Gene Cioning, Expression And Characterization Of A Prephenate Dehydrogenase From Uncultured Soil Microorganisms

Prephenate dehydrogenase (EC1.3.1.12) catalyzes the oxidative decarboxylation of prephenate to4-hydroxyphenylpyruvate in the presence of NAD(P)+, which plays a key role in the L-tyrosine biosynthesis metabolism. Currently, more and more research focus on the function and structure of the known prephenate dehydrogenase from Aquifex aeolicus、Streptococcus mutans, and Haemophilus influenzae. It’s interesting to mining for novel prephenate dehydrogenase from uncultured microorganisms.In this study, the direct sequencing strategy was used for screening a metagenomic library AL01from alkaline soil samples, the clone pGXAL10028may contain a novel gene encoding prephenate dehydrogenase, which was named pdhE. Bio-informatics analysis showed the gene pdhE was composed of609base pairs, which encoded a protein of203amino acid with molecular mass of22.3kDa, and the theoretical isoelectric point of5.31. At the nucleotide acid level, PdhE protein was found to share partial homology (82%) with a pdhE (GenBank accession number:FR856862.1) to the whole genome sequence of Novosphingobium sp. PP1Y; At the amino acid level, PdhE shared42%identical and65%similar with a prephenate dehydrogenase from Zymomonas mobilis (GenBank accession number:Q04983.2)pdhE gene was expressed using pETBlueTM expression system, and the recombinant protein PdhE was purified successfully with nickel affinity chromatography technology, but no catalytic activity was detected. Further biological information analysis showed that PdhE had a complete PDH core catalytic domain, but its amino end of the Rossmann β-α-β fold domain (coenzyme of NAD+/NADP+binding domain), which combined with coenzyme is not complete, that may affect the catalytic activity of the protein.Fusion PCR was applied to recombine E. coli K-12strains prephenate dehydrogenase Rossmann β-α-β fold domain coding DNA and pdhE. A novel recombinant gene was obtained, and was named pdhE-1.pdhE-1gene was expressed using pETBlueTM expression system, and the recombinant protein PdhE-1was purified with nickel affinity chromatography technology. The biochemical characterization of recombinant protein PdhE-1were found that the optimal temperature of was45℃, the optimal pH was8.0, and the specific activity was7.63U/mg. The kinetics parameters of recombinant protein PdhE-1were found that Km was0.87mmol/L, kcat may be10.08s-1as the substrate of prephenate, and the Km for coenzyme NAD+was0.13mmol/L. An important results had been found that the final metabolic production L-tyrosine (10mmol/L) did not show inhibition to recombinant protein PdhE-1. The functional characterization of a novel prephenate dehydrogenase from a soil metagenome will contribute to the L-tyrosine production, and also provide a technology reference to the research of novel target enzyme using the biochemical specificity of PdhE-1protein.