Isolation and biochemical characterization of a metagenome-derived 3-deoxy- d-arabino-heptulosonate-7-phosphate synthase gene from subtropical marine mangrove wetland sediments

摘要

3-Deoxy- d -arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in aromatic amino acid anabolism. A new I _(β)-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. Ba ~(2+) and Ca ~(2+) 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 cat) PEP)/ 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. Electronic supplementary material The online version of this article (10.1186/s13568-019-0742-4) contains supplementary material, which is available to authorized users.