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Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate.

ABSTRACT: Di-myo-inositol 1,1'-phosphate (DIP) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. Although the DIP biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. In this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipA and dipB) that remained missing. In Thermotoga maritima both candidate genes (in an originally misannotated locus TM1418) form an operon with the inositol-1-phosphate synthase encoding gene (TM1419). A predicted inositol-mono-phosphate cytidylyltransferase activity was directly confirmed for the purified product of T. maritima gene dipA cloned and expressed in Escherichia coli. The entire DIP pathway was reconstituted in E. coli by cloning of the TM1418-TM1419 operon in pBAD expression vector and confirmed to function in the crude lysate. (31)P NMR and MS analysis revealed that DIP synthesis proceeds via a phosphorylated DIP intermediate, P-DIP, which is generated by the dipB-encoded enzyme, now termed P-DIP synthase. This previously unknown intermediate is apparently converted to the final product, DIP, by an inositol monophosphatase-like phosphatase. These findings allowed us to revise the previously proposed DIP pathway. The genomic survey confirmed its presence in the species known to use DIP for osmoprotection. Among several newly identified species with a postulated DIP pathway, Aeropyrum pernix was directly proven to produce this osmolyte.

SUBMITTER: Rodionov DA

PROVIDER: S-EPMC1838593 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate.

Rodionov Dmitry A DA Kurnasov Oleg V OV Stec Boguslaw B Wang Yan Y Roberts Mary F MF Osterman Andrei L AL

Proceedings of the National Academy of Sciences of the United States of America 20070302 11

Di-myo-inositol 1,1'-phosphate (DIP) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. Although the DIP biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. In this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipA and dipB) that remained missing. In Thermotoga marit ...[more]

PMID: 17360515

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Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

Dataset Information

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate.

Publications

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate.

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