Dataset Information

Structural determinants of substrate recognition in the HAD superfamily member D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) .

ABSTRACT: The haloalkanoic acid dehalogenase (HAD) enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases, a cap domain functions in substrate recognition. However, for the HAD phosphatases that lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB of the HisB subfamily, was selected for structure-function analysis. Herein, the X-ray crystallographic structures of Escherichia coli GmhB in the apo form (1.6 A resolution), in a complex with Mg(2+) and orthophosphate (1.8 A resolution), and in a complex with Mg(2+) and d-glycero-d-manno-heptose 1beta,7-bisphosphate (2.2 A resolution) were determined, in addition to the structure of Bordetella bronchiseptica GmhB bound to Mg(2+) and orthophosphate (1.7 A resolution). The structures show that in place of a cap domain, the GmhB catalytic site is elaborated by three peptide inserts or loops that pack to form a concave, semicircular surface around the substrate leaving group. Structure-guided kinetic analysis of site-directed mutants was conducted in parallel with a bioinformatics study of sequence diversification within the HisB subfamily to identify loop residues that serve as substrate recognition elements and that distinguish GmhB from its subfamily counterpart, the histidinol-phosphate phosphatase domain of HisB. We show that GmhB and the histidinol-phosphate phosphatase domain use the same design of three substrate recognition loops inserted into the cap domain yet, through selective residue usage on the loops, have achieved unique substrate specificity and thus novel biochemical function.

SUBMITTER: Nguyen HH

PROVIDER: S-EPMC2844806 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Structural determinants of substrate recognition in the HAD superfamily member D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) .

Nguyen Henry H HH Wang Liangbing L Huang Hua H Peisach Ezra E Dunaway-Mariano Debra D Allen Karen N KN

Biochemistry 20100201 6

The haloalkanoic acid dehalogenase (HAD) enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases, a cap domain functions in substrate recognition. However, for the HAD phosphatases that lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB ...[more]

PMID: 20050614

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Project description:D-Glycero-d-manno-heptose-1,7-bisphosphate phosphatase (GmhB) is a member of the histidinol-phosphate phosphatase (HisB) subfamily of the haloalkanoic acid dehalogenase (HAD) enzyme superfamily. GmhB supports two divergent biochemical pathways in bacteria: the d-glycero-d-manno-heptose-1alpha-GDP pathway (in S-layer glycoprotein biosynthesis) and the l-glycero-d-manno-heptose-1beta-ADP pathway (in lipid A biosynthesis). Herein, we report the comparative analysis of substrate recognition in selected GmhB orthologs. The substrate specificity of the l-glycero-d-manno-heptose-1beta-ADP pathway GmhB from Escherichia coli K-12 was evaluated using hexose and heptose bisphosphates, histidinol phosphate, and common organophosphate metabolites. Only d-glycero-d-manno-heptose 1beta,7-bisphosphate (k(cat)/K(m) = 7 x 10(6) M(-1) s(-1)) and d-glycero-d-manno-heptose 1alpha,7-bisphosphate (k(cat)/K(m) = 7 x 10(4) M(-1) s(-1)) displayed physiologically significant substrate activity. (31)P NMR analysis demonstrated that E. coli GmhB selectively removes the C(7) phosphate. Steady-state kinetic inhibition studies showed that d-glycero-d-manno-heptose 1beta-phosphate (K(is) = 60 microM, and K(ii) = 150 microM) and histidinol phosphate (K(is) = 1 mM, and K(ii) = 6 mM), while not hydrolyzed, do in fact bind to E. coli GmhB, which leads to the conclusion that nonproductive binding contributes to substrate discrimination. High catalytic efficiency and a narrow substrate range are characteristic of a well-evolved metabolic enzyme, and as such, E. coli GmhB is set apart from most HAD phosphatases (which are typically inefficient and promiscuous). The specialization of the biochemical function of GmhB was examined by measuring the kinetic constants for hydrolysis of the alpha- and beta-anomers of d-glycero-d-manno-heptose 1beta,7-bisphosphate catalyzed by the GmhB orthologs of the l-glycero-d-manno-heptose 1beta-ADP pathways operative in Bordetella bronchiseptica and Mesorhizobium loti and by the GmhB of the d-glycero-d-manno-heptose 1alpha-GDP pathway operative in Bacteroides thetaiotaomicron. The results show that although each of these representatives possesses physiologically significant catalytic activity toward both anomers, each displays substantial anomeric specificity. Like E. coli GmhB, B. bronchiseptica GmhB and M. loti GmhB prefer the beta-anomer, whereas B. thetaiotaomicron GmhB is selective for the alpha-anomer. By determining the anomeric configuration of the physiological substrate (d-glycero-d-manno-heptose 1,7-bisphosphate) for each of the four GmhB orthologs, we discovered that the anomeric specificity of GmhB correlates with that of the pathway kinase. The conclusion drawn from this finding is that the evolution of the ancestor to GmhB in the HisB subfamily provided for specialization toward two distinct biochemical functions.

Dataset Information

Structural determinants of substrate recognition in the HAD superfamily member D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) .

Publications

Structural determinants of substrate recognition in the HAD superfamily member D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) .

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