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Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis.

ABSTRACT: We report the occurrence of an isomerase with a putative (betaalpha)(8)-barrel structure involved in both histidine and trypto-phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N'-[(5'-phosphoribosyl)-formimino]-5 amino-imidazole-4-carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene SCO2050 and its orthologue Rv1603 from M. tuberculosis complemented both hisA and trpF mutants of Escherichia coli. Expression of the E. coli trpF gene in the S. coelicolor mutant only complemented the tryptophan auxo-trophy, and the hisA gene only complemented the histidine auxotrophy. The discovery of this enzyme, which has a broad-substrate specificity, has implications for the evolution of metabolic pathways and may prove to be important for understanding the evolution of the (betaalpha)(8)-barrels.

SUBMITTER: Barona-Gomez F

PROVIDER: S-EPMC1315899 | biostudies-literature | 2003 Mar

REPOSITORIES: biostudies-literature

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Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis.

Barona-Gómez Francisco F Hodgson David A DA

EMBO reports 20030301 3

We report the occurrence of an isomerase with a putative (betaalpha)(8)-barrel structure involved in both histidine and trypto-phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N'-[(5'-phosphoribosyl)-formimino]-5 amino-imidazole-4-carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene ...[more]

PMID: 12634849

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Project description:BACKGROUND: Glycyrrhiza uralensis is one of the most popular medicinal plants in the world and is also widely used in the flavoring of food and tobacco. Due to limited genomic and transcriptomic data, the biosynthetic pathway of glycyrrhizin, the major bioactive compound in G. uralensis, is currently unclear. Identification of candidate genes involved in the glycyrrhizin biosynthetic pathway will significantly contribute to the understanding of the biosynthetic and medicinal chemistry of this compound. RESULTS: We used the 454 GS FLX platform and Titanium regents to produce a substantial expressed sequence tag (EST) dataset from the vegetative organs of G. uralensis. A total of 59,219 ESTs with an average read length of 409 bp were generated. 454 ESTs were combined with the 50,666 G. uralensis ESTs in GenBank. The combined ESTs were assembled into 27,229 unique sequences (11,694 contigs and 15,535 singletons). A total of 20,437 unique gene elements representing approximately 10,000 independent transcripts were annotated using BLAST searches (e-value <or= 1e-5) against the SwissProt, KEGG, TAIR, Nr and Nt databases. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. With respect to the genes related to glycyrrhizin metabolism, genes encoding 16 enzymes of the 18 total steps of the glycyrrhizin skeleton synthesis pathway were found. To identify novel genes that encode cytochrome P450 enzymes and glycosyltransferases, which are related to glycyrrhizin metabolism, a total of 125 and 172 unigenes were found to be homologous to cytochrome P450s and glycosyltransferases, respectively. The cytochrome P450 candidate genes were classified into 32 CYP families, while the glycosyltransferase candidate genes were classified into 45 categories by GO analysis. Finally, 3 cytochrome P450 enzymes and 6 glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis through an organ-specific expression pattern analysis based on real-time PCR. CONCLUSIONS: Using the 454 GS FLX platform and Titanium reagents, our study provides a high-quality EST database for G. uralensis. Based on the EST analysis, novel candidate genes related to the secondary metabolite pathway of glycyrrhizin, including novel genes encoding cytochrome P450s and glycosyltransferases, were found. With the assistance of organ-specific expression pattern analysis, 3 unigenes encoding cytochrome P450s and 6 unigenes encoding glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis.

Project description:Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.

Dataset Information

Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis.

Publications

Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis.

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