title: Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27.
abstract: The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
authors.forename: Licong
authors.initials: L
authors.lastname: Yi
authors.affiliation: National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production and Construction Corps, College of Life Science, Tarim University, Alar, Xinjiang 843300, People's Republic of China.
authors.forename: Lili
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State of Oceanic Administration, Xiamen 361005, People's Republic of China.
authors.forename: Zongze
authors.initials: Z
authors.lastname: Shao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China. Electronic address: lzd@mail.hzau.edu.cn.
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D053521:Alcanivoraceae / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005998:Glycine; D011993:Recombinant Fusion Proteins; D012765:Shikimic Acid; D000097797:Glyphosate; C058070:5-enolpyruvoylshikimate-3-phosphate
keywords: Alcanivorax sp; Cold adaption; Site-directed mutagenesis; aroA