Madan K. Kharel

Baeyer-Villiger C-C bond cleavage reaction in gilvocarcin and jadomycin biosynthesis.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical of the gilvocarcin-type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxyoxepinone intermediate, leading to important conclusions regarding the cleavage mechanism.

Enzymatic Total Synthesis of Defucogilvocarcin M and Its Implications for Gilvocarcin Biosynthesis

Gilvocarcin V (GV, 4) is the major metabolite of Streptomyces griseoflavus Go 3592 and various other Streptomyces species. GV is usually produced along with its minor congeners, gilvocarcin M (3) and gilvocarcin E (5), that vary with respect to their side chain at the C8-position.[1] Several analogues of GV (for example, 6–8, Scheme 1), which are collectively called the gilvocarcin group of natural products, have been isolated from different Streptomyces species. All of these analogues contain the characteristic, polyketide-derived benzo[d]naphtho[1,2-b]pyran-6-one chromophore, but different C-glycosidically linked sugar units Scheme 1).[2] Members of this group of natural products are well-known for strong antitumor activities,[3] a unique mode of action,[4] and remarkably low toxicities.[2c, 5] However, the inherent poor solubility of these molecules appears to be a major obstacle in their development as therapeutics. The chemical syntheses that have been developed so far are unsuitable for generating a library of analogues,[6] whereas combinatorial biosynthetic efforts have shown more promise.[7] The continued advancement and successful implementation of such combinatorial biosynthetic and mutasynthetic approaches requires an in-depth knowledge of the biosynthetic pathway. Incorporation studies with isotope-labeled precursors[8] and genetic experiments[1a, 8d, 9] have revealed that the benzo[d]naphtho[1,2-b]pyran-6-one chromophore of the gilvocarcins is produced from a polyketide-derived angucyclinone intermediate through a complex oxidative rearrangement process. However, the details of the exact sequence of biosynthetic events and the enzymes that are involved have remained elusive. In this context, we herein report a complete, one-pot, enzymatic total synthesis of defucogilvocarcin M(1), a model compound that contains the unique chromophore common to all members of the gilvocarcin group of natural products.[10] The reconstitution of this pathway then enabled further investigation into the details of the oxidative rearrangement process of GV biosynthesis by systematic variation of the enzyme mixture used. For this approach we suggest the term “combinatorial biosynthetic enzymology”.

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D‐ravidosamine and D‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D‐ravidosamine and 5‐membered D‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.

Moromycins A and B, isolation and structure elucidation of C-glycosylangucycline-type antibiotics from Streptomyces sp. KY002.

Two new anticancer antibiotics of the angucycline class, moromycins A and B (1, 2), along with the known microbial metabolites saquayamycin B (3) and fridamycin D (4) were isolated from the ethyl acetate extract of a culture broth of the terrestrial Streptomyces sp. KY002. The structures consist of a tetrangomycin core and various C- and O-glycosidically linked deoxysugars. The chemical structures of the new secondary metabolites were elucidated by 1D and 2D NMR and by mass spectrometry. Moromycin B (2) showed significant cytotoxicity against H-460 human lung cancer and MCF-7 human breast cancer cells.

Frenolicins C–G, Pyranonaphthoquinones from Streptomyces sp. RM-4-15

Appalachian active coal fire sites were selected for the isolation of bacterial strains belonging to the class actinobacteria. A comparison of high-resolution electrospray ionization mass spectrometry (HRESIMS) and ultraviolet (UV) absorption profiles from isolate extracts to natural product databases suggested Streptomyces sp. RM-4-15 to produce unique metabolites. Four new pyranonaphthoquinones, frenolicins C-F (1-4), along with three known analogues, frenolicin (6), frenolicin B (7), and UCF76-A (8), were isolated from the fermentation of this strain. An additional new analogue, frenolicin G (5), along with two known compounds, deoxyfrenolicin (9) and UCF 13 (10), were isolated from the fermentation supplied with 18 mg/L of scandium chloride, the first example, to the best of our knowledge, wherein scandium chloride supplementation led to the confirmed production of new bacterial secondary metabolites. Structures 1-5 were elucidated on the basis of spectral analysis and chemical modification. While frenolicins are best known for their anticoccidial activity, the current study revealed compounds 6-9 to exhibit moderate cytotoxicity against the human lung carcinoma cell line (A549) and thereby extends the anticancer SAR for this privileged scaffold.

Inactivation of the Ketoreductase gilU Gene of the Gilvocarcin Biosynthetic Gene Cluster Yields New Analogues with Partly Improved Biological Activity

Four new analogues of the gilvocarcin‐type aryl‐C‐glycoside antitumor compounds, namely 4′‐hydroxy gilvocarcin V (4′‐OH‐GV), 4′‐hydroxy gilvocarcin M, 4′‐hydroxy gilvocarcin E and 12‐demethyl‐defucogilvocarcin V, were produced through inactivation of the gilU gene. The 4′‐OH‐analogues showed improved activity against lung cancer cell lines as compared to their parent compounds without 4′‐OH group (gilvocarcins V and E). The structures of the sugar‐containing new mutant products indicate that the enzyme GilU acts as an unusual ketoreductase involved in the biosynthesis of the C‐glycosidically linked deoxysugar moiety of the gilvocarcins. The structures of the new gilvocarcins indicate substrate flexibility of the post‐polyketide synthase modifying enzymes, particularly the C‐glycosyltransferase and the enzyme responsible for the sugar ring contraction. The results also shed light into biosynthetic sequence of events in the late steps of biosynthetic pathway of gilvocarcin V.

Inactivation of gilGT, Encoding a C-Glycosyltransferase, and gilOIII, Encoding a P450 Enzyme, Allows the Details of the Late Biosynthetic Pathway to Gilvocarcin V to be Delineated

Resequencing of the gilGT gene, which encodes a putative glycosyltransferase (GT) that is 495 amino acids (aa) long, from the Streptomyces griseoflavus Gö3592 gilvocarcin V (GV) gene cluster, revealed that the previously reported gilGT indeed contains two genes. These are the larger gilGT, which encodes the C‐glycosyltransferase GilGT (379 aa), and the smaller gilV gene, which encodes an enzyme of unknown function (116 aa). The gene gilV is located immediately upstream of gilGT in the GV gene cluster. In‐frame deletion of gilGT created a mutant that accumulated defucogilvocarcin E (defuco‐GE). The result proves the function of GilGT as a C‐glycosyltransferase. Deletion of gilOIII, which is located immediately downstream of gilGT, led to a mutant that accumulated gilvocarcin E (GE). This confirms that the corresponding P450 enzyme, GilOIII, is involved in the vinyl‐group formation of GV. Cross‐feeding experiments in which GE, defuco‐GE, and defucogilvocarcin V (defuco‐GV) were fed to an early blocked mutant of the GV biosynthetic pathway, showed that neither GE nor any of the defuco‐ compounds was an intermediate of the pathway.

GilR, an Unusual Lactone-Forming Enzyme Involved in Gilvocarcin Biosynthesis

Last at last: The terminal step of the gilvocarcin V (GV) biosynthetic pathway is an unusual lactone formation. Here we show that the enzyme, GilR, dehydrogenates the hemiacetal moiety of pregilvocarcin V to the lactone found in GV by using covalently bound FAD.

Enzymatic Total Synthesis of Rabelomycin, an Angucycline Group Antibiotic

A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, starting from acetyl-CoA and malonyl-CoA, using a mixture of polyketide synthase (PKS) enzymes of the gilvocarcin, ravidomycin, and jadomycin biosynthetic pathways. The in vitro results were compared to in vivo catalysis using analogous sets of enzymes.

The Crystal Structure and Mechanism of an Unusual Oxidoreductase, GilR, Involved in Gilvocarcin V Biosynthesis

GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.