Enzyme-Catalysed Spiroacetal Formation in Polyketide Antibiotic Biosynthesis (original) (raw)
Related papers
Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation
Nature Chemical Biology, 2011
Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.
Microbiology, 2007
Spiramycin, a 16-membered macrolide antibiotic used in human medicine, is produced by Streptomyces ambofaciens; it comprises a polyketide lactone, platenolide, to which three deoxyhexose sugars are attached. In order to characterize the gene cluster governing the biosynthesis of spiramycin, several overlapping cosmids were isolated from an S. ambofaciens gene library, by hybridization with various probes (spiramycin resistance or biosynthetic genes, tylosin biosynthetic genes), and the sequences of their inserts were determined. Sequence analysis showed that the spiramycin biosynthetic gene cluster spanned a region of over 85 kb of contiguous DNA. In addition to the five previously described genes that encode the type I polyketide synthase involved in platenolide biosynthesis, 45 other genes have been identified. It was possible to propose a function for most of the inferred proteins in spiramycin biosynthesis, in its regulation, in resistance to the produced antibiotic or in the provision of extender units for the polyketide synthase. Two of these genes, predicted to be involved in deoxysugar biosynthesis, were inactivated by gene replacement, and the resulting mutants were unable to produce spiramycin, thus confirming their involvement in spiramycin biosynthesis. This work reveals the main features of spiramycin biosynthesis and constitutes a first step towards a detailed molecular analysis of the production of this medically important antibiotic.
Mechanistic insights into the biosynthesis of polyketide antibiotics
2006
Anthracyclines are a group of aromatic polyketide compounds with significant medical importance due to their antineoplastic properties. Doxorubicin and daunorubicin, members of this family are among the two most commonly used anticancer drugs. These compounds exhibit severe side effects like cardiotoxicity and multi-drug resistance. A promising approach towards the production of modified anthracyclines with improved toxicity profiles appears to be combinatorial biosynthesis, including the redesign of biosynthetic enzymes; however, structural and mechanistic information of the biosynthetic enzymes is necessary for the redesigning approach. 2.2.3 The overall fold of AknOx 2.2.4 AknOx belongs to PCMH superfamily 2.2.4.1 The conserved F-domain and FAD binding features in PCMH superfamily 2.2.4.2 Diversity in the structure of the substrate binding domain is observed in PCMH superfamily 2.2.5 FAD binding site in AknOx 2.2.6 Covalent flavinylation in AknOx and other flavoenzymes 2.2.6.1 Categories of covalent flavinylation observed in different flavoenzymes 2.2.6.2 The flavinylation observed in AknOx and PCMH superfamily 2.2.7 Ligand binding features and active site 2.2.8 Catalytic mechanism of AknOx 2.2.9 Comparison of catalytic properties in PCMH superfamily 3 Conclusions 3.1 SnoaL and AknH 3.2 AknOx References Acknowledgements
Post-PKS Tailoring Steps of the Spiramycin Macrolactone Ring in Streptomyces ambofaciens
Antimicrobial Agents and Chemotherapy, 2013
Spiramycins are clinically important 16-member macrolide antibiotics produced by Streptomyces ambofaciens. Biosynthetic studies have established that the earliest lactonic intermediate in spiramycin biosynthesis, the macrolactone platenolide I, is synthesized by a type I modular polyketide synthase (PKS). Platenolide I then undergoes a series of post-PKS tailoring reactions yielding the final products, spiramycins I, II, and III. We recently characterized the post-PKS glycosylation steps of spiramycin biosynthesis in S. ambofaciens. We showed that three glycosyltransferases, Srm5, Srm29, and Srm38, catalyze the successive attachment of the three carbohydrates mycaminose, forosamine, and mycarose, respectively, with the help of two auxiliary proteins, Srm6 and Srm28. However, the enzymes responsible for the other tailoring steps, namely, the C-19 methyl group oxidation, the C-9 keto group reduction, and the C-3 hydroxyl group acylation, as well as the timing of the post-PKS tailoring reactions, remained to be established. In this study, we show that Srm13, a cytochrome P450, catalyzes the oxidation of the C-19 methyl group into a formyl group and that Srm26 catalyzes the reduction of the C-9 keto group, and we propose a timeline for spiramycin-biosynthetic post-PKS tailoring reactions.
A two-step sulfation in antibiotic biosynthesis requires a type III polyketide synthase
Nature Chemical Biology, 2013
MraY translocase, an essential enzyme involved in peptidoglycan biosynthesis. We have recently identified analogs that are decorated with a sulfate group at the 2-hydroxy of the aminoribosyl moiety, and we now report an unprecedented two-step sulfation mechanism during the biosynthesis of CPZs. A type III polyketide synthase (PKS) known as Cpz6 is employed in the biosynthesis of a group of new triketide pyrones that are subsequently sulfated by an unusual 3-phosphoadenosine-5-phosphosulfate (PAPS)-dependent sulfotransferase (Cpz8) to yield phenolic sulfate esters, which serve as sulfate donors for a PAPS-independent arylsulfate sulfotransferase (Cpz4) to generate sulfated CPZs. This finding is to our knowledge the first demonstration of genuine sulfate donors for an arylsulfate sulfotransferase and the first report of a type III PKS to generate a chemical reagent in bacterial sulfate metabolism.
Journal of the American Chemical Society, 2017
Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV), which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, s...
The mechanisms of two diterpene cyclases from streptomycetes—one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolenea nd one with the knownp roduct tsukubadiene—were investigated in detail by isotope labeling experiments.A lthough the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions,b efore they diverge towardst he individual products,w hichi sr eflected in the close phylogenetic relationship of the enzymes.
Journal of Bacteriology, 2008
Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of D-tetronitrose, L-amicetose, and L-digitoxose may begin with D-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ⌬chlD3 and ⌬chlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.
ChemBioChem, 2018
Armeniaspirols are potent antibiotics containing an unusual spiro[4.4]non-8-ene moiety. We describe the cloning and functional analysis of the armeniaspirol biosynthetic gene cluster. Gene inactivation studies and subsequent isolation of previously unknown biosynthetic intermediates shed light on intriguing biosynthetic details. Remarkably, deletion of ams15, encoding a protein bearing a flavin-binding domain, led to the accumulation of several non-spiro intermediates with varying numbers of chlorine substitutions on the pyrrole moiety. The trichloropyrrole and dichloropyrrole species were converted by Streptomyces albus expressing Ams15 into dichlorinated and monochlorinated spiro derivatives, respectively. In addition, in vitro conversion of these non-spiro intermediates into des-N-methyl spiro intermediates by the cell lysate of the same recombinant strain proved Ams15 to be responsible for spiro formation through oxidative dehalogenation.
A highly convergent total synthesis of the spiroacetal macrolide (+)-milbemycinβ1
Tetrahedron, 1989
A highly convergent synthesis of the macrolide natural product milbemycin PI is reported. The key features of this synthesis include the introduction of the Cl l-Cl5 side chain by selective ring opening of a symmetrical 1,4-pentane bis-epoxide (3) followed by reaction with the anion derived from the 2,3-rruns-dimethyl-6phenylsulphonyl pyran (2) to afford the "northern" Cl l-C25 fragment (33) of milbemycin PI. Coupling of the derived Cl l-C25 aldehyde unit (37) with a Cl-Cl0 southern xone fragment (5) was achieved via a novel deconjugative vinyl sulphone anion sequence to give a product containing all the carbon substituents of the natural product. Final manipulations involved macrolactonisation and subsequent introduction of the important 3.4 double bond by selenoxide syn-elimination. Methylation of the C-5 hydroxyl group was accomplished as the penultimate step with methyl iodide and silver (I) oxide under ultrasonication. As targets for organic synthesis the milbemycinsl and avermectins2 have attracted special attention owing to their exceedingly potent antiparasitic and insecticidal activity. 3 The presence of the architecturally interesting spiroacetal in these compounds has led to the development of many new methods for the preparation of this unit.4 Several fragment syntheses, coupling strategies and analogue syntheses have also been reported.5,6 Our continued interest in these molecules7 has led us into much new chemistry,8 some of which is exploited below in the total synthesis of milbemycin PI. 9 Our route to (1) employs a highly convergent approach, bringing together key structural elements as illustrated in Scheme 1. The choice of the various coupling fragments was governed by several important factors. The synthesis of tetrahydropyranyl phenylsulphone (2) utilizes methodology developed by our group for the formation of carboncarbon bonds at the 2-position of cyclic ethers, to allowing subsequent elaboration to sphoacctals via intermediate enol ethers.11 The symmetrical bis-epoxide (3) is an ideal coupling fragment in that it is doubly activated towards ring opening, facilitating the introduction of the relevant side chains. Furthermore, its stereogenic centres are common to all milbemycins and avermectins. Use of the Cll-Cl5 iodide (4), via its cuprate, ensures the correct E-geometry for the C-14,15 double bond, a cause of problems in other milbemycin syntheses. Finally, the Cl-s. v. LEY et al. Cl0 "southern" unit (5) was designed to effect a deconjugative anion coupling via a Julia type reaction to establish the required E,E-1,3-diene portion of milbemycin pl. The choice of (5) as a 3,Cdihydro derivative was deliberate; others have noted problems.l2 such as conjugation or epimerisation at C-2, if the 3Pdouble bond is present at an early stage of the synthesis. This strategy for milbemycin PI synthesis is very versatile and in principle could be applied to many members of the milbemycin or avermecdn classes.