Dual Carbamoylations By Carbamoyltransferase Asm21 In The Biosynthesis Of Ansamitocin

Maytansinoids are potent antitumor agents that exert their cytotoxicity by disrupting microtubule assembly. Antibody conjugates of maytansinoids exhibit increased tumour selectivity and longer circulation half-life and are currently in different stages of clinical development. Ansamitocins, produced by Actinosynnema pretiosum ssp. auranticum ATCC 31565, are maytansinoids of microbial origin. In the biosynthesis of ansamitocin P-3, 3-amino-5-hydroxybenzoic acid (AHBA) is used as a starter unit, and the incorporation of seven PKS extender units gives a 19-membered macrolactam, proansamitocin, which further undergoes a series of post-PKS modifications, including O- and N-methylation, chlorination, epoxidation, O-carbamoylation, and O-acylation.Recently, ansamitocinosides P-1, P-2 and P-3 (AGP-1, AGP-2 and AGP-3) were isolated from A. pretiosum, which carry aβ-D-glucosyl moiety attached to the amide nitrogen in place of the N-methyl group of ansamitocins. Meanwhile, a series of polar ansacarbamitocins with a glucosyl moiety and three carbamoyl groups were isolated from Amycolatopsis sp. CP2808. Moreover, two new ansamitocin derivatives produced by mutational biosynthesis were assigned to be N-β-D-glucopyranosylated at the macrolactam amide. In addition, a novel ansamitocinoside with carbamoyl substitution at the C-4 hydroxyl group of the N-β-D-glucosyl moiety (ACGP-3) was identified from Actinosynnema pretiosum by our co-workers. Asm25 has been proved to be the dedicated N-glycosyltransferase for the sugar attachment. All these findings raised the question which genes and enzymes are responsible for the carbamoylation of the glucose hydroxyl group.The construction of numerous mutants of A. pretiosum ATCC 31565 in the previous work allowed convenient identification ofthe relevant functional carbamoyltransferase gene(s). Through biotransformation, the carbamoyltransferase gene asm21 was suggested to be responsible for the carbamoylation of the glucosyl moiety. Moreover, based on sequence homology, gene asm21 in the biosynthetic gene cluster of ansamitocin was assigned the putative function of introducing the cyclic carbinolamide group. Therefore Asm21 was proved to have dual carbamoylation activityon both the polyketide backbone and the glucosyl moiety in the biosynthesis of ansamitocin. This is quite unique and interesting because the dual actions by one carbamoyltransferase have not been reported before.Through gene inactivation and complementation, the involvement of asm21 in ansamitocin biosynthesis was confirmed. Besides, the functional length of gene asm21 was refined based on complementation results. In addition to the previously identified 19-chloroproansamitocin, three novel compounds (14-β-hydroxy-20-O-methyl-19-chloroiso proansamitocin,14- -hydroxy-20-O-methyl-19-chloroisoproansamitocin and 20-O-methyl-19-chloroproansamitocin) lacking the C-7 carbamoyl group were isolated from the culture of BLQ16 (asm21 mutant) on solid YMG medium and characterized by NMR. Among them, 20-O-methyl- 19-chloroproansamitocin was the major product and chosen to examine the enzymatic properties of Asm21 using carbamoyl phosphate as another substrate in the presence of Mg2+ and ATP.Asm21 was optimally active at 37oC and pH 8.5 - 9.0 and showed the highest activity when supplied with 5 - 10 mM Mg2+. It showed higher affinity towards 20-O-methyl- 19-chloroproansamitocin with a Km of 25.2±8.7μM compared to 19-chloroproansamitocinwith a Km of 78.7±18.8μM, indicating that 20-O-methyl-19- chloroproansamitocin is a preferred substrate for Asm21.Asm25 was obtained as inclusion body in the previous report, and herein this study, soluble protein was expressed and purified with a different vector. Pure AGP-3 was therefore prepared through large scale incubation of PND-3 and UDP-glucose catalyzed by purified Asm25. Purified Asm21 also catalyzed the conversion of AGP-3 into ACGP-3, verifying that it does have dual carbamoylation activity on both the polyketide backbone and the sugar moiety. Moreover, Asm21 has a Km of 135.3±38.4μM for AGP-3, much higher than those for 20-O-methyl- 19-chloroproansamitocin or 19-chloroproansamitocin, which indicates a favored binding toward the polyketide backbone rather than the glucosyl moiety in the reaction site of Asm21. However, the overall catalytic efficiency (kcat /Km) for AGP-3 is higher than the other two substrates. Hence, the catalytic constants indicate that Asm21 displays a preference for carbamoylation of the glucosyl moiety over the polyketide backbone. Furthermore, the dual carbamoylations and N-glycosylation were precisely demonstrated in vivo.This work represents the first biochemical characterization of an O-carbamoyltransferase with very high substrate flexibility during ansamitocin biosynthesis. Remarkably, the enzyme catalyzes not only the C-7 carbamoylation of the macrolactam backbone, accepting a variety of ansamitocin structures as substrates, but also the carbamoylation of the C-4 hydroxyl group of the N-glucosyl moiety in ansamitocinoside P-3. Thus, the ansamitocin biosynthetic pathway could be extended through the tandem catalysis of the N-glycosyltransferase Asm25 and the O-carbamoyltransferase Asm21 by cultivating the strain on solid medium. Due to its broad substrate range, Asm21 can be used to generate O-carbamoylated derivatives of many ansamycins as potential drug candidates.The clinically important family of AHBA (3-amino-5-hydroxybenzoic acid) containing natural products, including ansamycins or other antibiotics, possesses potent antibacterial, antifungal or antitumor activities. The biosynthetic pathway of AHBA has been disclosed in the biosynthesis of rifamycin. AHBA synthase RifK catalyzes the last step converting aminoDHS into AHBA, and this protein shares high identity with its homologs in other ansamycin producers. On the other hand, the C7N-aminocyclitol family of natural products, exemplified by anti-diabetic acarbose and anti-fungal validamycin A, contain a C7N aminocyclitol in their core structure. The biosynthesis of these compounds is universally initiated with the cyclization of sedoheptulose 7-phosphate, catalyzed by a sugar phosphate cyclase (ValA for validamycin A and AcbC for acarbose), to give 2-epi-5-epi-valiolone, which then undergoes different routes to yield various final products. The high similarities shared by AHBA synthases and cyclases isolated from different gene clusters enabled us to design degenerate primers using CODEHOP to screen a pool of Actinomycete strains. As a result, seven AHBA positive and three cyclase positive strains were obtained from the laboratory library. The amplified DNA fragments were cloned and sequenced to confirm the correctness and homology with the probe。The deduced animo acid sequence of onecyclase positive strain, Streptomyces sp. CS, showed high similarity to ValA and AcbC. Subsequently a fosmid genomic library was constructed and 13 fosmids were fished out with the cyclase primers. One of them (16H9) was fully sequenced, and more than 20 ORFs were identified using Frameplot 3.0 and BLASTp. Interestingly, ORF16 - 20, which showed high homology to AcbC, AcbM, AcbL, AcbN, and AcbO respectively, are clustered as in acarbose biosynthetic gene cluster. These five enzymes probably catalyze the first five reactions of the biosynthesis of acarbose to generate 1-epi- valienol 7-phosphate, whose chemical structure is quite similar to CSS, a polyhydroxyl derivative of C7 cyclohexane previously isolated from Streptomyces sp.CS. The production of CSS was abolished by inactivation of CS-valA, indicating that this gene cluster is responsible for its biosynthesis. This was further confirmed by a successful heterologous expression of the CSS gene cluster in S. albus J1074. This gene to compound mining strategy provides a new and pratical way for the discovery of new antibiotics.