Dataset Information

Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor. Mechanism and stereochemistry of the enzymatic formation of epi-isozizaene.

ABSTRACT: Epi-isozizaene synthase from Streptomyces coelicolor catalyzes the multistep cyclization of farnesyl diphosphate (2, FPP) to the tricyclic sesquiterpene hydrocarbon (+)-epi-isozizaene (3), which is converted in turn to the antibiotic albaflavenone (1) in a two-step, cytochrome P450-catalyzed oxidation. Competitive incubation of deuterated and nondeuterated samples of (3S)-NPP and (3RS)-NPP followed by GC-MS analysis of the degree of deuteration in the resulting labeled epi-isozizaene established that (3R)-NPP is the natural cyclization intermediate. Incubation of (3RS)-(Z)-[1-(2)H]NPP (4b) with epi-isozizaene synthase gave [11(anti)-(2)H]epi-isozizaene (3b), indicating that the S(N)' cyclization of 4 involves the predicted anti stereochemistry, consistent with the inference from earlier experiments with chirally deuterated FPP. Incubation of separate samples of [12,12,12-(2)H(3)]FPP (2d) and [13,13,13-(2)H(3)]FPP (2e) gave epi-isozizaenes 3d and 3e, thereby establishing the stereochemical course of the cyclization of the proposed intermediate acorenyl cation 6, as well as the stereochemistry of the successive 1,2-methyl migration and deprotonation that generate the final product. Further insights into the mechanism and the role of the enzyme came from site-directed mutagenesis of active site residues in two universally conserved Mg(2+)-binding domains and the identification of six minor sesquiterpene products 9-13 and 15 produced by the wild-type and mutant proteins. The aberrant products are believed to result from derailment and premature quenching of the normal intermediates of the cationic cyclization cascade.

SUBMITTER: Lin X

PROVIDER: S-EPMC2690639 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor. Mechanism and stereochemistry of the enzymatic formation of epi-isozizaene.

Lin Xin X Cane David E DE

Journal of the American Chemical Society 20090501 18

Epi-isozizaene synthase from Streptomyces coelicolor catalyzes the multistep cyclization of farnesyl diphosphate (2, FPP) to the tricyclic sesquiterpene hydrocarbon (+)-epi-isozizaene (3), which is converted in turn to the antibiotic albaflavenone (1) in a two-step, cytochrome P450-catalyzed oxidation. Competitive incubation of deuterated and nondeuterated samples of (3S)-NPP and (3RS)-NPP followed by GC-MS analysis of the degree of deuteration in the resulting labeled epi-isozizaene established ...[more]

PMID: 19385616

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Project description:Many microorganisms, including bacteria of the class Streptomycetes, produce various secondary metabolites including antibiotics to gain a competitive advantage in their natural habitat. The production of these compounds is highly coordinated in a population to expedite accumulation to an effective concentration. Furthermore, as antibiotics are often toxic even to their producers, a coordinated production allows microbes to first arm themselves with a defense mechanism to resist their own antibiotics before production commences. One possible mechanism of coordination among individuals is through the production of signaling molecules. The gamma-butyrolactone system in Streptomyces coelicolor is a model of such a signaling system for secondary metabolite production. The accumulation of these signaling molecules triggers antibiotic production in the population. A pair of repressor-amplifier proteins encoded by scbA and scbR mediates the production and action of one particular gamma-butyrolactone, SCB1. Based on the proposed interactions of scbA and scbR, a mathematical model was constructed and used to explore the ability of this system to act as a robust genetic switch. Stability analysis shows that the butyrolactone system exhibits bistability and, in response to a threshold SCB1 concentration, can switch from an OFF state to an ON state corresponding to the activation of genes in the cryptic type I polyketide synthase gene cluster, which are responsible for production of the hypothetical polyketide. The switching time is inversely related to the inducer concentration above the threshold, such that short pulses of low inducer concentration cannot switch on the system, suggesting its possible role in noise filtering. In contrast, secondary metabolite production can be triggered rapidly in a population of cells producing the butyrolactone signal due to the presence of an amplification loop in the system. S. coelicolor was perturbed experimentally by varying concentrations of SCB1, and the model simulations match the experimental data well. Deciphering the complexity of this butyrolactone switch will provide valuable insights into how robust and efficient systems can be designed using "simple" two-protein networks.

Project description:Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Dataset Information

Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor. Mechanism and stereochemistry of the enzymatic formation of epi-isozizaene.

Publications

Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor. Mechanism and stereochemistry of the enzymatic formation of epi-isozizaene.

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