Functional identification of sll1383 from Synechocystis sp PCC 6803 as L-myo-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization |
| |
Authors: | Barunava Patra Krishnarup Ghosh Dastidar Susmita Maitra Jyotirmoy Bhattacharyya Arun Lahiri Majumder |
| |
Affiliation: | (1) Plant Molecular and Cellular Genetics, Bose Institute, P-1/12. CIT Scheme VII M, Kolkata, 700054, India |
| |
Abstract: | The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V max (∼0.033 μmol Pi/min/mg protein) was low. Li+ was found to be an inhibitor (IC50 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na+, K+ NH4+) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg2+ dependent, which can be substituted partially by Mn2+. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed. |
| |
Keywords: | Synechocystis sp PCC6803 Inositol monophosphatase L-myo-inositol 1-phosphate synthase MIPS IMPase |
本文献已被 PubMed SpringerLink 等数据库收录! |
|