Allosteric Inhibition of Acinetobacter baumannii ATP Phosphoribosyltransferase by Protein:Dipeptide and Protein:Protein Interactions

Abstract

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to generate N 1 -(5-phospho-β-D-ribosyl)-ATP (PRATP) and pyrophosphate. Catalytic (HisG S ) and regulatory (HisZ) subunits assemble in a hetero-octamer where HisZ activates HisG S and mediates allosteric inhibition by histidine. In Acinetobacter baumannnii, HisG S is necessary for the bacterium to persist in the lung during pneumonia. Inhibition of ATPPRT is thus a promising strategy for specific antibiotic development. Here, A. baumannii ATPPRT is shown to follow a rapid equilibrium random kinetic mechanism, unlike any other ATPPRT. Histidine noncompetitively inhibits ATPPRT. Binding kinetics indicates histidine binds to free ATPPRT and to ATPPRT:PRPP and ATPPRT:ATP binary complexes with similar affinity following a twostep binding mechanism, but with distinct kinetic partition of the initial enzyme:inhibitor complex. The dipeptide histidine-proline inhibits ATPPRT competitively and likely uncompetitively, respectively, against PRPP and ATP. Rapid kinetics analysis shows His-Pro binds to the ATPPRT:ATP complex via a two-step binding mechanism. A related HisZ that shares 43% sequence identity with A. baumannii HisZ is a tight-binding allosteric inhibitor of A. baumannii HisG S . These findings lay the foundation for inhibitor design against A. baumannii ATPPRT.

Allosteric rescue of catalytically impaired ATP phosphoribosyltransferase variants links protein dynamics to active-site electrostatic preorganisation

“…28 AbHisZ was produced as previously reported. 26 All PaHisGS mutants were expressed and purified by the same protocol as PaHisGS. 28 Partial charges for the ligand PRPP, were calculated in vacuo at the HF/6-31G* level of theory using Gaussian 16 Rev.…”

“…and PaHisGS share 43% and 69% sequence identity with their orthologues from the pathogenic bacterium Acinetobacter baumannii, AbHisZ and AbHisGS, respectively, but PaHisZ has been shown to be a potent allosteric inhibitor of AbHisGS. 26 We thus hypothesised that AbHisZ could inhibit WT-PaHisGS. However, addition of AbHisZ activated catalysis by WT-PaHisGS (Fig.…”

“…22,23 ATPPRT is the focus of synthetic biology efforts to enable the production of histidine in bacteria 16,24 and a promising drug target against some pathogenic bacteria. [25][26][27] Short-form ATPPRTs form an intricate allosteric system comprising catalytic (HisGS) and regulatory (HisZ) subunits assembled as a heterooctamer with a tetrameric core of HisZ sandwiched by two dimers of HisGS. [28][29][30][31][32][33] HisGS on its own is catalytically active and insensitive to inhibition by histidine.…”

“…28,34 Binding of HisZ, which has no catalytic power of its own, to form the ATPPRT holoenzyme, allosterically activates catalysis by HisGS. 26,28,31,34 However, HisZ also harbours the pocket where histidine binds and allosterically inhibits ATPPRT. Thus, the regulatory protein plays a dual role, as allosteric activator of catalysis in the absence of histidine and mediator of allosteric inhibition in the presence of histidine.…”

Computer-Based Identification of Potential Druggable Targets in Multidrug-Resistant Acinetobacter baumannii: A Combined In Silico, In Vitro and In Vivo Study

“…Previous studies have reported identifying potential drug targets in A. baumannii by the subtractive proteomics approach [ 20 , 21 , 22 , 49 ]. Some of the 68 shortlisted drug targets in the current study have been reported in previous studies [ 20 , 21 , 22 , 50 , 51 , 52 , 53 ]. However, it is worth mentioning that our study is the first one to proceed with further in vitro and in vivo validation.…”