19504-70-2Relevant articles and documents
Divergence of biochemical function in the HAD superfamily: D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB)
Wang, Liangbing,Huang, Hua,Nguyen, Henry H.,Allen, Karen N.,Mariano, Patrick S.,Dunaway-Mariano, Debra
experimental part, p. 1072 - 1081 (2010/11/21)
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-1α-GDP pathway (in S-layer glycoprotein biosynthesis) and the L-glycero-D-manno- heptose-1β-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-1β-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 1β,7-bisphosphate (kcat/K m=7 × 106 M-1 s-1) and D-glycero-D-manno-heptose 1α,7-bisphosphate (kcat/Km = 7 × 104 M-1 s-1) displayed physiologically significant substrate activity. 31P 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 1β-phosphate (Kis = 60 μM, and Kii = 150 μM) and histidinol phosphate (Kis = 1 mM, and Kii = 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 α- and β-anomers of D-glycero-D-manno-heptose 1β,7-bisphosphate catalyzed by the GmhB orthologs of the L-glycero-D-manno-heptose 1β-ADP pathways operative in Bordetella bronchiseptica and Mesorhizobium loti and by the GmhB of the D-glycero-D-manno-heptose 1α-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 β-anomer, whereas B. thetaiotaomicron GmhB is selective for the α-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.
