NDLI: Cloning and characterization of the glycoside hydrolases that remove xylosyl groups from 7-ß-xylosyl-10-deacetyltaxol and its analogues.

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Cloning and characterization of the glycoside hydrolases that remove xylosyl groups from 7-ß-xylosyl-10-deacetyltaxol and its analogues.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Cheng, Hai-Li Zhao, Rui-Yu Chen, Tian-Jiao Yu, Wen-Bo Wang, Fen Cheng, Ke-Di Zhu, Ping
Description	Country affiliation: China Author Affiliation: Cheng HL ( State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.)
Abstract	Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-ß-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-ß-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related ß-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of ß-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1-1 and Lxyl-p1-2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional ß-d-xylosidase/ß-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1-1, G-Lxyl-p1-2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km of up to 1.07 s(-1)mm(-1) against 7-ß-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-ß-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-ß-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-ß-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-ß-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.
File Format	HTM / HTML
ISSN	15359476
e-ISSN	15359484
DOI	10.1074/mcp.M113.030619
Journal	Molecular & Cellular Proteomics
Issue Number	8
Volume Number	12
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2013-08-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Proteomics Shiitake Mushrooms Enzymology Taxoids Metabolism Xylosidases Amino Acid Sequence Cloning, Molecular Dna, Complementary Genetics Dna, Fungal Fungal Proteins Molecular Sequence Data Rna, Fungal Yeasts Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Medicine Analytical Chemistry Molecular Biology Biochemistry

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