Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Abstract

Campylobacter jejuni is a Gram-negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain-Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram-negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O-antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP-3-acetamido-3,6-dideoxy-d-glucose and dTDP-3-acetamido-3,6-dideoxy-d-galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5'-phosphate dependent aminotransferase. Here, we describe the first three-dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP-3-amino-3,6-dideoxy-d-galactose or dTDP-3-amino-3,6-dideoxy-d-glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C-4' carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.

Keywords: Campylobacter jejuni; WbpE; WlaRG; aminotransferase; dTDP-3-amino-3,6-dideoxy-d-galactose; dTDP-3-amino-3,6-dideoxy-d-glucose; lipooligosaccharide; pyridoxal 5′-phosphate.

Scheme 1

Pathways leading to the formation of either dTDP‐Fuc3NAc or dTDP‐Qui3NAc.

Figure 1

Ribbon representation of the apo form of WlaRG. The local twofold rotational axis that relates subunits 1 and 2 lies in the plane of the paper as indicated by the black arrow. The subunit:subunit interface is extensive with a total buried surface area of ∼4800 Ų. All figures were prepared with the software package PyMOL.16

Figure 2

Structure of the WlaRG internal aldimine. Shown in (a) is the electron density corresponding to the internal aldimine presented in stereo. The map was calculated with (F _o − F _c) coefficients and contoured at 3σ. The ligand was not included in the X‐ray coordinate file used to calculate the omit map, and thus there is no model bias. A close‐up view of the binding pocket is presented in stereo in (b). Those residues whose labels are marked with asterisks belong to subunit 2 of the dimer. Ordered water molecules are depicted as red spheres. The dashed lines indicate possible hydrogen bonding interactions.

Figure 3

Structure of the WlaRG external aldimine with PLP and dTDP‐Qui3N. The observed electron density for the external aldimine is shown in stereo in (a). The map was calculated as described in figure legend 2. A close‐up stereo view of the protein region surrounding the ligand is shown in (b). Those residues whose labels are marked with asterisks belong to subunit 2 of the dimer. Water molecules are displayed as red spheres, and possible hydrogen bonding interactions are indicated by the dashed lines.

Figure 4

Structure of the WlaRG external aldimine with PLP and dTDP‐Fuc3N. The electron density shown in (a) was calculated as described in figure legend 2. A stereo view of the protein region surrounding the ligand is displayed in (b). Water molecules are depicted as red spheres and possible hydrogen bonding interactions are indicated by the dashed lines. Those residues whose labels are marked with asterisks belong to subunit 2 of the dimer.

Figure 5

Structure of the WbpE external aldimine with PLP and UDP‐3‐amino‐GlcNAcA. We calculated omit maps to 1.9 Å resolution using (F _o − F _c) coefficients and the structure factors deposited in the Protein Data Bank under accession no. 3NUB. Shown in (a) and (b) are the observed electron densities for the WbpE external aldimines in subunits 1 and 2. The maps were contoured at ∼3σ. Note the incorrect stereochemistries about the C‐2 and C‐3 ribose carbons. Displayed in (c) is the observed electron density for the structure of the WbpE external aldimine determined in our laboratory. The map was calculated to 1.3 Å resolution with (F _o − F _c) coefficients and contoured at 3σ.

Scheme 2

Reaction catalyzed by WbpE.

Figure 6

Close‐up stereo view of the WbpE active site. Those residues whose labels are marked with asterisks belong to subunit 2 of the dimer. Water molecules are depicted as red spheres and possible hydrogen bonding interactions are indicated by the dashed lines.

Figure 7

Comparison of WlaRG with QdtB and WbpE. A superposition of the active sites for WlaRG and QdtB with bound external aldimines is displayed in (a). Residues belonging to WlaRG are highlighted in purple and blue whereas those corresponding to QdtB are colored in white. A superposition of the active sites for WlaRG and WbpE with bound external aldimines is presented in (b). Residues belonging to WlaRG are highlighted in purple and blue whereas those corresponding to WbpE are colored in white.