Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutants resistant to feedback inhibition by histidine

• Published in: Biochimie 2012-03, Vol.94 (3), p.829-838
• Main Authors: Zhang, Yun, Shang, Xiuling, Deng, Aihua, Chai, Xin, Lai, Shujuan, Zhang, Guoqiang, Wen, Tingyi
• Format: Article
• Language: English
• Subjects: Allosteric properties; ATP; ATP phosphoribosyltransferase; ATP Phosphoribosyltransferase - genetics; ATP Phosphoribosyltransferase - metabolism; Complementation; Condensation; Corynebacterium glutamicum; Corynebacterium glutamicum - enzymology; Data processing; Electrostatic properties; Enzymes; Feedback inhibition; Gene disruption; Gram-positive bacteria; Histidine; Histidine - metabolism; Mutagenesis; Mutagenesis, Site-Directed; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Polarity; Site-directed mutagenesis
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2011.11.015

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, HisGCg, from Corynebacterium glutamicum, including site-directed mutagenesis of the histidine-binding site for the first time. Gene disruption and complementation experiments showed that HisGCg is essential for histidine biosynthesis. HisGCg activity was noncompetitively inhibited by histidine and the α-amino group of histidine were found to play an important role for its binding to HisGCg. Homology-based modeling predicted that four residues (N215, L231, T235 and A270) in the C-terminal domain of HisGCg may affect the histidine inhibition. Mutating these residues in HisGCg 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 Ki value. As expected, overexpressing a hisGCg 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 HisGCg, 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. ► HisGCg is indispensable for histidine biosynthesis in Corynebacterium glutamicum. ► Alkaline pH decreases the inhibitory effect of histidine. ► Asn215, Leu231, and Thr235 in the C domain are critical for histidine binding. ► The change of electrostatic potential of HisGCg results in releasing allosteric inhibition.