Biochemical and structural characterization of WlbA from Bordetella pertussis and Chromobacterium violaceum: enzymes required for the biosynthesis of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid.

The incidence of whooping cough has risen steadily in the last few years with several states, including Ohio and California, witnessing their largest outbreaks in 25 and 60 years, respectively. Whereas the disease is often mild in adults, it can result in significant morbidity and mortality rates in children under the age of two. The causative agent of whooping cough is Bordetella pertussis, a Gram-negative bacterium that infects humans by colonizing lung epithelial cells. As observed in most other Gram-negative bacteria, B. pertussis has a lipopolysaccharide or LPS, which serves as the major structural component for its outer membrane. The LPS is composed of lipid A, the core polysaccharide, and the O-antigen. A rather rare diacetylated sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid or ManNAc3NAcA, is found in the O-antigen of B. pertussis, and it has also been observed in the LPS of Pseudomonas aeruginosa serogroup O2 (1). The physiological importance of the O-antigen in B. pertussis is underscored by the fact that it confers resistance to complement-mediated cell lysis (2), and it also prevents clearance of the bacterium by the pulmonary surfactant protein A (3). Five enzymes are required for the production of UDP-ManNAc3NAcA in both B. pertussis and P. aeruginosa as indicated in Scheme 1 (4–6). The focus of this investigation is on the enzyme WlbA, which catalyzes the second step in the pathway, namely the oxidation of UDP-N-acetyl-d-glucosaminuronic acid (UDP-GlcNAcA) to UDP-2-acetamido-2-deoxy-3-oxo-d-glucuronic acid (UDP-3-keto-GlcNAcA). The enzyme is NAD-dependent, and a previous biochemical study of the protein from P. aeruginosa PAO1 demonstrated that it employs α-ketoglutarate to regenerate the oxidized cofactor necessary for the next round of catalysis (6). Indeed, α-ketoglutarate is absolutely required for catalysis by the P. aeruginosa enzyme (6). We recently reported the three-dimensional structures of WlbA from both P. aeruginosa and Thermus thermophilus in complex with NAD(H), with NAD(H) and α-ketoglutarate, and with NAD(H) and UDP-GlcNAcA. The enzymes from both bacterial species assumed unusual tetrameric quaternary structures with the NAD(H) moieties closely situated to one another as can be seen in Figure 1 (7). The individual subunits of the tetramers displayed overall folds similar to that reported for glucose-fructose oxidoreductase from Zymomonas mobilis (8). Strikingly, the UDP-linked substrates adopted highly unusual curved conformations in the active site clefts of the tetramers. In addition to the structural studies, we showed that these two enzymes catalyzed their reactions via ping-pong mechanisms (7). Figure 1 Structure of WlbA from P. aeruginosa. A stereo-view of the WlbA tetramer is shown with the individual subunits colored in green, wheat, purple, and blue. The NAD(H) cofactors are displayed as space-filling representations. This figure and Figures 3– ... Scheme 1 Proposed pathway for the biosynthesis of UDP-ManNAc3NAcA. Given that the WlbA enzymes from P. pseudomonas and B. pertussis catalyze the same reactions in the biosynthesis of UDP-ManNAc3NAcA (4), we anticipated that the structure of the B. pertussis enzyme would adopt a similar molecular architecture to that shown in Figure 1. We also assumed that the B. pertussis WlbA would use α-ketoglutarate to regenerate the oxidized cofactor. To our surprise, all attempts to solve the structure of the B. pertussis enzyme in the presence of α-ketoglutarate failed. Likewise, we were never able to demonstrate the recycling mechanism with the B. pertussis enzyme in vitro. Here we report the structure of the B. pertussis enzyme in complex with NAD(H) and its UDP-linked substrate. Additionally, we describe the structure of WlbA from Chromobacterium violaceum, a Gram-negative bacterium that produces the antibiotic violacein (9). The B. pertussis and C. violaceum enzymes share an identity of 73% and a similarity of 86%. Details concerning the kinetic and structural data are presented.