Crystal Structure of GDP-Mannose Dehydrogenase:  A Key Enzyme of Alginate Biosynthesis in P. aeruginosa^,

Abstract

The enzyme GMD from Pseudomonas aeruginosa catalyzes the committed step in the synthesis of the exopolysaccharide alginate. Alginate is a major component of P. aeruginosa biofilms that protect the bacteria from the host immune response and antibiotic therapy. The 1.55 A crystal structure of GMD in ternary complex with its cofactor NAD(H) and product GDP-mannuronic acid reveals that the enzyme forms a domain-swapped dimer with two polypeptide chains contributing to each active site. The extensive dimer interface provides multiple opportunities for intersubunit communication. Comparison of the GMD structure with that of UDP-glucose dehydrogenase reveals the structural basis of sugar binding specificity that distinguishes these two related enzyme families. The high-resolution structure of GMD provides detailed information on the active site of the enzyme and a template for structure-based inhibitor design.

Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

“…This also correlates well with the results of WbpO (pH 8.5), another dehydrogenase that was previously characterized by our group (31). The preference for basic conditions may be due to the involvement of a cysteine residue, since the mechanisms for both AlgD (34) and Ugd (35) involve nucleophilic attack by a cysteine to form a thiosemiacetal intermediate, and a basic pH would help to stabilize the thiolate anion that would be required (typical pK a for a cysteine side chain is ϳ9.0). The most likely candidate, based on amino acid alignment with AlgD, is Cys 273 , which aligns with Cys 268 from the active site of AlgD.…”

“…Subsequent analysis using gel filtration, dynamic light scattering and analytical ultracentrifugation (data not shown) substantiated the interpretation that WbpA is a trimer in solution. These results are surprising since dehydrogenases that have been crystallized to date have been shown as dimeric, tetrameric or hexameric structures (33,34,45,46). However, when visualized by TEM and negative staining, images of WbpA oligomers with 3-fold symmetry could not be discerned.…”

“…in which data collected earlier suggested a trimeric structure in solution (44); however, when the three-dimensional crystal structure of the enzyme was solved, the protein was found to exist as a dimer of dimers (34).…”

“…For example, the residues that are conserved in WbpA include the majority of those which are responsible for contacts with NADH and the nucleotide sugar substrate in AlgD, as well as those postulated to be involved in catalysis (Glu 165 , Lys 218 , Cys 273 , and Asp 277 in WbpA) (34). There are also similarities that can be seen not only by superimposition of the predicted structure of WbpA with the crystal structure of AlgD, but also in comparing the electron micrographs of WbpA with the structure of AlgD.…”

Activated Sugar Precursors: Biosynthetic Pathways and Biological Roles of an Important Class of Intermediate Metabolites in Bacteria

“…GMdh is a member of the NAD-dependent 4-electron transfer dehydrogenases. This protein family also includes UDP-glucose dehydrogenases (UGD) (Snook et al, 2003). Both the GMdh and UGD enzyme activities are mechanistically similar, using a unique active site to catalyze the two-step conversion of an alcohol group to the corresponding acid, via a thiohemiacetal intermediate.…”