title: Cloning and characterization of the glycoside hydrolases that remove xylosyl groups from 7-β-xylosyl-10-deacetyltaxol and its analogues.
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-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-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 β-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 β-d-xylosidase/β-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-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-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-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.
authors.affiliation: 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.
authors.forename: Hai-Li
authors.initials: HL
authors.lastname: Cheng
authors.forename: Rui-Yu
authors.initials: RY
authors.lastname: Zhao
authors.forename: Tian-Jiao
authors.initials: TJ
authors.lastname: Chen
authors.forename: Wen-Bo
authors.initials: WB
authors.lastname: Yu
authors.forename: Fen
authors.initials: F
authors.lastname: Wang
authors.forename: Ke-Di
authors.initials: KD
authors.lastname: Cheng
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D012331:RNA, Fungal / Q000235:genetics; D020942:Shiitake Mushrooms / Q000201:enzymology* / Q000235:genetics; D043823:Taxoids / Q000378:metabolism*; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: C100478:7-xylosyl-10-deacetyltaxol; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D043823:Taxoids; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase