title: Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutants resistant to feedback inhibition by histidine.
abstract: ATP phosphoribosyltransferase (ATP-PRT) catalyzes the condensation of ATP and PRPP at the first step of histidine biosynthesis and is regulated by a feedback inhibition from product histidine. Here, we report the genetic and biochemical characterization of such an enzyme, HisG(Cg), from Corynebacterium glutamicum, including site-directed mutagenesis of the histidine-binding site for the first time. Gene disruption and complementation experiments showed that HisG(Cg) is essential for histidine biosynthesis. HisG(Cg) activity was noncompetitively inhibited by histidine and the α-amino group of histidine were found to play an important role for its binding to HisG(Cg). Homology-based modeling predicted that four residues (N215, L231, T235 and A270) in the C-terminal domain of HisG(Cg) may affect the histidine inhibition. Mutating these residues in HisG(Cg) did not cause significant change in the specific activities of the enzyme but resulted in the generation of mutant ones resistant to histidine inhibition. Our data identified that the mutant N215K/L231F/T235A resists to histidine inhibition the most with 37-fold increase in K(i) value. As expected, overexpressing a hisG(Cg) gene containing N215K/L231F/T235A mutations in vivo promoted histidine accumulation to a final concentration of 0.15 ± 0.01 mM. Our results demonstrated that the polarity change of electrostatic potential of mutant protein surface prevents histidine from binding to the C-terminal domain of HisG(Cg), resulting in the release of allosteric inhibition. Considering that these residues were highly conserved in ATP-PRTs from different genera of Gram-positive bacteria the mechanism by histidine inhibition as exhibited in Corynebacterium glutamicum probably represents a ubiquitously inhibitory mechanism of ATP-PRTs by histidine.
authors.affiliation: Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiuling
authors.initials: X
authors.lastname: Shang
authors.forename: Aihua
authors.initials: A
authors.lastname: Deng
authors.forename: Xin
authors.initials: X
authors.lastname: Chai
authors.forename: Shujuan
authors.initials: S
authors.lastname: Lai
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Zhang
authors.forename: Tingyi
authors.initials: T
authors.lastname: Wen
mesh_terms: D001276:ATP Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D006639:Histidine; D001276:ATP Phosphoribosyltransferase