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

Date Published: July 25, 2017

Publisher: Public Library of Science

Author(s): Shannon E. Hill, Elaine Nguyen, Chiamaka U. Ukachukwu, Dana M. Freeman, Stephen Quirk, Raquel L. Lieberman, Titus J. Boggon.


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.

Partial Text

Enzymes involved in folate biosynthesis in bacteria are targets of antimicrobial agents that have been used in the clinic since the 1930s [1, 2]. The development of new folate-like antimicrobials has been aided by the availability of genes encoding these enzymes and emergence of new biochemical and structural details. One addition to the E. coli pathway, dihydroneopterin triphosphate pyrophosphatase (DHNTPase), was first isolated in 1974 [3] but its biological role in folate biosynthesis was confirmed only 30 years later [4, 5]. DHNTPase catalyzes the hydrolysis of 7,8-dihydroneopterin triphosphate, the product of GTP cyclohydrolase, to 7,8-dihydroneopterin monophosphate and pyrophosphate [4]. DHNTPase can now be included in an antibiotic strategy that, like classical sulfa-class antibiotics [1], simultaneously targets multiple enzymes in the folate biosynthetic pathway.




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