Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.
Bottom Line: We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis.Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity.Our work provides insight into the evolution and functional diversification of an ancient enzyme family.
Affiliation: From the Structural Plant Biology Laboratory, Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland and.Show MeSH
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Mentions: We investigated the contributions of the two metal ion centers to TTM substrate binding and catalysis. A conceptual problem with the analysis of our plant and bacterial tripolyphosphatases is that they catalyze the asymmetric cleavage of a symmetric substrate (Fig. 2) (25, 26). It is thus difficult to assess in crystal structures, which terminal phosphate represents the γ-phosphate that is being hydrolyzed (Fig. 3, A and B). We thus structurally characterized a mammalian TTM ThTPase, which was previously shown to specifically hydrolyze thiamine triphosphate (ThTP) into ThDP and Pi (19–21, 43). We synthesized ThTP from ThDP and produced co-crystal structures of mouse ThTPase with its substrate at pH 6, where ThTPase catalytic activity is minimal (20). Consequently, we found an intact ThTP molecule bound in the tunnel center of ThTPase (Fig. 5A). The thiamine portion of the substrate binds to a pocket generated by the tunnel walls and the C-terminal plug helix, with the thiazole ring making a stacking interaction with Trp-53 and with Met-195 from the plug helix (Fig. 5A). The ThTP triphosphate moiety binds in the same conformation as outlined for the PPPi-bound structures of AtTTM3 and ygiF above. Our substrate-bound mouse ThTPase structure supports an earlier docking model of human ThTPase (21). We next solved a crystal structure of mouse ThTPase in the presence of ThTP and Mg2+ in a second crystal form grown at pH 9.0, where substrate hydrolysis can occur (20). Indeed, we found a product complex trapped in the active site of the enzyme, with a ThDP molecule and an orthophosphate located in the tunnel center (Fig. 5A). ThDP is coordinated by Arg-55 and Arg-57 in the substrate binding site but no longer allows for the coordination of a Mg2+/Mn2+ ion in metal binding site 1, possibly because the missing γ-phosphate would be required for Mg2+/Mn2+ coordination (Fig. 5A). The γ-phosphate in our structure apparently has been hydrolyzed, and the resulting Pi has slightly moved away from the tunnel center (Fig. 5A). It is now found coordinated by Arg-125 and in direct contact with a Mn2+ ion located in metal binding site 2, reinforcing the notion that this metal ion may play a crucial role in catalysis (Fig. 5A).
Affiliation: From the Structural Plant Biology Laboratory, Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland and.