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 (KmPEP = 19.58 μM, VmaxPEP = 29.02 μM min−1, and kcatPEP/KmPEP = 0.88 s−1 μM−1) under optimal reaction conditions. The enzymatic property of Aro1A indicates its potential in aromatic amino acid industrial production.
Highlights
3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in aromatic amino acid anabolism
DAHPSs can be classified as type I or type II according to their molecular dimension: type I is < 40 kDa, and type II is 50 kDa (Gosset et al 2001; Jensen et al 2002)
The target gene in this study was from a clone named pUME11 and was annotated as a DAHPS gene based on the sequence analysis
Summary
3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in aromatic amino acid anabolism. 3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is a key rate-limiting enzyme in the synthesis of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan (Herrmann 1995). Type II DAHPS includes DAHPSs from vegetation and certain microorganisms such as M. tuberculosis, Corynebacterium glutamicum, and Helicobacter pylori These representatives are inhibited as types Iα and Iβ DAHPS. T DAHPS type, PF polymer form synthases, and alkaline proteases were identified using function-based and sequence-based screening strategies (Leis et al 2015; Mewis et al 2013; Niehaus et al 2011; Seow et al 1997; Yang et al 2016; Yun et al 2004) Most of these enzymes have new physio-biochemical characteristics and provide rich research materials for the improvement of industrial enzymes and for the further investigation of enzyme structures and functions
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