Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate

Abstract

Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.

Template‐based protein structure prediction in CASP11 and retrospect of I‐TASSER in the last decade

“…(D,E)]. Both 3g79A and 2y0cA consist of two dinucleotide binding Rossmann‐like folds in the N‐ and C‐terminal domains, where the subtle difference in the intermediate linker domain results in a small twist on the domain orientation . However, the I‐TASSER potential was unable to distinguish the domain orientations where nearly equal numbers of the structure decoys have been generated by the I‐TASSER simulations, for the different domain orientations, as indicated by the similar rank of consensus score shown in Table .…”

Analysis of nucleotide diphosphate sugar dehydrogenases reveals family and group‐specific relationships

“…This helix also contributes to intersubunit contacts responsible for formation of homodimers, as seen in PaGDPMDH (Fig. B) and Burkholdaria cepacia UDPGDH . Motif 2 contains the fully conserved residues Gly257{408}, Gly258{409}, Cys260{411} and Asp264{415}.…”

Burkholderia cenocepacia K56‐2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation

“…The results showed that the distribution of fluorescence is similar in all cases. As a negative control, we conducted a colocalization experiment using BceC, an intracellular Burkholderia enzyme involved in exopolysaccharide synthesis (Rocha et al, ). The Pearson's correlation coefficient (Rr; 0.124 for x – y plane and 0.177 for side view; Figure S3) was significantly lower than the one determined for the pair TNFR1‐BcaA, validating our analysis.…”

“…The results showed that the distribution of fluorescence is similar in all cases. As a negative control, we conducted a colocalization experiment using BceC, an intracellular Burkholderia enzyme involved in exopolysaccharide synthesis (Rocha et al, 2011).…”