Biochemical analysis and structure determination of bacterial acetyltransferases responsible for the biosynthesis of UDP-N,N'-diacetylbacillosamine.
UDP-N,N'-diacetylbacillosamine (UDP-diNAcBac) is a unique carbohydrate produced by a number of bacterial species and has been implicated in pathogenesis. The terminal step in the formation of this important bacterial sugar is catalyzed by an acetyl-CoA (AcCoA)-dependent acetyltransferase in both N- and O-linked protein glycosylation pathways. This bacterial acetyltransferase is a member of the left-handed β-helix family and forms a homotrimer as the functional unit. Whereas previous endeavors have focused on the Campylobacter jejuni acetyltransferase (PglD) from the N-linked glycosylation pathway, structural characterization of the homologous enzymes in the O-linked glycosylation pathways is lacking. Herein, we present the apo-crystal structures of the acetyltransferase domain (ATD) from the bifunctional enzyme PglB (Neisseria gonorrhoeae) and the full-length acetyltransferase WeeI (Acinetobacter baumannii). Additionally, a PglB-ATD structure was solved in complex with AcCoA. Surprisingly, this structure reveals a contrasting binding mechanism for this substrate when compared with the AcCoA-bound PglD structure. A comparison between these findings and the previously solved PglD crystal structures illustrates a dichotomy among N- and O-linked glycosylation pathway enzymes. Based upon these structures, key residues in the UDP-4-amino and AcCoA binding pockets were mutated to determine their effect on binding and catalysis in PglD, PglB-ATD, and WeeI. Last, a phylogenetic analysis of the aforementioned acetyltransferases was employed to illuminate the diversity among N- and O-linked glycosylation pathway enzymes.
From the Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Michael J
MJ
Morrison
From the Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. Electronic address: imper@mit.edu.
Barbara
B
Imperiali
D000105:Acetyl Coenzyme A; D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040981:Acinetobacter baumannii / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006031:Glycosylation; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D014539:Uridine Diphosphate Sugars / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
C502751:2,4-diacetamido-2,4,6-trideoxyglucopyranose; D001426:Bacterial Proteins; D014539:Uridine Diphosphate Sugars; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D000117:Acetylglucosamine
Acetyl-coenzyme A; Acetyltransferase; Bacillosamine; Bacterial Pathogenesis; Enzyme Kinetics; Glycoprotein Biosynthesis; Protein Crystallization; UDP-diNAcBac