This Article Full Text (PDF) Other Versions of this Article: JB.00465-07v1 189/15/5405    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Rodrigues, M. V. Articles by Santos, H. Search for Related Content PubMed PubMed Citation Articles by Rodrigues, M. V. Articles by Santos, H.

 Previous Article  |  Next Article 

J. Bacteriol. doi:10.1128/JB.00465-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Biology Division and Chemistry Division, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal; Biochemistry Department, Universidade de Coimbra, 3001-401 Coimbra, Portugal

^* To whom correspondence should be addressed. Email: santos{at}itqb.unl.pt.

	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 NMR (N. Borges et al., J. Bacteriol. 188:8128-35, 2006). Herein, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in E. coli of the putative genes for CTP:L-myo-inositol-1-phosphate cytidylyltransferase and DIPP synthase from several (hyper)thermophiles (Archaeoglobus 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 catalysed 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 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 C1 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 with previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.