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Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.

Martinez J, Truffault V, Hothorn M - J. Biol. Chem. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the Structural Plant Biology Laboratory, Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland and.

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TTM proteins use a two-metal catalytic mechanism.A, close-up view of the mouse ThTPase tunnel center with either the ThTP substrate bound (transparent gray) or the ThDP/Pi products post-catalysis (in yellow, in bond representation). The thiamine part of ThTP is buried in a pocket close to the C-terminal plug helix of the TTM domain formed by Tyr-79 and Met-195; the thiazole ring makes a stacking interaction with Trp-53 (in yellow, in bond representation). The product Pi is coordinated by a Mn2+ ion bound to site 2 and by Arg-125. B, close-up of the ygiF active site (in yellow, in bond representation) bound to PPPi and two Mn2+ ions (magenta spheres) in sites 1 and 2. The Mn2+ ion in site 2 coordinates a water molecule (red sphere), which is well positioned to act as nucleophile. Structural superposition with a product-bound class IV adenylate cyclase (PDB ID 3N10) reveals the O3′ of cAMP in the same position as the water molecule in ygiF. This position is also occupied by an oxygen atom of the product Pi located in the AtTTM3 post-catalysis complex. C, the suggested mechanism for acidic-patch containing TTM proteins. The metal ion in site 1 coordinates the triphosphate moiety of the substrate to the tunnel center by interacting with a conserved Glu residue. Three additional glutamates form metal binding site 2, which coordinates and polarizes a water molecule to attack the γ-phosphate of the substrate. Conserved basic residues in the tunnel center are involved in substrate binding and potentially stabilize the pyrophosphate leaving group.
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Figure 5: TTM proteins use a two-metal catalytic mechanism.A, close-up view of the mouse ThTPase tunnel center with either the ThTP substrate bound (transparent gray) or the ThDP/Pi products post-catalysis (in yellow, in bond representation). The thiamine part of ThTP is buried in a pocket close to the C-terminal plug helix of the TTM domain formed by Tyr-79 and Met-195; the thiazole ring makes a stacking interaction with Trp-53 (in yellow, in bond representation). The product Pi is coordinated by a Mn2+ ion bound to site 2 and by Arg-125. B, close-up of the ygiF active site (in yellow, in bond representation) bound to PPPi and two Mn2+ ions (magenta spheres) in sites 1 and 2. The Mn2+ ion in site 2 coordinates a water molecule (red sphere), which is well positioned to act as nucleophile. Structural superposition with a product-bound class IV adenylate cyclase (PDB ID 3N10) reveals the O3′ of cAMP in the same position as the water molecule in ygiF. This position is also occupied by an oxygen atom of the product Pi located in the AtTTM3 post-catalysis complex. C, the suggested mechanism for acidic-patch containing TTM proteins. The metal ion in site 1 coordinates the triphosphate moiety of the substrate to the tunnel center by interacting with a conserved Glu residue. Three additional glutamates form metal binding site 2, which coordinates and polarizes a water molecule to attack the γ-phosphate of the substrate. Conserved basic residues in the tunnel center are involved in substrate binding and potentially stabilize the pyrophosphate leaving group.

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).


Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.

Martinez J, Truffault V, Hothorn M - J. Biol. Chem. (2015)

TTM proteins use a two-metal catalytic mechanism.A, close-up view of the mouse ThTPase tunnel center with either the ThTP substrate bound (transparent gray) or the ThDP/Pi products post-catalysis (in yellow, in bond representation). The thiamine part of ThTP is buried in a pocket close to the C-terminal plug helix of the TTM domain formed by Tyr-79 and Met-195; the thiazole ring makes a stacking interaction with Trp-53 (in yellow, in bond representation). The product Pi is coordinated by a Mn2+ ion bound to site 2 and by Arg-125. B, close-up of the ygiF active site (in yellow, in bond representation) bound to PPPi and two Mn2+ ions (magenta spheres) in sites 1 and 2. The Mn2+ ion in site 2 coordinates a water molecule (red sphere), which is well positioned to act as nucleophile. Structural superposition with a product-bound class IV adenylate cyclase (PDB ID 3N10) reveals the O3′ of cAMP in the same position as the water molecule in ygiF. This position is also occupied by an oxygen atom of the product Pi located in the AtTTM3 post-catalysis complex. C, the suggested mechanism for acidic-patch containing TTM proteins. The metal ion in site 1 coordinates the triphosphate moiety of the substrate to the tunnel center by interacting with a conserved Glu residue. Three additional glutamates form metal binding site 2, which coordinates and polarizes a water molecule to attack the γ-phosphate of the substrate. Conserved basic residues in the tunnel center are involved in substrate binding and potentially stabilize the pyrophosphate leaving group.
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Figure 5: TTM proteins use a two-metal catalytic mechanism.A, close-up view of the mouse ThTPase tunnel center with either the ThTP substrate bound (transparent gray) or the ThDP/Pi products post-catalysis (in yellow, in bond representation). The thiamine part of ThTP is buried in a pocket close to the C-terminal plug helix of the TTM domain formed by Tyr-79 and Met-195; the thiazole ring makes a stacking interaction with Trp-53 (in yellow, in bond representation). The product Pi is coordinated by a Mn2+ ion bound to site 2 and by Arg-125. B, close-up of the ygiF active site (in yellow, in bond representation) bound to PPPi and two Mn2+ ions (magenta spheres) in sites 1 and 2. The Mn2+ ion in site 2 coordinates a water molecule (red sphere), which is well positioned to act as nucleophile. Structural superposition with a product-bound class IV adenylate cyclase (PDB ID 3N10) reveals the O3′ of cAMP in the same position as the water molecule in ygiF. This position is also occupied by an oxygen atom of the product Pi located in the AtTTM3 post-catalysis complex. C, the suggested mechanism for acidic-patch containing TTM proteins. The metal ion in site 1 coordinates the triphosphate moiety of the substrate to the tunnel center by interacting with a conserved Glu residue. Three additional glutamates form metal binding site 2, which coordinates and polarizes a water molecule to attack the γ-phosphate of the substrate. Conserved basic residues in the tunnel center are involved in substrate binding and potentially stabilize the pyrophosphate leaving group.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: From the Structural Plant Biology Laboratory, Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland and.

Show MeSH
Related in: MedlinePlus