Isolation and biochemical characterization of a metagenome-derived 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase gene from subtropical marine mangrove wetland sediments
Huaxian Zhao,
Hua Gao,
Kai Ji,
Bing Yan,
Quanwen Li,
Shuming Mo,
Minggang Zheng,
Qian Ou,
Bo Wu,
Nan Li,
Chengjian Jiang
Affiliations
Huaxian Zhao
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Hua Gao
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Kai Ji
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Bing Yan
Guangxi Key Laboratory of Mangrove Conservation and Utilization, Guangxi Mangrove Research Center, Guangxi Academy of Sciences
Quanwen Li
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Shuming Mo
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Minggang Zheng
The First Institute of Oceanography, State Oceanic Administration of China
Qian Ou
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Bo Wu
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Nan Li
Key Laboratory of Environment Change and Resources Use in Beibu Gulf (Guangxi Teachers Education University), Ministry of Education
Chengjian Jiang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University
Abstract 3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in aromatic amino acid anabolism. A new Iβ-type DAHPS gene (aro1A) was identified in a metagenomic library from subtropical marine mangrove sediment. The gene encoded a polypeptide composed of 272 amino acids and had a maximum similarity of 52.4% to a known DAHPS at the amino acid level. Multiple sequence alignment, homologous modeling, and molecular docking showed that Aro1A had the typical (β/α)8 barrel-shaped catalytic structural domain of DAHPS. The motifs and amino acid residues involved in the combination of substrates and metal ligand were highly conservative with the known DAHPS. The putative DAHPS gene was subcloned into a pET-30a(+) vector and was overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein was purified to homogeneity. The maximum activity for the recombinant Aro1A protein occurred at pH 8.0 and 40 °C. Ba2+ and Ca2+ stimulated the activity of Aro1A protein. The enzyme showed high affinity and catalytic efficiency (K m PEP = 19.58 μM, V max PEP = 29.02 μM min−1, and k catPEP/K m PEP = 0.88 s−1 μM−1) under optimal reaction conditions. The enzymatic property of Aro1A indicates its potential in aromatic amino acid industrial production.
