Identification of a metagenome-derived prephenate dehydrogenase gene from an alkaline-polluted soil microorganism
2013
Jiang, Chengjian | Yin, Bangqiao | Tang, Mingze | Zhao, Gaochao | He, Jianjun | Shen, Peihong | Wu, Bo
A novel prephenate dehydrogenase gene designated pdhE-1 was cloned by sequence-based screening of a plasmid metagenomic library from uncultured alkaline-polluted microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis indicated that PdhE-1 and other putative prephenate dehydrogenases were closely related. The putative prephenate dehydrogenase gene was subcloned into pETBlue-2 vector and overexpressed in Escherichia coli BL21(DE3) pLacI. The recombinant protein was purified to homogeneity. The maximum activity of the PdhE-1 protein occurred at pH 8.0 and 45 °C using prephenic acid as the substrate. The prephenate dehydrogenase had an apparent K ₘ value of 0.87 mM, a V ₘₐₓ value of 41.5 U/mg, a k cₐₜ value of 604.8/min and a k cₐₜ/K ₘ value of 1.16 × 10⁴/mol/s. L-Tyrosine did not obviously inhibit the recombinant PdhE-1 protein. The identification of a metagnome-derived prephenate dehydrogenase provides novel material for studies and application of proteins involved in tyrosine biosynthesis.
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