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PMID:21672638
| Citation |
Fujii, T, Shimizu, M, Doi, Y, Fujita, T, Ito, T, Miura, D, Wariishi, H and Takaya, N (2011) Novel fungal phenylpyruvate reductase belongs to d-isomer-specific 2-hydroxyacid dehydrogenase family. Biochim. Biophys. Acta 1814:1669-76 |
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| Abstract |
We discovered the phenyllactate (PLA)-producing fungal strain Wickerhamia fluorescens TK1 and purified phenylpyruvate reductase (PPR) from fungal cell-free extracts. The PPR used both NADPH and NADH as cofactors with more preference for the former. The enzyme reaction as well as the fungal culture produced optically active d-PLA. The gene for the PPR (pprA) was cloned and expressed in Escherichia coli cells. Purified preparations of both native and recombinant PPR used hydroxyphenylpyruvate, glyoxylate and hydroxypyruvate as substrates but not pyruvate, oxaloacetate or benzoylformate. The predicted PPR protein had sequence similarity to proteins in the d-isomer-specific 2-hydroxyacid dehydrogenase family. Phylogenetic analyses indicated that the predicted PPR protein together with fungal predicted proteins constitutes a novel group of glyoxylate/hydroxypyruvate reductases. The fungus efficiently converted phenylalanine and phenylpyruvate to d-PLA. These compounds up-regulated the transcription of pprA, suggesting that it plays a role in fungal phenylalanine metabolism. |
| Links |
PubMed Online version:10.1016/j.bbapap.2011.05.024 |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0016616: oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor |
ECO:0000314: |
F |
Figure 1: Time dependent changes in PLA levels were monitored using HPLC. The PLA concentration linearly increased (Fig. 1A), indicating that the strain produced PLA at a constant rate. Adding PPA, a potential PLA substrate, to the culture medium increased the amount of PLA produced by strain TK1 (Fig. 1B). After 48-h of culture, 1.5 mM PPA was consumed and 0.8 mM of PLA accumulated in the medium,indicating that 53% of the PPA was converted to PLA. When cultured in MM containing phenylalanine, strain TK1 consumed phenylalanine and produced PLA (Fig. 1C). After the initial 24-h of incubation, 5.0 mMphenylalanine was consumed and 5.7 mMPLA was generated,indicating that most of the added phenylalanine was converted to PLA. We detected phenylalanine aminotransferase (PAT) (1.9±0.2 μmol min−1 mg−1) and PPR (see below) activities in the fungal cell-free extracts. These results together with the higher levels of PLA being generated from either PPA or phenylalanine than from glucose when added as a carbon source, indicated that the fungus generates PLA via PPA and/or phenylalanine (Fig. 1D), and that biosynthesized PPA limits PLA production from glucose. |
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References
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