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

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 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.

VTC4 Is a Bifunctional Enzyme That Affects Myoinositol and Ascorbate Biosynthesis in Plants

“…The apparent V max for VTC4 with D-Ins 3-P was 4.0 units and that for L-Gal 1-P was 5.4 units. In general, these kinetic parameters are comparable to those found for the Lilium IMP (K m of 78 mM for D-Ins 3-P; Loewus and Loewus, 1982) and kiwifruit VTC4 (K m of 150 mM for L-Gal 1-P and 330 mM for D-Ins 3-P; Laing et al, 2004; Table II), yet they differ from the previously reported values for the Synechocystis (Patra et al, 2007) and barley (Hordeum vulgare) enzymes (Fu et al, 2008), which have lower K m values (Table II). Furthermore, the ratio of V max to K m provides one measure of discriminating substrate preference, and we found that there is a 1.7-fold difference in the ratios, indicating a small difference between L-Gal 1-P and D-Ins 3-P (Table II).…”

Barley (Hordeum vulgare L.) inositol monophosphatase: gene structure and enzyme characteristics

“…Recently Patra et al (2007) isolated sequences encoding an IMPase from the cyanobacterium Synechocystis sp. PCC6803, and studied the catalytic properties of the recombinant protein.…”

Occurrence of myo-inositol-1-phosphate phosphatase in pteridophytes: characteristics of the enzyme from the reproductive pinnules of Dryopteris filix-mas (L.) Schott

“…Chhetri et al (2005Chhetri et al ( , 2006 have already established the occurrence of the prime enzyme of myo-inositol biosynthesis, MIPS, in pteridophytes. Therefore, the presence of MIPP adds (Eisenberg Jr. 1967, Attwood et al, 1988Gee et al, 1988;Honchar et al, 1989;Leech et al, 1993;Pollack et al, 1994;Caselli et al, 1996, Fujimoto et al, 1996, Nigou and Besra, 2002Wang et al, 2006;Patra et al, 2007). However, its kinetic parameter, thermo-tolerance and calciumdependent inhibition at lower concentrations, differ substantially in pteridophytic species.…”

“…In pteridophytes, Benaroya et al (2004) and Chettri et al (2005Chettri et al ( , 2006 have recently documented the occurrence and characterization of L-myo-inositol-1-phosphate synthase. So far, work with regard to the participation of the subsequent enzyme in this metabolic sequence, myo-inositol-1-phosphate phosphatase (MIPP), has been carried out principally in animal systems (Eisenberg Jr, 1967;Attwood et al, 1988;Gee et al, 1988;Honchar et al, 1989;Leech et al, 1993;Pollack et al, 1994;Kwok and Lo, 1994;Fujimoto et al, 1996;Caselli et al, 1996), in archaea (Wang et al, 2006), in bacteria (Nigou and Besra, 2002), and in cyanobacteria (Patra et al, 2007). However, only a few reports are available of this enzyme from plant systems (Loewus and Loewus, 1983;Gumber et al, 1984).…”