| The haloalkanoic acid dehalogenase superfamily is a large superfamily of proteins which share a conserved active-site core domain. The superfamily is further divided into four subfamilies C0, C1, C2a, and C2b, based on the topology and insertion site of a cap domain into the core that provides substrate specificity. To further understand structure/function relationships between subfamilies, X-ray crystallographic and kinetic analyses were performed on beta-phosphoglucomutase (beta-PGM) and NagD, respectively C1 and C2a subfamily members.;beta-PGM catalyzes the interconversion of beta-D-glucose-1-phosphate (beta-G1P) and beta-D-glucose-6-phosphate. We report the 1.90 A resolution X-ray crystal structure of the two-domain beta-PGM in the active-site open conformation and compare it to the previously reported structures of beta-PGM. Together with kinetic studies these results demonstrate that the coupling of cap domain closure to ligand binding and acid-base catalysis via Asp 10 with cap closure, allows phospho-beta-PGM to discriminate between beta-G1P and water as the phosphoryl group acceptor.;Previously, the intermediate pentavalent-phosphorane had been reported based on crystallographic data. Dispute arose which called into question the validity of the data interpretation, stating instead that MgF3 - species could be a possible interpretation of the observed electron density. Control crystals of beta-PGM in complex with alpha-galactose-1-phosphate were grown in the presence and the absence of NH4F and refined to 1.97A and to 1.90 A resolutions respectively, and were indistinguishable. Bradford protein and Malachite Green phosphate assays were performed on solutions from the beta-PGM-alpha-Gal1P complex and beta-PGM-beta-D-glucose-6-phosphate-1-phosphorane complex. Control crystals contained the anticipated 1:1 ratio of enzyme to phosphorus while the test crystals contained a 1:2 ratio. This is direct chemical evidence for the original beta-PGM-betaD-glucose-6-phosphate-1-phosphorane structure.;For an analysis of the C2a subfamily, NagD from the E. coli K-12 was selected. The structure of the NagD protein was solved to 1.80 A. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates (kcat/Km = 3.12 x 104 and 1.28 x 104 M-1 s-1 for UMP and GMP, respectively). Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain identifying a probable substrate specificity loop. |
