Molecular Structure ofWlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesisof 2,3-Diacetamido-2,3-dideoxy-D-mannuronic Acid

摘要

The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP- GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 A resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA.WlbB adopts a trimeric quaternary structure and belongs to the LβH superfamily of N-acyltransferases. Each subunit contains 27 β-strands, 23 of which formthe canonical left-handed β-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between Oδ1 of Asn 84 and the sugar C-30 amino group and the second between the backbone amide group of Arg 94 and the sugar C-50 carboxylate. The sugar C-30 amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.rnMembranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required forrnthe biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, isrnan N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-rnGlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here wernreport the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternaryrnstructures were determined to 1.43 A resolution: one in which the protein was complexed with acetyl-CoArnand UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA.WlbB adopts arntrimeric quaternary structure and belongs to the LβH superfamily of N-acyltransferases. Each subunitrncontains 27 β-strands, 23 of which formthe canonical left-handed β-helix. There are only two hydrogen bondsrnthat occur between the protein and the GlcNAc3NA moiety, one between Oδ1rnof Asn 84 and the sugar C-30rnamino group and the second between the backbone amide group of Arg 94 and the sugar C-50rncarboxylate.rnThe sugar C-30rnamino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Givenrnthat there are no protein side chains that can function as general bases within the GlcNAc3NA bindingrnpocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as thernproton acceptor required for catalysis.