A mechanistic interrogation of ATP phosphoribosyltransferase from Acinetobacter baumannii

Abstract

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyses the first step in the histidine biosynthesis pathway, and it has been identified as a promising novel antimicrobial target against carbapenem-resistant Acinetobacter baumannii. Short-form ATPPRTs contain independent catalytic (HisGₛ) and regulatory (HisZ) subunits assembled into a hetero-octameric complex, where HisZ allosterically activates HisGₛ and harbours the histidine binding site to mediate negative-feedback inhibition by histidine. This work aimed to elucidate the underpinnings of ATPPRT catalysis and inhibition, informing future antibiotic discovery efforts. Steady-state kinetic characterisation of A. baumannii HisGS (AbHisGₛ) and ATPPRT (AbATPPRT) showed a unique mechanism amongst other ATPPRTs. Stopped-flow spectrofluorometry and data fitting by numerical integration allowed a detailed mechanism of inhibition by histidine and novel allosteric inhibitor scaffolds to be established. The rate-limiting steps of AbHisGₛ and AbATPPRT were uncovered via pre-steady-state kinetics, solvent viscosity effects, and divalent metal effects. Results reflected that of previously characterised short-form ATPPRTs, with a switch from rate-limiting interconversion of ternary complexes to product release upon allosteric activation by HisZ. At low temperatures, AbATPPRT product release is governed by a rate-limiting enzyme-product binary complex isomerisation – the rate of which is decreased further upon perturbation of the bond vibrational frequencies by heavy-isotope substitution of AbHisGₛ. A model for the dynamic control of product release was established: rapid femtosecond- picosecond-timescale dynamics, involving the highly conserved Arg70 of AbHisGₛ, govern slower millisecond-timescale physical motions controlling product release, and thus catalytic turnover. This work contributes to a greater understanding of the dynamic control in multi-subunit allosteric systems, and ultimately paves the way for inhibitor design against AbATPPRT.

Date of Award	12 Jun 2024
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Rafael Guimaraes da Silva (Supervisor) & John B. O. Mitchell (Supervisor)