Biosynthesis of the macrolide antibiotic chlorothricin: basic building blocks

Holzbach, Rainer; Pape, H.; Hook, Derek J.; Kreutzer, Ernest F.; Chang, Ching‐Jer; Floss, Heinz G.

doi:10.1021/bi00596a029

Cited by 24 publications

(23 citation statements)

References 19 publications

(23 reference statements)

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“…A similar labeling pattern was found in the biosynthetic study of CHL (10,22), and the three remaining carbons at corresponding positions were confirmed to be derived from the glycerol (17), suggesting that a common strategy involves the incorporation of a glycerol-derived 3-C unit with a polyketide chain to form the aglycones in spirotetronate biosynthesis. In 2006, we reported the cloning and characterization of the chl gene cluster as the first genetic model to propose the biosynthetic pathway in this family (11).…”

supporting

confidence: 75%

Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics

et al. 2008

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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.

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supporting

confidence: 75%

Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics

et al. 2008

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“…Earlier biosynthetic studiese 6,7) have established the biosynthetic origin for most of the carbon frameworks of these components, as summarized in Scheme 1. The modified 6-methylsalicylic acid comes from 4 acetate-malonate units via the polyketide pathway, with contribution of the additional 0-methyl group by methionine, and the two 2,6-dideoxyhexose moieties are derived directly from glucose.…”

Section: Chlorothricin (I) Is An Unusual Macrolide Antibiotic Producementioning

confidence: 99%

Further studies on the biosynthesis of chlorothricin.

Lee

Keller

et al. 1986

J. Antibiot.

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Feeding experiments with [U-13C3]-and (2R)-[1-2H2]glycerolshowed that glycerol is incorporated intact into carbon atoms 22, 23 and 24 of the aglycone of chlorothricin. C-1 of glycerol gives rise to C-22 with retention of one atom of deuterium, which occupies the H-22R position. A mechanism for the assembly of the aglycone is proposed which invokes phosphoenolpyruvate as the direct precursor of the 3-carbon moiety and a Baeyer-Villiger oxidation as the mode of formation of the macrocyclic lactone functionality. A feeding experiment with [1,2-13C2]succinate suggests that the propionate units of the aglycone polyketide are formed entirely via the methylmalonyl-CoA mutase reaction. The formation of the two 2,6-dideoxy-D-rhamnose moieties of chlorothricin from glucose was shown to involve replacement of the 2-hydroxyl group of the sugar by hydrogen with inversion of configuration at C-2. This contrasts with the retention stereochemistry observed earlier for the analogous formation of the 2,6-dideoxyhexose moiety of the antibiotic granaticin.Chlorothricin (I) is an unusual macrolide antibiotic produced by Streptomyces antibioticus1-5) Its structure consists of three types of components, a modified 6-methylsalicylic acid, two identical 2,6-dideoxyhexose moieties and the aglycone, chlorothricolide (II). Earlier biosynthetic studiese 6,7) have established the biosynthetic origin for most of the carbon frameworks of these components, as summarized in Scheme 1. The modified 6-methylsalicylic acid comes from 4 acetate-malonate units via the polyketide pathway, with contribution of the additional 0-methyl group by methionine, and the two 2,6-dideoxyhexose moieties are derived directly from glucose. The aglycone is predominantly of polyketide origin, being made up of ten acetate and two propionate units which account for all but three carbon atoms of chlorothricolide. While carbon atoms 25 and 26 of the tetronic acid moiety are derived from an intact acetate unit7), C-24, C-23 and the adjacent C-22 are not labeled by either acetate or propionate. The origin of these three carbon atoms, and with it the overall mode of formation of chlorothricolide, is the subject of this paper. In addition, we report on the metabolic route by which the propionate units are generated from primary metabolites, and on a stereochemical aspect of the conversion of glucose into the 2,6-dideoxyhexose moieties. Results Precursor of the Three Missing Carbon AtomsIn our previous publication7) we had hypothesized that carbon atoms 22, 23 and 24 of chlorothricolide originate from a molecule of oxalacetic acid which undergoes Q-decarboxylation. However, a feeding experiment with [1,4-13C2]succinic acid, expected to be a direct precursor of oxalacetic acid,

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“…It was reported that the aglycone of chlorothricin is synthesized via the polyketide route and one of its hypothetical intermediates has the same basic skeletal structure as that of tetrocarcin A. 6 ) Although acetate and propionate could not stimulate the production of tetrocarcin A, it is very likely that tetrocarcin A is synthesized via the polyketide route and experiments to substantiate these hypotheses are in progress using labeled acetate and propionate by 14C and 13e. It is very interesting that L-Ieucine and its corresponding keto acid (a-ketoisocaproic acid) stimulated the production of tetrocarcin A, but retarded the growth of the organism.…”

Section: Discussionmentioning

confidence: 99%

“…It has been shown that chlorothricin is synthesized via the polyketide route from acetic and propionic acid. 6 ) Therefore the effects of these organic acids and others on tetrocarcin A production were studied. As shown in Table VI, acetic acid, propionic acid and other organic acid did not promote tetrocarcin A production but were slightly effective for growth of the organism.…”

Section: Effects Of Organic Acidsmentioning

confidence: 99%