Abstract

Abstract

The gene valC, which encodes an enzyme homologous to the 2-epi-5-epi-valiolone kinase (AcbM) of the acarbose biosynthetic pathway, was identified in the validamycin A biosynthetic gene cluster. Inactivation of valC resulted in mutants that lack the ability to produce validamycin A. Complementation experiments with a replicating plasmid harboring full-length valC restored the production of validamycin A, thus suggesting a critical function of valC in validamycin biosynthesis. In vitro characterization of ValC revealed a new type of C7-cyclitol kinase, which phosphorylates valienone and validone—but not 2-epi-5-epi-valiolone, 5-epi-valiolone, or glucose—to afford their 7-phosphate derivatives. The results provide new insights into the activity of this enzyme and also confirm the existence of two different pathways leading to the same end-product: the valienamine moiety common to acarbose and validamycin A.

Feeding experiments using isotopically labeled precursors remain crucial elements in the study of natural product biosynthesis. Through careful isotope tracer studies, a number of previously unrecognized biosynthetic pathways have now become obvious. One of the remarkable examples is the discovery of the non-mevalonate pathway in plants, bacteria, algae, and plasmodium, whereas others include the discovery of a new shunt pathway that connects the mevalonate pathway and branched-chain fatty acids in myxobacteria and the involvement of an unusual HMG-CoA synthase-like mechanism in polyketide biosynthesis. Significant progress has also been made in the biosynthetic studies of aminocyclitol-derived natural products and other bioactive secondary metabolites. This review highlights some of the more recent discoveries of novel mechanisms and metabolic pathways involved in natural product biosynthesis based on isotope tracer experiments. Copyright © 2007 John Wiley & Sons, Ltd.

Sugar phosphate cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, for example, aromatic amino acids, and clinically relevant secondary metabolites, for example, aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically labeled cyclitols revealed that cetoniacytone A, a unique C₇N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2-epi-5-epi-valiolone, a product of SPC. By using heterologous probes from the 2-epi-5-epi-valiolone synthase class of SPCs, an SPC homologue gene, cetA, was isolated from the cetoniacytone producer. cetA is closely related to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actinoplanes sp. strain A40644. Recombinant expression of cetA and BE-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-5-epi-valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that might regulate the biosynthesis of a novel set of secondary metabolites.

Toward antitubercular drugs. Rif-Orf20, a homologue of M. tuberculosis PapA5 protein, is an acetyltransferase that is responsible for the conversion of DMDARSV to DMRSV in rifamycin B biosynthesis. This enzyme, together with PapA5, represents a new class of acyltransferases. The enzyme also utilizes propionyl-CoA as substrate to give rise to a new analogue of rifamycin that appears, from spectroscopic results, to have the structure 1.

A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched-chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched-chain α-keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched-chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl-CoA is derived from 3,3-dimethylacrylyl-CoA (DMA-CoA). Here we show that DMA-CoA is an isomerization product of 3-methylbut-3-enoyl-CoA (3MB-CoA). This compound is directly derived from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate. Incubation of cell-free extracts of a bkd mutant with HMG-CoA gave product(s) with the molecular mass of 3MB-CoA or DMA-CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L-leucine as precursor.