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--xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7--xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related -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 -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 -D-xylosidase/-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/K m of up to 1.07 s ؊1 mM ؊1 against 7--xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7--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--xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7--xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7--xylosyltaxanes and contribute to the growing database of glycoside hydrolases. Molecular &
The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5'-untranslated region (UTR), 3'-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS-PAGE and western blot. GC-MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.
The coxsackievirus B4 (CVB4) belongs to human enterovirus B species within the family Picornaviridae. Here we report a novel complete genome sequence of a recombinant CVB4 strain, CVB4/GX/10, which was isolated from a patient with a fatal case of hand, foot, and mouth disease in China. The complete genome consists of 7,293 nucleotides, excluding the 3= poly(A) tail, and has an open reading frame that maps between nucleotide positions 742 and 7293 and encodes a 2,183-amino-acid polyprotein. Phylogenetic analysis based on different genome regions reveals that CVB4/GX/10 is closest to a CVB4 strain, EPIHFMD-CLOSE CONTACT-16, in the 5= half (VP4ϳ2B) of the genome, although it is closer to a Chinese CVB5 strain, CVB5/Henan/2010, in the 3= half (2Cϳ3D) of the genome. Furthermore, similar bootscan analysis based on the whole genomes demonstrates that recombination has possibly occurred within the 2C domain and that CVB4/GX/10 is a possible progeny of intertypic recombination of the CVB4 strain EPIHFMD-CLOSE CONTACT-16 and CVB5/Henan/2010 that occurred during their cocirculation and evolution, which is a relatively common phenomenon in enteroviruses.
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