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.
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 approximately 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) ( approximately 0.033 mumol Pi/min/mg protein) was low. Li(+) was found to be an inhibitor (IC(50) 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na(+), K(+) NH (4) (+) ) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg(2+) dependent, which can be substituted partially by Mn(2+). However, unlike other IMPase(s), the enzyme is optimally active at approximately 42 degrees 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.
Patra
Barunava
B
Plant Molecular and Cellular Genetics, Bose Institute, P-1/12. CIT Scheme VII M, Kolkata, 700054, India.
Ghosh Dastidar
Krishnarup
K
Maitra
Susmita
S
Bhattacharyya
Jyotirmoy
J
Majumder
Arun Lahiri
AL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008094:Lithium; D008274:Magnesium; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D046939:Synechocystis / Q000378:metabolism*; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008094:Lithium; D008274:Magnesium