title: Structure and reactivity of LpxD, the N-acyltransferase of lipid A biosynthesis.
abstract: The external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable LPS. The biosynthesis of LPS relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine in Chlamydia trachomatis. Our crystallographic study reveals that LpxD is a homotrimer, each subunit of which is constructed from a novel combination of an N-terminal uridine-binding domain, a core lipid-binding domain, and a C-terminal helical extension. Highly conserved residues dominate nucleotide binding. Phe-43 and Tyr-49 form pi-stacking interactions with uracil, and Asn-46 and His-284 form hydrogen bonds with the phosphate groups. These interactions place the glucosamine moiety at the catalytic center formed by two adjacent subunits. Here His-247 and His-284 contribute to a mechanism involving nucleophilic attack by the amine of one substrate on the carbonyl carbon of an acyl carrier protein thioester conjugate. Serendipitously, our study reveals a fatty acid (FA) binding groove near the catalytic center. MS elucidated the presence of a FA mixture binding to LpxD, with palmitic acid the most prevalent. The placement of UDP-N-acetylglucosamine and the FA provides details of N-acyltransferase ligand interactions and allows for a description of structure and reactivity at an early stage of LPS assembly.
authors.affiliation: Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Lori
authors.initials: L
authors.lastname: Buetow
authors.forename: Terry K
authors.initials: TK
authors.lastname: Smith
authors.forename: Alice
authors.initials: A
authors.lastname: Dawson
authors.forename: Stewart
authors.initials: S
authors.lastname: Fyffe
authors.forename: William N
authors.initials: WN
authors.lastname: Hunter
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000502:physiology*; D000595:Amino Acid Sequence; D002244:Carbon / Q000737:chemistry; D002692:Chlamydia trachomatis / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D005227:Fatty Acids / Q000378:metabolism; D006860:Hydrogen Bonding; D008050:Lipid A / Q000096:biosynthesis*; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D008050:Lipid A; D014537:Uridine Diphosphate N-Acetylglucosamine; D002244:Carbon; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase