Mechanistic enzymology of short-form ATP phosphoribosyltransferase and bifunctional phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase

Abstract

Adenosine 5′-triphosphate phosphoribosyltransferase (ATPPRT) catalyses the first reaction of the histidine biosynthetic pathway. Short-form ATPPRT, denoted HisGₛ, is allosterically activated by HisZ, which also mediates histidine feedback inhibition. A HisGₛ variant that exhibits a high rate of reaction in the absence of HisZ is desirable for industrial histidine biosynthesis. First, this work sought to define the mechanism underpinning allosteric activation of Psychrobacter arcticus HisGₛ by HisZ. Knowledge of this mechanism could drive mutagenic strategies to improve turnover by mimicking allosteric activation. The second and third steps of the histidine biosynthetic pathway are catalysed by HisE and HisI, respectively. These enzymes may be encoded by a single polypeptide chain, termed HisIE. HisIE is a possible target for novel antimicrobial development in several multi-drug resistant organisms, including carbapenem-resistant Acinetobacter baumannii Secondly, this work sought to initiate biochemical characterisation of HisIE from A. baumannii.

This work established binding of HisZ shifts the rate-limiting step of reactions catalysed by HisGₛ from interconversion between ternary complexes to product release. Characterisation of HisGₛ variants indicated two conserved arginine residues, R32 and R56, are required for stabilisation of the transition state. A model of allosteric activation of HisGₛ by HisZ is proposed in which binding of HisZ modifies the conformational sampling of these arginine residues such that conformations with these residues in position to stabilise the transition state are sampled more frequently. This work also documents the first reported characterisations of the steady-state kinetic parameters and the rate-limiting steps of a bifunctional HisIE.

Ultimately, this work suggests efforts to engineer a constitutively activated short-form HisGₛ for industrial histidine biosynthesis should seek to introduce mutations which narrow the conformational sampling of R32 and R56. Furthermore, this work has paved the way for future characterisation of the catalytic mechanism of HisIE with a view towards novel antimicrobial development.