title: Bifunctional CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper)thermophiles.
abstract: 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.
authors.affiliation: Biology Division, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal.
authors.forename: Marta V
authors.initials: MV
authors.lastname: Rodrigues
authors.forename: Nuno
authors.initials: N
authors.lastname: Borges
authors.forename: Mafalda
authors.initials: M
authors.lastname: Henriques
authors.forename: Pedro
authors.initials: P
authors.lastname: Lamosa
authors.forename: Rita
authors.initials: R
authors.lastname: Ventura
authors.forename: Chantal
authors.initials: C
authors.lastname: Fernandes
authors.forename: Nuno
authors.initials: N
authors.lastname: Empadinhas
authors.forename: Christopher
authors.initials: C
authors.lastname: Maycock
authors.forename: Milton S
authors.initials: MS
authors.lastname: da Costa
authors.forename: Helena
authors.initials: H
authors.lastname: Santos
mesh_terms: D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D019843:Archaeal Proteins / Q000235:genetics; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D002247:Carbon Isotopes / Q000378:metabolism; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D050939:Gene Fusion; D023281:Genomics; D007295:Inositol Phosphates / Q000096:biosynthesis* / Q000378:metabolism; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D004269:DNA, Bacterial; D007295:Inositol Phosphates; C075843:di-myo-inositol-1,1'-phosphate; C002647:inositol 1-phosphate; D003570:Cytidine Triphosphate; D014166:Transferases