Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii
| Autor: | Guo-Jhang Ma, Ping-Du Lee, Lu-Sheng Hsieh, Chien-Chih Yang |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Ammonia-Lyases
Molecular Sequence Data Bambusa Gene Expression Phenylalanine Plant Science Phenylalanine ammonia-lyase Horticulture Bambusa oldhamii medicine.disease_cause Biochemistry Substrate Specificity medicine Cloning Molecular Tyrosine Site-directed mutagenesis Molecular Biology Escherichia coli Phenylalanine Ammonia-Lyase Base Sequence biology Molecular mass General Medicine Hydrogen-Ion Concentration biology.organism_classification Immunohistochemistry Molecular biology humanities Molecular Weight Open reading frame Mutagenesis Site-Directed |
| Zdroj: | Phytochemistry. 71:1999-2009 |
| ISSN: | 0031-9422 |
| DOI: | 10.1016/j.phytochem.2010.09.019 |
| Popis: | Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 μM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells. |
| Databáze: | OpenAIRE |
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