Crystal structure and acyl chain selectivity of Escherichia coli LpxD, the N-acyltransferase of lipid A biosynthesis.
LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxyacyl-ACP-dependent N-acylation of UDP-3-O-(acyl)-alpha-D-glucosamine, and is a target for new antibiotic development. Here we report the 2.6 A crystal structure of the Escherichia coli LpxD homotrimer (EcLpxD). As is the case in Chlamydia trachomatis LpxD (CtLxpD), each EcLpxD chain consists of an N-terminal uridine-binding region, a left-handed parallel beta-helix (LbetaH), and a C-terminal alpha-helical domain. The backbones of the LbetaH domains of the two enzymes are similar, as are the positions of key active site residues. The N-terminal nucleotide binding domains are oriented differently relative to the LbetaH regions, but are similar when overlaid on each other. The orientation of the EcLpxD tripeptide (residues 303-305), connecting the distal end of the LbetaH and the proximal end of the C-terminal helical domains, differs from its counterpart in CtLpxD (residues 311-312); this results in a 120 degrees rotation of the C-terminal domain relative to the LbetaH region in EcLpxD versus CtLpxD. M290 of EcLpxD appears to cap the distal end of a hydrophobic cleft that binds the acyl chain of the R-3-hydroxyacyl-ACP donor substrate. Under standard assay conditions, wild-type EcLpxD prefers R,S-3-hydroxymyristoyl-ACP over R,S-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of E. coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42 degrees C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual R-3-hydroxymyristate at positions 2 and 2'.
Bartling
Craig M
CM
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Raetz
Christian R H
CR
D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000215:Acylation; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002692:Chlamydia trachomatis / Q000201:enzymology; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008050:Lipid A / Q000096:biosynthesis* / Q000737:chemistry; D009227:Myristic Acids / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008050:Lipid A; D009227:Myristic Acids; C009893:beta-hydroxymyristic acid; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase; D000581:Amidohydrolases; C097307:UDP-3-O-acyl-N-acetylglucosamine deacetylase