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Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoids of ent-kauranol and ent-atiserene biosynthetic origin. C7 oxidation in the B-ring plays a key biosynthetic role in generating structural complexity known for ent-kaurane and ent-atisane derived diterpenoids. While all three oxidation patterns, α-hydroxyl, β-hydroxyl, and ketone, at C7 are seen in both the ent-kaurane and ent-atisane derived diterpenoids, their biosynthetic origins remain largely unknown. We previously established that PTM and PTN are produced by a single biosynthetic machinery, featuring cryptic C7 oxidations at the B-rings that transform the ent-kauranol and ent-atiserene derived precursors into the characteristic PTM and PTN scaffolds. Here, we report a three-enzyme cascade affording C7 α-hydroxylation in PTM and PTN biosynthesis. Combining in vitro and in vivo studies, we show that PtmO3 and PtmO6 are two functionally redundant α-ketoglutarate-dependent dioxygenases that generate a cryptic C7 β-hydroxyl on each of the ent-kauranol and ent-atiserene scaffolds and PtmO8 and PtmO1, a pair of NAD(+)/NADPH-dependent dehydrogenases, subsequently work in concert to invert the C7 β-hydroxyl to α-hydroxyl via a C7 ketone intermediate. PtmO3 and PtmO6 represent the first dedicated C7 β-hydroxylases characterized to date, and, together with PtmO8 and PtmO1, provide an account for the biosynthetic origins of all three C7 oxidation patterns that may shed light on other B-ring modifications in bacterial, plant, and fungal diterpenoid biosynthesis. Given their unprecedented activities in C7 oxidations, PtmO3, PtmO6, PtmO8, and PtmO1 enrich the growing toolbox of novel enzymes that could be exploited as biocatalysts to rapidly access complex diterpenoid natural products.

Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoid natural products that target bacterial and mammalian fatty acid synthases. PTM and PTN feature varying diterpene-derived ketolides that are linked to the same 3-amino-2,4-dihydroxybenzoic acid moiety. As a result, PTM is a selective inhibitor for FabF/FabB, while PTN is a dual inhibitor of FabF/FabB and FabH. We previously determined that the PTM cassette, consisting of five genes found in the ptm, but not ptn, gene cluster, partitions the biosynthesis of the PTM and PTN diterpene-derived ketolides. We now report investigation of the PTM cassette through the construction of diterpene production systems in E. coli and genetic manipulation in the PTM-PTN dual overproducer Streptomyces platensis SB12029, revealing two genes, ptmT3 and ptmO5, that are responsible for the biosynthetic divergence between the PTM and PTN diterpene-derived ketolides. PtmT3, a type I diterpene synthase, was determined to be a (16R)-ent-kauran-16-ol synthase, the first of its kind found in bacteria. PtmO5, a cytochrome P450 monooxygenase, is proposed to catalyze the formation of the characteristic 11S,16S-ether ring found in PTM. Inactivation of ptmO5 in SB12029 afforded the ΔptmO5 mutant SB12036 that accumulated nine PTM and PTN congeners, seven of which were new, including seven 11-deoxy-16R-hydroxy-PTM congeners. The two fully processed PTM analogues showed antibacterial activities, albeit lower than that of PTM, indicating that the ether ring, or minimally the stereochemistry of the hydroxyl group at C-16, is crucial for the activity of PTM.

Nonheme diiron monooxygenases make up a rapidly growing family of oxygenases that are rarely identified in secondary metabolism. Herein, we report the in vivo, in vitro, and structural characterizations of a nonheme diiron monooxygenase, PtmU3, that installs a C-5 β-hydroxyl group in the unified biosynthesis of platensimycin and platencin, two highly functionalized diterpenoids that act as potent and selective inhibitors of bacterial and mammalian fatty acid synthases. This hydroxylation sets the stage for the subsequent A-ring cleavage step key to the unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 adopts an unprecedented triosephosphate isomerase (TIM)-barrel structural fold for this class of enzymes and possesses a noncanonical diiron active site architecture with a saturated six-coordinate iron center lacking a μ-oxo bridge. This study reveals the first member of a previously unidentified superfamily of TIM-barrel fold enzymes for metal-dependent dioxygen activation, with the majority predicted to act on CoA-linked substrates, thus expanding our knowledge of nature’s repertoire of nonheme diiron monooxygenases and TIM-barrel fold enzymes.

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases. The regio- and stereospecificity of the ether oxygen atom in PTM, which PTN does not have, strongly contribute to the selectivity and potency of PTM. We previously reported the biosynthetic origin of the 11 S,16 S-ether moiety by characterizing the diterpene synthase PtmT3 as a (16 R)- ent-kauran-16-ol synthase and isolating 11-deoxy-16 R-hydroxylated congeners of PTM from the Δ ptmO5 mutant. PtmO5, a cytochrome P450, was proposed to catalyze formation of the ether moiety in PTM. Here we report the in vitro characterization of PtmO5, revealing that PtmO5 stereoselectively hydroxylates the C-11 position of the ent-kaurane scaffold resulting in an 11 S,16 R-diol intermediate. The ether moiety, the oxygen of which originates from the P450-catalyzed hydroxylation at C-11, is formed via cyclization of the diol intermediate. This study provides mechanistic insight into ether formation in natural product biosynthetic pathways.

Platensimycin (1) is the flagship member of a new and growing class of antibiotics with promising antibacterial properties against drug resistant bacteria. The total syntheses of platensimycin and its congeners, platensimycins B1 (7) and B3 (9), platensic acid (2), methyl platensinoate (3), platensimide A (4), homoplatensimide A (5), and homoplatensimide A methyl ester (6) (Figure 1) are described. The convergent strategy developed toward these target molecules involved construction of their cage-like core followed by attachment of the various side chains through amide bond formation. In addition to a racemic synthesis, two asymmetric routes to the core structure are described: one exploiting a rhodium-catalyzed asymmetric cycloisomerization, and another employing a hypervalent iodine-mediated dearomatizing cyclization of an enantiopure substrate. The final two bonds of the core structure were forged through a samarium diiodide-mediated ketyl radical cyclization and an acid-catalyzed etherification. The rhodium-catalyzed asymmetric reaction involving a terminal acetylene was developed as a general method for the asymmetric cycloisomerization of terminal enynes.

Peptide dendrimers incorporating 3,5-diaminobenzoic acid 1 as a branching unit (B) were prepared by solid-phase synthesis of ((Ac-A(3))(2)B-A(2))(2)B-Cys-A(1)-NH(2) followed by disulfide bridge formation. Twenty-one homo- and heterodimeric dendrimers were obtained by permutations of aspartate, histidine, and serine at positions A(1), A(2), and A(3). Two dendrimers catalyzed the hydrolysis of 7-hydroxy-N-methyl-quinolinium esters (2-5), and two other dendrimers catalyzed the hydrolysis of 8-hydroxy-pyrene-1,3,6-trisulfonate esters (10-12). Enzyme-like kinetics was observed in aqueous buffer pH 6.0 with multiple turnover, substrate binding (K(M) = 0.1-0.5 mM), rate acceleration (k(cat)/k(uncat)10(3)), and chiral discrimination (E = 2.8 for 2-phenylpropionate ester 5). The role of individual amino acids in catalysis was investigated by amino acid exchanges, highlighting the key role of histidine as a catalytic residue, and the importance of electrostatic and hydrophobic interactions in modulating substrate binding. These experiments demonstrate for the first time selective catalysis in peptide dendrimers.

A systematic study of C-H insertion reactions with variously substituted and conformationally constrained substrates was carried out. High selectivity in the insertion between two competing C-H bonds caused by a strong stereoelectronic effect of an oxygen substituent was achieved. This regioselective C-H insertion reaction was employed as a platform to develop a concise asymmetric synthesis of platensimycin.

Chorismate is an important and central metabolite branching off to the biosyntheses of aromatic amino acids and p-aminobenzoic acid (pABA), a component of the vitamin folic acid. Here we report on a novel variation of a unified catalytic mechanism in Bacillus subtilis pABA biosynthesis that includes the formation of a new intermediate, 2-amino-2-deoxyisochorismate (ADIC), thus significantly differing from the mechanism in Escherichia coli. In B. subtilis, chorismate is converted to ADIC, which is catalyzed by aminodeoxychorismate synthase (ADCS). In a second step, ADIC is converted to aminodeoxychorismate (ADC) by addition of ammonia to C4, also catalyzed by ADCS. The third step is the aminodeoxychorismate lyase-catalyzed elimination of pyruvate from ADC. To our knowledge, B. subtilis aminodeoxychorismate synthase is the first enzyme exhibiting ADIC synthase activity in primary metabolism. We further provide evidence that pABA biosynthesis via ADIC might be a common mechanism for several other microorganisms.

Fatty acids are essential for survival of bacteria and are synthesized by a series of enzymes including the elongation enzymes, beta-ketoacyl acyl carrier protein synthase I/II (FabF/B). Inhibition of fatty acid synthesis is one of the new targets for the discovery and development of antibacterial agents. Platensimycin (1a) is a novel broad spectrum Gram-positive antibiotic produced by Streptomyces platensis. It was discovered by target-based whole-cell screening strategy using antisense differential sensitivity assay. It inhibits bacterial growth by selectively inhibiting condensing enzyme FabF of the fatty acid synthesis pathway and was isolated by a two-step process, a capture step followed by reversed-phase HPLC. The structure was elucidated by 2D NMR methods and confirmed by X-ray crystallographic analysis of a bromo derivative. It was determined that potential reactivity of the enone moiety does not play a key role in the biological activity of platensimycin. However, cyclohexenone ring conformation renders for the stronger binding interaction with the enzyme. The isolation, structure elucidation, derivatization, and biological activity of 6,7-dihydroplatensimycin are described.

The biosynthesis of 3-amino-5-hydroxybenzoic acid (AHBA), precursor of the ansamycin and mitomycin antibiotics, proceeds by the aminoshikimate pathway from 3,4-dideoxy-4-amino-d-arabino-heptulosonic acid 7-phosphate (aminoDAHP). Identification of RifN, product of one of three genes from the rifamycin biosynthetic gene cluster known to be essential for aminoDAHP formation, as a specific kanosamine (3-deoxy-3-amino-d-glucose) 6-kinase establishes kanosamine and its 6-phosphate as specific intermediates in AHBA formation. This suggests a hypothetical reaction sequence for aminoDAHP formation, and thus for the early steps of AHBA biosynthesis, starting from UDP-d-glucose and introducing the nitrogen by oxidation and transamination at C-3.

Platensimycin is a novel Gram-positive broad spectrum antibiotic that inhibits bacterial growth by specifically and potently inhibiting the condensing enzyme (FabF) of the fatty acid biosynthesis pathway. It is produced by several strains of Streptomyces platensis and comprises a 3-amino-2,4-dihydroxybenzoic acid and C-17 tetracyclic enone moieties linked by an amide bond. Feeding experiments using 13C precursors suggest that the aminobenzoic acid unit is derived from the TCA cycle, and the tetracyclic enone moiety of platensimycin in S. platensis is derived from the non-mevalonate MEP terpenoid pathway by oxidative excision of three carbons from ent-kaurene.

The chemical synthesis and biological evaluation of carbaplatensimycin, the carbon analogue of the recently reported antibiotic platensimycin has been accomplished. Carbaplatensimycin exhibited similar potency to the natural product as an antibacterial agent against a variety of strains, including methicilin- and vancomycin-resistant bacteria.

A mesoporous metal-organic framework possessing permanent porosity has been synthesized and characterized for the first time.

Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.