In vivo evaluation of the interaction between the Escherichia coli IGP synthase subunits using the Bacterial Two-Hybrid system

Abstract

<jats:title>Abstract</jats:title> <jats:p>Histidine biosynthesis is one of the most characterized metabolic routes for its antiquity and its central role in cellular metabolism; indeed, it represents a cross-road between nitrogen metabolism and de novo synthesis of purines. This interconnection is due to the activity of imidazole glycerol phosphate synthase, a heterodimeric enzyme constituted by the products of two his genes, hisH and hisF, encoding a glutamine amidotransferase and a cyclase, respectively.</jats:p> <jats:p>Despite their interaction was suggested by several in vitro experiments, their in vivo complex formation has not been demonstrated. On the contrary, the analysis of the entire Escherichia coli interactome performed using the yeast two hybrid system did not suggest the in vivo interaction of the two IGP synthase subunits.</jats:p> <jats:p>The aim of this study was to demonstrate the interaction of the two proteins using the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system. Data obtained demonstrated the in vivo interaction occurring between the proteins encoded by the E. coli hisH and hisF genes; this finding might also open the way to pharmaceutical applications through the design of selective drugs toward this enzyme.</jats:p>

The Histidine Biosynthetic Genes in the Superphylum Bacteroidota-Rhodothermota-Balneolota-Chlorobiota: Insights into the Evolution of Gene Structure and Organization

“…It is quite interesting that, in almost all genomes analyzed, one of the most shared mini-operons contains the three genes hisH , hisA , and hisF that code for enzymes catalyzing the two steps involved in the cross connection of histidine biosynthesis, nitrogen metabolism, and the de novo synthesis of purines. As reported in the Introduction, it is known that HisH and HisF interact both in vitro and in vivo to form the bifunctional enzyme IGPS [ 48 ]. Thus, the possibility that the three proteins HisA, HisH, and HisF might act in a concerted manner forming a metabolon-like complex, which, in turn, should require the three proteins to be colocalized in the same cellular microenvironment, cannot be excluded a priori.…”

Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses

“…The heterodimeric enzyme imidazole glycerol phosphate (IGP) synthase (spot M25) catalyzes histidine biosynthesis which plays an important role in cellular metabolism by interconnecting nitrogen metabolism and purine synthesis ( Chioccioli et al, 2020 ). This enzyme was upregulated under all the stress treatments and could be responsible for providing tolerance to P. pentosaceus M41 through growth and repair by increased histidine biosynthesis.…”

Effect of Non-Lethal Selection on Spontaneous Revertants of Frameshift Mutations: The Escherichia coli hisF Case

“…The E. coli FB8 strain (wild-type E. coli K12 UTH1038 [27]) and E. coli FB182 (hisF892) strain [23] were used in the present work. E. coli FB182 carries a single nucleotide deletion in position 718 or 719 of the hisF gene, causing a frame shift and the formation of a premature stop codon, resulting in a shorter (243 aa vs. 258 aa of the wild-type E. coli HisF protein) and non-functioning enzyme [24].…”

“…However, some mutations could partially affect the correct functionality of the revertant HisF protein and, consequently, mutants' ability to grow in the total absence of histidine in the culture medium. A second hypothesis relies on the fact that HisF can perform its activity when it interacts with the product of the hisH gene, forming the heterodimeric enzyme imidazole glycerol phosphate synthase (IGPS) [24]; hence, in principle, the slow growth of these revertants in the absence of histidine might be due to a weaker interaction of HisF protein with the HisH counterpart. However, at least in T. maritima, it is known that the HisH subunit docks onto the N-terminal face of the HisF subunit, located opposite the C-terminal's active site face [45].…”

“…To the best of our knowledge, very little is known about the reversion of frameshift mutations resulting in amino acid auxotrophy and the correlation with the structure of the protein encoded by the mutated gene, as well as the growth rate in the absence of the amino acid whose biosynthetic pathway has been altered. For this reason, we investigated the different mutations restoring the prototrophy in an auxotrophic E. coli mutant (strain FB182 [23]) harboring the hisF892 single nucleotide deletion [24], causing a frame shift and resulting in the inability of E. coli cells to grow in the absence of histidine. The hisF gene, involved in the histidine biosynthetic pathway, was chosen as the subject of this study since it encodes a protein whose structure has been clearly elucidated in recent years, especially in the thermophilic bacterium Thermotoga maritima.…”