Gilvocarcin V (GV) belongs to the angucycline class of antibiotics that possesses remarkable anticancer and antibacterial activities with low toxicity. Gilvocarcin exhibits its light induced anticancer activity by mediating crosslinking between DNA and histone H3. When photo-activated by near-UV light, the C8 vinyl group forms a [2+2] cycloadduct with thymine residues of double stranded DNA. D-fucofuranose is considered essential for histone H3 interactions. However, the poor water solubility has rendered it difficult to develop gilvocarcin as a drug. We aim to design novel gilvocarcin analogues with improved pharmaceutical properties through chemo-enzymatic synthesis and mutasynthesis. Previous studies have characterized many biosynthetic genes encoding the gilvocarcin biosynthetic skeleton. Despite these previous findings the exact functions of many other key genes are yet to be fully understood. Prior gene inactivation and cross-feeding experiments have revealed that the first isolable tetracyclic aromatic product undergoes a series of steps involving C–C bond cleavage followed by two O-methylations, a penultimate C-glycosylation and final lactone formation in order to fully develop the gilvocarcin structure. To provide a deeper understanding of these complex biochemical transformations, three specific aims were devised: 1) synthesis of the proposed intermediate and in vitro enzyme reactions revealed GilMT and GilM’s roles in gilvocaric biosynthesis; 2) utilizing in vitro studies the enzyme responsible for the C–C bond cleavage and its substrate were determined; 3) a small series of structural analogues of the intermediate from the gilvocarcin pathway was generated via chemical synthesis and fed to the mixture of the enzymes, GilMT and GilM. These reaction mixtures were then analyzed to establish the diversity of substrates tolerated by the enzymes.

Gilvocarcin V (GV) and ravidomycin (RMV) exhibit excellent antitumor activities in the presence of near-UV light at low concentration maintaining a low in vivo cytotoxicity. Although, the exact molecular mechanism for in vivo actions of these antibiotics has yet to be determined, a [2+2] cycloaddition reaction of the vinyl side chain with DNA thymidine residues in addition to the inhibition of topoisomerase II and DNAhistone H3 cross-linking are reported for the GV’s mechanism of action. Such activities have made these molecules interesting candidates for the biosynthetic investigation to generate analogues with improved activity/solubility. Previous biosynthetic studies have suggested that the GV biosynthetic pathway involves a number of synchronously occurring transformations leading to the oxidative C-C bond cleavage and other intriguing biosynthetic reactions, such as the vinyl side chain formation, methylations, Cglycosylation and dehydrogenation. Although gene inactivation results identified many candidate genes whose corresponding enzymes are involved in these biochemical transformations, their exact functional roles and the identity of their natural substrates remained elusive. To provide more insights into these complex biochemical tranfrormations, three specific aims were set up. Specific aim 1 was to clone and characterize the RMV biosynthetic gene cluster. Through the comparison of GV cluster with the RMV cluster, the genes encoding the biosynthesis of sugar and tetracyclic aromatic moieties were identified. RavGT, the sole glycosyltransferase of the RMV cluster has demonstrated to have unprecedented sugar donor substrate flexibility, transferring an amino-pyranose sugar as well as a neutral furanose sugar. Specific aim 2 was to characterize all of the TDP-D-ravidosamine biosynthetic enzymes. The aim also included to a one-pot enzymatic synthetic protocol for the routine production of TDP-D-ravidosamine. Specific aim 3 focussed on a total enzymatic synthesis of defucogilvocarcin M (defucoGM), the polyketide-derived core of GV and RMV. This aim clearly identified the minimal enzymes required to biosynthesize the complex architecture of defucoGM from the simple building blocks acetate and malonate. In addition, the GV-pathway enzyme GilR was fully characterized. Through in vitro studies, GilR was shown to catalyze the dehydrogenation of hemiacetal moiety of the penultimate intermediate pregilvocarcin V to the lactone moiety of GV at the last step.

Gilvocarcin V (GV), the principal product of Streptomyces griseoflavus Gö 3592 and other Streptomyces spp., is the most prominent member of a distinct class of antitumor antibiotics that share a polyketide derived coumarin-based aromatic core. GV and other members of this class including polycarcin V from Streptomyces polyformus, often referred to as gilvocarcin-like aryl C-glycosides, are particularly interesting because of their potent bactericidal, virucidal and antitumor activities at low concentrations while maintaining low in vivo toxicity. Although the precise molecular mechanism of GV bioactivity is unknown, gilvocarcin V has been shown to undergo a photoactivated [2+2] cycloaddition of its vinyl side chain with thymine residues of DNA in near-UV or visible blue light. In addition, GV was shown to selectively crosslink histone H3 with DNA, thereby effectively disrupting normal cellular processes such as transcription. Furthermore, GVs ability to inhibit topoisomerase II has also been attributed as a mechanism of action for gilvocarcin V activity. The excellent antitumor activity, as well as an unprecedented structural architecture, has made GV an ideal candidate for biosynthetic studies toward the development of novel analogues with improved pharmacological properties. Previous biosynthetic research has identified several candidate genes responsible for key steps during the biosynthesis of gilvocarcin V including an oxygenase cascade leading to C-C bond cleavage, methylations, lactone formation, C-glycosylation and vinyl side chain formation. In this study, we further examined two critical biosynthetic transformations essential for the bioactivity of gilvocarcin V, namely starter unit incorporation and C-glycosylation, through the following specific aims: 1) creation of functional chimeric C-glycosyltransferases through domain swapping of gilvocarcin-like glycosyltransferases and identification and evaluation of the donor substrate flexibility of PlcGT, the polycarcin V pathway specific C-glycosyltransferase; 2) creation of a library of O-methylated-L-rhamnose analogues of polycarcin V for structure activity relationship studies; 3) identification of the role of GilP and GilQ in starter unit specificity during gilvocarcin V biosynthesis; and 4) creation of a plasmid based approach in which selective gilvocarcin biosynthetic genes were utilized to produce important gilvocarcin intermediates for further in vivo and in vitro experimentation.

Type II polyketide synthase (PKS) produced natural products have proven to be an excellent source of pharmacologically relevant molecules due to their rich biological activities and chemical scaffolds. Type II-PKS manufactured polyketides share similar polycyclic aromatic backbones leaving their diversity to stem from various chemical additions and alterations facilitated by post-PKS tailoring enzymes. Evidence suggests that post-PKS tailoring enzymes form complexes in order to facilitate the highly orchestrated process of biosynthesis. Thus, protein-protein interactions between these enzymes must play crucial roles in their structures and functions. Despite the importance of these interactions little has been done to study them. In the mithramycin (MTM) biosynthetic pathway the Baeyer−Villiger monooxygenase (BVMO) MtmOIV and the ketoreductase MtmW form one such enzyme pair that catalyze the final two steps en route to the final product. MtmOIV oxidatively cleaves the fourth ring of the mithramycin intermediate premithramycin B (PreB) via a Baeyer−Villiger reaction, generating MTM’s characteristic tricyclic aglycone core and highly functionalized pentyl side chain at position 3. This Baeyer−Villiger reaction precedes spontaneous lactone ring opening, decarboxylation, and the final step of MTM biosynthesis, a reduction of the 4′- keto group catalyzed by the ketoreductase MtmW. Another example of co-dependent post-PKS tailoring enzymes from the gilvocarcin biosynthetic pathway is composed of GilM and GilR. These two enzymes form an unusual synergistic tailoring enzyme pair that does not function sequentially. GilM exhibits dual functionality by catalyzing the reduction of a quinone intermediate to a hydroquinone and stabilizes O-methylation and hemiacetal formation. GilM mediates its reductive catalysis through the aid of GilR that provides its covalently bound FADH(2) for the GilM reaction, through which FAD is regenerated for the next catalytic cycle. A few steps later, following glycosylation related events unique to each gilvocarcin derivative, GilR dehydrogenates the hemiacetal moiety created by GilM to establish the formation of a lactone and the final gilvocarcin chromophore. To achieve a better understanding of post-type II PKS tailoring enzymes and their protein-proteininteractions for the benefit of future combinatorial biosynthetic efforts two specific aims were devised. Specific aim 1 was to investigate the structure of MtmOIV and the role of active site residues in its catalytic mechanism. Specific aim 2 was to integrate the function of GilM and its protein-protein interactionswith GilR that lead to their synergistic activity and sharing of GilR’s bicovalently bound FAD moiety.

Natural products provide some of the most potent anticancer agents and offer a template for new drug design or improvement with the advantage of an enormous chemical space. The overall goal of this thesis research is to enhance the chemical space of two natural products in order to generate novel drugs with better in vivo bioactivities than the original natural products. Polycarcin V (PV) is a gilvocarcin-type antitumor agent with similar structure and comparable bioactivity with the principle compound of this group, gilvocarcin V (GV). Modest modifications of the polyketide-derived tetracyclic core of GV had been accomplished, but the most challenging part was to modify the sugar moiety. In order to solve this problem, PV was used as an alternative lead-structure for modification because its sugar moiety offered the possibility of enzymatic O-methylation. We produced four PV derivatives with different methylation patterns for cytotoxicity assays and provided important structure-activity-relationship information. Mithramycin (MTM) is the most prominent member of the aureolic acid type anticancer agents. Previous work in our laboratory generated three MTM analogues, MTM SA, MTM SK, and MTM SDK by inactivating the mtmW gene. We developed new MTM analogues by coupling many natural and unnatural amino acids to the C-3 side chain of MTM SA via chemical semi-synthesis and successfully made some compounds with both improved bioactivity and in vivo tolerance than MTM. Some of them were consequently identified as promising lead-structures against Ewing’s sarcoma. The potential of selectively generating novel MTM analogues led us to focus on a key enzyme in the biosynthetic pathway of mithramycin, MtmC. This protein is a bifunctional enzyme involved in the biosynthesis of TDP-D-olivose and TDP-D-mycarose. We clarified its enzymatic mechanisms by X-ray diffraction of several crystal complexes of MtmC with its biologically relevant ligands. Two more important post-PKS tailoring enzymes involved in the biosynthesis of the MTM side chains, MtmW and MtmGIV, are currently under investigation. This would not only give us insight into this biosynthetic pathway but also pave the way to develop potentially useful MTM analogues by engineered enzymes.