Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

Michael J Morrison,Barbara Imperiali

doi:10.1074/jbc.m113.510560

Michael J Morrison, Barbara Imperiali

Open Access

https://doi.org/10.1074/jbc.m113.510560

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Abstract

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.

Highlights

A connection between glycoproteins containing N,NЈ-diacetylbacillosamine and pathogenicity has previously been shown in Campylobacter jejuni
Structure of the N. gonorrhoeae Acetyltransferase PglB-ATD— PglB from N. gonorrhoeae is a bifunctional enzyme containing an N-terminal phosphoglycosyltransferase domain and a C-terminal acetyltransferase domain (ATD) that are homologous to the C. jejuni enzymes PglC and PglD, respectively [5]
The membrane-bound phosphoglycosyltransferase domain was removed based upon a Clustal Omega alignment with PglD, leaving behind the acetyltransferase domain referred to as PglB-ATD

Summary

Background

A connection between glycoproteins containing N,NЈ-diacetylbacillosamine and pathogenicity has previously been shown in Campylobacter jejuni. To further our understanding of acetyltransferases from the different UDP-diNAcBac biosynthetic pathways and to gain insight into the divergent nature of N- and O-linked protein glycosylation in prokaryotes, acetyltransferases from N. gonorrhoeae (PglB-ATD) and A. baumannii (WeeI) were investigated To this effect, these enzymes were purified and crystallized, and the structures were solved to high resolution. Based upon this structural comparison, a series of active site mutations was carried out on all three acetyltransferases, and the enzymes were characterized kinetically for both AcCoA and UDP-4-amino substrates to gain insight into the catalytic mechanism These studies suggest that each enzyme catalyzes the acetyltransferase reaction with identical substrates, key residues within the binding pockets lead to a diverse set of catalytic efficiencies. PglB-ATD from the O-linked glycosylation pathway shares a more common ancestral lineage with the PglD (N-linked) when compared with WeeI (O-linked)

EXPERIMENTAL PROCEDURES

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DISCUSSION