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Isovaleryl coenzyme A (IV-CoA) is an important precursor for iso-fatty acids and lipids. It acts in the development of myxobacteria, which can produce this compound from acetyl-CoA through alternative IV-CoA biosynthesis (aib). A central reaction of aib is catalyzed by AibA/AibB, which acts as a cofactor-free decarboxylase despite belonging to the family of CoA-transferases. We developed an efficient expression system for AibA/AibB that allowed the determination of high-resolution crystal structures in complex with different ligands. Through mutational studies, we show that an active-site cysteine previously proposed to be involved in decarboxylation is not required for activity. Instead, AibA/AibB seems to induce an intramolecular decarboxylation by binding its substrate in a hydrophobic cavity and forcing it into a bent conformation. Our study opens opportunities for synthetic biology studies, since AibA/AibB may be suitable for the production of isobutene, a precursor of biofuels and chemicals. [ABSTRACT FROM AUTHOR]

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis- and trans-acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl-C2 unit is derived from phenylpyruvate (phenyl-C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl-C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl- S-acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin. [ABSTRACT FROM AUTHOR]

Take a detour: An alternative pathway to synthesize isovaleryl coenzyme A (CoA) has recently been suggested in myxobacteria, which is highly active when leucine is limited. Each enzymatic step of this unprecedented route has now been characterized and a novel 3‐methylglutaconyl CoA decarboxylase identified that has apparently evolved from CoA transferases. [ABSTRACT FROM AUTHOR]

Summary: The thuggacins are macrolide antibiotics that are active against Mycobacterium tuberculosis, the causative agent of tuberculosis. Distinct variants of these structures are produced by the myxobacteria Sorangium cellulosum So ce895 and Chondromyces crocatus Cm c5, which differ in side chain structure and modification by hydroxylation. We report here a comparative analysis of the biosynthetic gene clusters in these strains, which reveals the mechanistic basis for this architectural diversity. Although the polyketide-nonribosomal peptide cores of the molecules are highly similar, the underlying biosynthetic machineries exhibit an unexpected degree of divergence. Furthermore, the S. cellulosum gene cluster contains a crotonyl-CoA reductase (CCR) homolog not present in C. crocatus, which likely participates in assembling the unusual hexyl side chain of the So ce895 thuggacins, whereas the distinct hydroxylation pattern may result from variable action of a conserved FMN-dependent monooxygenase. Indeed, inactivation of the monooxygenase gene in C. crocatus resulted in production of both mono- and di-deshydroxy thuggacin derivatives, providing direct evidence for the role of this enzyme in the pathway. Finally, integration of a Tn5-derived npt promotor upstream of the thuggacin cluster in C. crocatus led to a significant increase in thuggacin production. [Copyright &y& Elsevier]