Molecular basis for the allosteric activation mechanism of the heterodimeric imidazole glycerol phosphate synthase complex

Jan Philip Wurm,Sihyun Sung,Matthias Wilmanns,Remco Sprangers,Reinhard Sterner,Andrea Christa Kneuttinger,Enrico Hupfeld

doi:10.1038/s41467-021-22968-6

Abstract

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood. Here, we elucidate the molecular basis of the long range, allosteric activation of HisFH. We establish that the catalytically active HisFH conformation is only formed when the substrates of both HisH and HisF are bound. We show that in this conformation an oxyanion hole in the HisH active site is established, which rationalizes the observed 4500-fold allosteric activation compared to the inactive conformation. In solution, the inactive and active conformations are in a dynamic equilibrium and the HisFH turnover rates correlate with the population of the active conformation, which is in accordance with the ensemble model of allostery.

Highlights

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism
HisF and HisH (HisFH) belongs to the class I of glutamine amidotransferases (GATs) that play a central role in metabolism as they incorporate nitrogen derived from glutamine into a variety of metabolites[4,5,6,7,8]
To monitor conformational changes of the catalytic histidine hH178 we prepared deuterated HisFH complexes, in which the HisH histidines are labeled at the C2/H2 position (Supplementary Fig. 3)

Summary

Introduction

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. The HisFH complex catalyzes the synthesis of the histidine precursor imidazole glycerol phosphate (ImGP) and the purine precursor 5aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N′-[(5′-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and glutamine (Gln)[9,10] (Fig. 1a) This reaction takes place in two main steps and starts with the hydrolysis of Gln to glutamate (Glu) and ammonia in the glutaminase subunit HisH. The amide proton points away from the catalytic site and the carbonyl oxygen of hV51 blocks the amide proton hL85 in the oxyanion hole (see below), which explains why the catalytic activity of HisH in the absence of an activating event is very low It has been suggested[21] that the allosteric activation of the glutaminase reaction entails the formation of an oxyanion hole in HisH similar to the ones observed in other class I GATs

Methods

Results

Conclusion