Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.

Congential disorder of glycosylation type 1a (CDG-1a) is a congenital disease characterized by severe defects in nervous system development. It is caused by mutations in ␣-phosphomannomutase (of which there are two isozymes, ␣-PMM1 and ␣-PPM2).

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the R/ core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight -turn in the helix-loophelix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and -phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.

The β-phosphoglucomutase (β-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of β-glucose 1-phosphate (βG1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active-site to mediate phosphoryl transfer from β-glucose 1,6-(bis)phosphate (βG1,6bisP) to βG1P. Herein we explore the mechanism by which hydrolysis of the β-PGM phosphoAsp8 is avoided during the time that the active site must remain open to solvent in order to allow the exchange of the bound product G6P with the substrate βG1P. Based on structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 of the domain-domain linker, to a functional position where it forms a hydrogen bond to the substrate leaving-group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of the Asp10 by Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by βG1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert βG1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray structure of the T16P mutant at 2.7 Å resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core-domain active site. The His20 and Lys76 site- * Address correspondence to Debra Dunaway-Mariano email: dd39@unm.edu, phone: 505-277-3383, fax: 505-277-2609 and Karen N. Allen, phone: 617-358-5544, fax: 617-358-5554, email: drkallen@bu.edu. c present address: Department of Chemistry, Boston University, Boston, MA 02215, USA 1 Abbreviations used are: α-PGM, α-phosphoglucomutase; α-PGM/PMM, dual specificity α-phosphoglucomutase/α-phosphomannomutase; β-PGM, β-phosphoglucomutase; E, β-PGM -Mg 2+ ; E-P, phospho-β-PGM-Mg 2+ ; βG1P, β-D-glucose 1-phosphate; βG1,6bisP, β-D-glucose 1,6-(bis)phosphate; αG1P, α-D-glucose 1-phosphate; αG1,6bisP, α-D-glucose 1,6-(bis)phosphate; PEP, phosphoenol pyruvate; SA, specific activity. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 August 6. Published in final edited form as:Biochemistry. Phosp...