Table 1.
Proteins encoded within RGP01 island of A. baumannii Dl279779.
Table 2.
Diffraction data statistics for Ab-WbjB a.
Table 3.
Refinement statistics for Ab-WbjB (PDB 4j2o).
Fig 1.
Components of polysaccharide biosynthesis cluster RGP01.
(A) Gene conservation between the RGP01 island in A. baumannii D1279779 and related components of selected A. baumannii strains (clonal lineage indicated, right). For each component gene, locus tag and transcription direction are indicated. Colour is used to categorize functional annotations (from NCBI), with flanking chromosomal regions in black. Alignment with elements of the O-antigen biosynthesis cluster in P. aeruginosa is incorporated at top (red), with % sequence identity indicated for each orthologous pair. (B) Five-step biosynthesis of UDP-L-FucNAc by enzymes WbjB/CapE, WbjC/CapF, and WbjD/CapG as demonstrated in P. aeruginosa and S. aureus [43, 44].
Fig 2.
Crystal structure of Ab-WbjB (PDB 4j2o) at 2.65Å.
(A) Hexamer structure incorporates a trimer of dimers: A-E (cyan, yellow), B-D (orange, pink) and C-F (violet, green). Six bound NADP+ molecules are shown (red) as CPK spheres. (B) Magnification of chain A (left) shows its two-domain architecture: N-terminal Rossmann (cyan) and C-terminal (pale cyan) domains; (right) B-factor putty representation from high (red) to low (blue) values. Polypeptide backbone for which density is absent is dashed. (C) Coenzyme NADP+ (yellow) and surrounding side-chains (chain A, cyan; chain D, pink) at the A-D interface within the Ab-WbjB hexamer. Interatomic distances compatible with hydrogen bonding of NADP+ to K40 sidechain is shown (dotted yellow). Inset shows the 2Fo-Fc SA-omit map (grey mesh) contoured at 2.0σ for coenzyme NADP+ (chain A).
Fig 3.
Sequence alignment of Ab-WbjB and its structural relatives.
Sequences of structural homologs of Ab-WbjB (r.m.s.d <3.0 Å); S. aureus CapE, H. pylori FlaA1, and Pyrococcus horikoshii dehydratase PyDH, are shown. Strictly conserved residues (bold) and dinucleotide binding/active site motifs characteristic of SDR enzymes (red) are emphasised. Residue M134, replacing the usual catalytic tyrosine is indicated (▽). Sequences are aligned in T-Coffee [50]. Secondary structure elements of Ab-WbjB crystal structure are overlaid to outline N-terminal Rossmann (dark) and C-terminal (light) domains.
Fig 4.
Structural comparison of Ab-WbjB with CapE and FlaA1.
(A) Backbone Cα trace of Ab-WbjB (PDB 4j2o, cyan) overlaid with those of holo CapE + UDP-xylo-sugar (PDB 4g5h, pink) and holo FlaA1 + UDP-GlcNAc (PDB 2gn4, green). Magnification (right) depicts coenzyme NADP+ and substrate/substrate analogues (sticks) bound to respective structures. Cartoons of the various SDR structures (bottom) are shown with coenzyme (yellow sticks) and substrate/substrate analogue (blue sticks). Backbone for polypeptide segment connecting strands β9 - βF is highlighted (red) in each case to emphasize the variable region (B) Surface representation of Ab-WbjB, CapE (PDB 4g5h) and FlaA1 (PDB 2gn4) coloured according to residue charge (generated in PyMOL [39]).
Fig 5.
Definition of Ab-WbjB substrate chemistry.
(A) ITC response for titration 25°C of Ab-WbjB (100 μM) with UDP-GlcNAc (1 mM) and GlcNAc (1 mM). All titrations were carried out in HEPES buffer (50mM, pH 7.5) with 200 mM NaCl. Curve of best fit is drawn by Origin 7 software. (B) Binding enthalpies for Ab-WbjB (red) and M134A-WbjB (green) with specified hexose sugars. Enthalpy values derived across triplicate ITC measurements are indicated. (C) Active site with coenzyme for (left) FlaA1 [31] and (right) Ab-WbjB. In both cases, substrate UDP-GlcNAc is shown (black) as observed in FlaA1(PDB 2gn4) and modelled in Ab-WbjB from coordinates of PDB 2gn4. Side-chains conserved in both enzymes are shown (grey), as well as tyrosine of the canonical Y-x3-K motif (green).