Dataset Information

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin.

ABSTRACT: A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.

SUBMITTER: Dairi T

PROVIDER: S-EPMC99688 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin.

Dairi T T Hamano Y Y Kuzuyama T T Itoh N N Furihata K K Seto H H

Journal of bacteriology 20011001 20

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstre ...[more]

PMID: 11567009

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Project description:Thermostability is the key to maintain the structural integrity and catalytic activity of enzymes in industrial biotechnological processes, such as terpene cyclase-mediated generation of medicines, chiral synthons, and fine chemicals. However, affording a large increase in the thermostability of enzymes through site-directed protein engineering techniques can constitute a challenge. In this paper, we used ancestral sequence reconstruction to create a hyperstable variant of the ent-copalyl diphosphate synthase PtmT2, a terpene cyclase involved in the assembly of antibiotics. Molecular dynamics simulations on the μs timescale were performed to shed light on possible molecular mechanisms contributing to activity at an elevated temperature and the large 40 °C increase in melting temperature observed for an ancestral variant of PtmT2. In silico analysis revealed key differences in the flexibility of a loop capping the active site, between extant and ancestral proteins. For the modern enzyme, the loop collapses into the active site at elevated temperatures, thus preventing biocatalysis, whereas the loop remains in a productive conformation both at ambient and high temperatures in the ancestral variant. Restoring a Pro loop residue introduced in the ancestral variant to the corresponding Gly observed in the extant protein led to reduced catalytic activity at high temperatures, with only moderate effects on the melting temperature, supporting the importance of the flexibility of the capping loop in thermoadaptation. Conversely, the inverse Gly to Pro loop mutation in the modern enzyme resulted in a 3-fold increase in the catalytic rate. Despite an overall decrease in maximal activity of ancestor compared to wild type, its increased thermostability provides a robust backbone amenable for further enzyme engineering. Our work cements the importance of loops in enzyme catalysis and provides a molecular mechanism contributing to thermoadaptation in an ancestral enzyme.

Project description:BackgroundIsoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II).ResultsSearches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria.ConclusionsOur analyses support a single origin of the DXR-II family through functional divergence, in which constitutes an exceptional model of acquisition and maintenance of redundant gene functions between non-homologous genes as a result of convergent evolution. Subsequently, although old episodic events of HGT could not be excluded, the results supported a prevalent role of gene loss in explaining the distribution of DXR-II in specific pathogenic eubacteria. Our results highlight the importance of the functional characterization of evolutionary shortcuts in isoprenoid biosynthesis for screening specific antibacterial drugs and for regulating the production of isoprenoids of human interest.

Dataset Information

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin.

Publications

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin.

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