Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

Abstract

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

Cations in motion: QM/MM studies of the dynamic and electrostatic roles of H+ and Mg2+ ions in enzyme reactions

“…Many enzymes use the so-called two-metal ion catalytic mechanism [59], where a second metal ion coordinates the incoming nucleophile thereby activating them for easier deprotonation [60]. Additionally, two ions may also be present to activate the leaving group, such as for kinases, and occasionally even three ions are required for activity in some phosphate catalytic enzymes such as alkaline phosphatases [61,62], aminoacyl ligases [63] and nudix hydrolases [64,65]. Polarization can also be achieved with the help of positively charged active site residues.…”

“…Accordingly, they also play a role in drug discovery to bind ligands directly (Figure 3). Less commonly, Mn 2+ and Zn 2+ , occasionally even Cu 2+ or Co 2+ can also act as the catalytic ion [65,70]. Importantly, Ca 2+ ions typically inhibit phosphate catalytic enzymes, which also underlines the key role of Mg 2+ as a Lewis acid.…”

Revitalizing antifolates through understanding mechanisms that govern susceptibility and resistance

“…33,34 Nudix enzymes hydrolyze substrates through the use of a nucleophilic substitution and require a metal ion for coordination of GTP, though the specific metal varies between Nudix enzymes. [32][33][34][35] Since dihydroneopterin triphosphate pyrophosphatases have only been recently discovered, inhibitors of this enzyme have yet to be identified. Nevertheless, deletion of nudB in E. coli has been shown to dramatically enhance susceptibility to both SMX and TMP.…”

Structures of MERS1, the 5′ processing enzyme of mitochondrial mRNAs in Trypanosoma brucei

“…However, it does harbor a putative Nudix box, which is a signature sequence found in a large family of hydrolases (Mildvan et al 2005;Lin et al 2008;Srouji et al 2017). Nudix hydrolases, which are widely distributed from bacteria to humans, are named for their conserved overall function, which is the hydrolysis of nucleoside diphosphates linked to other moieties, X (Mildvan et al 2005;McLennan 2006;Kraszewska 2008;Lin et al 2008;Hong et al 2014;Hill et al 2017;Srouji et al 2017). The Nudix box is a 23 residue signature sequence, GX 5 EX 7 REUXEEXGU (where X is any residue and U corresponds to hydrophobic residues, usually Ile, Leu, or Val) that forms a loop-helix-loop motif (Mildvan et al 2005;McLennan 2006).…”