A ThDP-dependent enzymatic carboligation reaction involved in Neocarazostatin A tricyclic carbazole formation

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (31570033 to Y. Y.) and the Leverhulme Trust-Royal Society Africa Award (AA090088 to K. K and H. D.). Open access via RSC Gold 4 Gold.Peer reviewedPublisher PD

Substrate entering and product leaving trajectories predict an engulfing dynamic for the major conformational change of the β-lactam acylase

It is still a major challenge to acquire insight into the conformational changes between the ground state and the transition state of an enzyme, although conformational fluctuation within interconverting conformers has been widely investigated (1-4). Here, we utilize different enzymatic reactions in b-lactam acylase to figure out the substrate/product trajectories in the enzyme, thereby probing the overall conformational changes in transition state. First, an auto-proteolytic intermediate of cephalosporin acylase (EC 3.5.1.11) with partial spacer segment was identified. As a final proteolytic step, the deletion of this spacer segment was revealed to be a first-order reaction, suggesting an intramolecular Ntn mechanism for the auto-proteolysis. Accordingly, the different proteolytic sites in the acylase precursor indicate a substrate entering pathway along the spacer peptide. Second, bromoacyl-7ACA can interact with penicillin G acylase (EC 3.5.1.11) in two distinguish aspects, to be hydrolyzed as a substrate analogue and to affinity alkylate the conserved Trpb4 as a product analogue. The kinetic correlation between these two reactions suggests a channel opening from Serb1 to Trpb4, responsible for the main product leaving. These two reaction trajectories relaying at the active centre, together with the crystal structures (5-10), predict an engulfing dynamic involving pocket constriction and channel opening

Characterization of the Biosynthetic Gene Cluster for Benzoxazole Antibiotics A33853 Reveals Unusual Assembly Logic

SummaryA33853, which shows excellent bioactivity against Leishmania, is a benzoxazole-family compound formed from two moieties of 3-hydroxyanthranilic acid and one 3-hydroxypicolinic acid. In this study, we have identified the gene cluster responsible for the biosynthesis of A33853 in Streptomyces sp. NRRL12068 through genome mining and heterologous expression. Bioinformatics analysis and functional characterization of the orfs contained in the gene cluster revealed that the biosynthesis of A33853 is directed by a group of unusual enzymes. In particular, BomK, annotated as a ketosynthase, was found to catalyze the amide bond formation between 3-hydroxypicolinic and 3-hydroxyanthranilic acid during the assembly of A33853. BomJ, a putative ATP-dependent coenzyme A ligase, and BomN, a putative amidohydrolase, were further proposed to be involved in the benzoxazole formation in A33853 according to gene deletion experiments. Finally, we have successfully utilized mutasynthesis to generate two analogs of A33853, which were reported previously to possess excellent anti-leishmanial activity

Mechanism of Thioesterase-Catalyzed Chain Release in the Biosynthesis of the Polyether Antibiotic Nanchangmycin

SummaryThe polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site-directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates

Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant Bacillus subtilis and Candida maltosa

<p>Abstract</p> <p>Background</p> <p>Xylose mother liquor has high concentrations of xylose (35%-40%) as well as other sugars such as L-arabinose (10%-15%), galactose (8%-10%), glucose (8%-10%), and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem.</p> <p>Results</p> <p>We designed a novel strategy in which <it>Bacillus subtilis </it>and <it>Candida maltosa </it>were combined and used to convert xylose in this mother liquor to xylitol, a product of higher value. First, the xylose mother liquor was detoxified with the yeast <it>C. maltosa </it>to remove furfural and 5-hydromethylfurfural (HMF), which are inhibitors of <it>B. subtilis </it>growth. The glucose present in the mother liquor was also depleted by this yeast, which was an added advantage because glucose causes carbon catabolite repression in <it>B. subtilis</it>. This detoxification treatment resulted in an inhibitor-free mother liquor, and the <it>C. maltosa </it>cells could be reused as biocatalysts at a later stage to reduce xylose to xylitol. In the second step, a recombinant <it>B. subtilis </it>strain with a disrupted xylose isomerase gene was constructed. The detoxified xylose mother liquor was used as the medium for recombinant <it>B. subtilis </it>cultivation, and this led to L-arabinose depletion and xylose enrichment of the medium. In the third step, the xylose was further reduced to xylitol by <it>C. maltosa </it>cells, and crystallized xylitol was obtained from this yeast transformation medium. <it>C. maltosa </it>transformation of the xylose-enriched medium resulted in xylitol with 4.25 g L^-1·h^-1volumetric productivity and 0.85 g xylitol/g xylose specific productivity.</p> <p>Conclusion</p> <p>In this study, we developed a biological method for the purification of xylose from xylose mother liquor and subsequent preparation of xylitol by <it>C. maltosa</it>-mediated biohydrogenation of xylose.</p

Dual Carbamoylations on the Polyketide and Glycosyl Moiety by Asm21 Result in Extended Ansamitocin Biosynthesis

SummaryCarbamoylation is one of the post-PKS modifications in ansamitocin biosynthesis. A novel ansamitocinoside with carbamoyl substitution at the C-4 hydroxyl group of the N-β-D-glucosyl moiety was identified from the ansamitocin producer, Actinosynnema pretiosum. Through biotransformation, the carbamoyltransferase gene asm21 was suggested to be responsible for the carbamoylation of the glucosyl moiety. Three new derivatives without the backbone carbamoyl group were isolated from an asm21 mutant and characterized by NMR spectroscopy. Among them, 18-O-methyl-19-chloroproansamitocin was the major product and the preferred substrate for macrolactam C-7 carbamoylation by Asm21. However, Asm21 exhibited higher catalytic efficiency toward the glucosyl moiety. Furthermore, the dual carbamoylations and N-glycosylation were precisely demonstrated in vivo. This work represents the first biochemical characterization of an O-carbamoyltransferase performing dual actions on both a polyketide backbone and a glycosyl moiety during ansamitocin biosynthesis