scholar). The availability of the plsB mutants enabled the cloning and extensive characterization of the membrane-bound enzyme that utilizes either acyl-ACP or acyl-CoA thioesters to acylate the 1-position of glycerol-phosphate (26Lightner V.A. Larson T.J. Tailleur P. Kantor G.D. Raetz C.R.H. Bell R.M. Modrich P. Membrane phospholipid synthesis in Escherichia coli: cloning of a structural gene (plsB) of the sn-glycerol-3-phosphate acyltransferase.J. Biol. Chem. 1980; 255: 9413-9420Abstract Full Text PDF PubMed Google Scholar, 27Green P.R. Merrill Jr., A.H. Bell R.M. Membrane phospholipid synthesis in Escherichia coli: purification, reconstitution, and characterization of sn-glycerol-3-phosphate acyltransferase.J. Biol. Chem. 1981; 256: 11151-11159Abstract Full Text PDF PubMed Google Scholar). PlsB is responsible for the selection of fatty acids incorporated into membrane phospholipids and is a key regulatory point in the pathway (4Cronan Jr., J.E. Rock C.O. Biosynthesis of membrane lipids.in: Neidhardt F.C. Curtis R. Gross C.A. In Escherichia coliSalmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 612-636Google Scholar, 5Rock C.O. Goelz S.E. Cronan Jr., J.E. Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol-3-phosphate.J. Biol. Chem. 1981; 256: 736-742Abstract Full Text PDF PubMed Google Scholar, 28Heath R.J. Jackowski S. Rock C.O. Guanosine tetraphosphate inhibition of fatty acid and phospholipid synthesis in Escherichia coli is relieved by overexpression of glycerol-3-phosphate acyltransferase (plsB).J. Biol. Chem. 1994; 269: 26584-26590Abstract Full Text PDF PubMed Google Scholar). The selectivity of PlsB for particular acyl chains is responsible for the positional asymmetry in the fatty acid composition of E. coli phospholipids (5Rock C.O. Goelz S.E. Cronan Jr., J.E. Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol-3-phosphate.J. Biol. Chem. 1981; 256: 736-742Abstract Full Text PDF PubMed Google Scholar), whereas the availability of glycerol-phosphate does not affect the positional distribution of acyl chains (29Goelz S.E. Cronan Jr., J.E. The positional distribution of fatty acids in Escherichia coli phospholipids is not regulated by sn-glycerol 3-phosphate levels.J. Bacteriol. 1980; 144: 462-464Crossref PubMed Google Scholar). In bacteria, there is a marked positional asymmetry in the incorporation of acyl chains into the 1- and 2-positions of glycerol-phosphate. The 1-position is occupied by 16:0 and 18:1 fatty acids, and the 2-position is occupied primarily by 16:1 and 18:1 fatty acids. However, the exclusion of 16:0 from the 2-position is not is not absolute, because fabA mutants that are unable to produce unsaturated fatty acids produce glycerolipids with 16:0 in both positions (30Jackson M.B. Cronan Jr., J.E. An estimate of the minimum amount of fluid lipid required for the growth of Escherichia coli.Biochim. Biophys. Acta. 1978; 512: 472-479Crossref PubMed Scopus (56) Google Scholar). A detailed biochemical analysis of the positional specificity of PlsB using native acyl-ACP substrates reveals that control is mainly exerted by the exclusion of 16:1 from the 1-position (5Rock C.O. Goelz S.E. Cronan Jr., J.E. Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol-3-phosphate.J. Biol. Chem. 1981; 256: 736-742Abstract Full Text PDF PubMed Google Scholar). There are four conserved blocks of amino acids in the PlsB class of acyltransferases. Much of what we know about these motifs has been learned from site-directed mutagenesis and kinetic analysis of the mammalian PlsB homolog in the Coleman laboratory and has been reviewed in detail (31Coleman R.A. Lee D.P. Enzymes of triacylglycerol synthesis and their regulation.Prog. Lipid Res. 2004; 43: 134-176Crossref PubMed Scopus (712) Google Scholar). Briefly, motif 1 is an HX4D sequence located at amino acid 306 (E. coli PlsB numbering) that directly participates in catalysis (32Heath R.J. Rock C.O. A conserved histidine is essential for glycerolipid acyltransferase catalysis.J. Bacteriol. 1998; 180: 1425-1430Crossref PubMed Google Scholar, 33Lewin T.M. Wang P. Coleman R.A. Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction.Biochemistry. 1999; 38: 5764-5771Crossref PubMed Scopus (233) Google Scholar). The invariant aspartate sets up a charge relay system with the histidine to facilitate the deprotonation of the hydroxyl group and promote its nucleophilic attack on the acyl donor. Motif 2 (348-GAFFIRRTF) and motif 3 (383-FVEGGRSRTG) are important for binding glycerol-phosphate by interacting with the phosphate group via the arginine residues underlined in the sequences. Motif 4 (417-ITLIPIYI) is also thought to be involved in catalysis based on the inactivation of PlsB when residues in this motif are mutated. However, a structural role for proline at this position cannot be ruled out. Experimentally verifying the correct folding of membrane proteins is a difficult undertaking. Understanding the function of specific residues in the acyltransferases is important because there are several human genetic disorders that involve mutations in acyltransferase genes, such as Barth syndrome (34Schlame M. Ren M. Barth syndrome, a human disorder of cardiolipin metabolism.FEBS Lett. 2006; 580: 5450-5455Crossref PubMed Scopus (249) Google Scholar), rhizomelic chondrodysplasia punctata type 2 (35Ofman R. Hettema E.H. Hogenhout E.M. Caruso U. Muijsers A.O. Wanders R.J. Acyl-CoA:dihydroxyacetonephosphate acyltransferase: cloning of the human cDNA and resolution of the molecular basis in rhizomelic chondrodysplasia punctata type 2.Hum. Mol. Genet. 1998; 7: 847-853Crossref PubMed Scopus (101) Google Scholar), and AGPAT-2-related lipodystrophy (36Agarwal A.K. Arioglu E. De A.S. Akkoc N. Taylor S.I. Bowcock A.M. Barnes R.I. Garg A. AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34.Nat. Genet. 2002; 31: 21-23Crossref PubMed Scopus (419) Google Scholar). PlsB has two membrane-spanning domains with the catalytic motifs separated in the C- and N-terminal halves that interact to affect catalysis (37Gonzalez-Baro M.R. Granger D.A. Coleman R.A. Mitochondrial glycerol phosphate acyltransferase contains two transmembrane domains with the active site in the N-terminal domain facing the cytosol.J. Biol. Chem. 2001; 276: 43182-43188Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 38Pellon-Maison M. Coleman R.A. Gonzalez-Baro M.R. The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity.Arch. Biochem. Biophys. 2006; 450: 157-166Crossref PubMed Scopus (17) Google Scholar). Although this suggests that the extracellular loop (or luminal loop in mammals) has little role in catalysis, modifications in this loop do result in a loss of activity, suggesting that it has a structural role in enzyme function. Plants have a variation on the bacterial acyl-ACP-dependent glycerol-phosphate acyltransferase that is a soluble protein localized in the plastid. These properties have allowed the determination of a high-resolution crystal structure of the glycerol-phosphate acyltransferase that informs us concerning the specific functions of the conserved amino acid blocks in this group of proteins (39Tamada T. Feese M.D. Ferri S.R. Kato Y. Yajima R. Toguri T. Kuroki R. Substrate recognition and selectivity of plant glycerol-3-phosphate acyltransferases (GPATs) from Cucurbita moscataSpinacea oleracea. Acta Crystallogr..D Biol. Crystallogr. 2004; 60: 13-21Crossref PubMed Scopus (43) Google Scholar, 40Slabas A.R. Kroon J.T. Scheirer T.P. Gilroy J.S. Hayman M. Rice D.W. Turnbull A.P. Rafferty J.B. Fawcett T. Simon W.J. Squash glycerol-3-phosphate (1)-acyltransferase. Alteration of substrate selectivity and identification of arginine and lysine residues important in catalytic activity.J. Biol. Chem. 2002; 277: 43918-43923Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 41Turnbull A.P. Rafferty J.B. Sedelnikova S.E. Slabas A.R. Schierer T.P. Kroon J.T. Simon J.W. Fawcett T. Nishida I. Murata N. et al.Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.Structure. 2001; 9: 347-353Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Motif 1 containing the HX4D motif is oriented with the carboxyl of the aspartate, which is hydrogen bonded to the histidine to leave the nonbonding electron pair on the histidine facing the active site, where the lone electron pair participates in abstracting a proton from the 1-position hydroxyl of glycerol-phosphate to activate this atom for nucleophilic attack on the acyl thioester. This configuration is reminiscent of the active site of serine hydrolases, where the histidine-aspartate pair activates the active site serine. The plant PlsB structure places the 1-position hydroxyl of glycerol-phosphate in the position of the serine hydroxyl of the hydrolases. These data strongly support the key catalytic role for the HX4D motif in acyltransferases. The phosphate of glycerol-phosphate is held in place in the plant acyltransferase structure by four basic residues arranged on two loops separated by a 42 residue spacer between each pair of basic residues. This organization is reminiscent of the 38 residue spacing between the twin arginine residues in PlsB located in motifs 2 and 3, supporting a role for these motifs in forming the phosphate binding pocket. One phosphate binding consensus sequence in plant PlsB is GGRxR, which matches the motif 3 sequence of