sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

Abstract

L-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of D-glucose 6-phosphate to L-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989-998, 2004). One of the forms with an approximately 50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as an approximately 65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified approximately 1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive approximately 65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.

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

“…Chatterjee et al (2004Chatterjee et al ( , 2006 identified two ORFs in Synechocystis that code for the first and the rate-limiting enzyme MIPS of the pathway. To further delineate the de novo inositol biosynthetic pathway in this cyanobacterium, we initiated a search for inositol monophosphatase, the second enzyme in this pathway and its structural gene.…”

Identification and organization of chloroplastic and cytosolic l-myo-inositol 1-phosphate synthase coding gene(s) in Oryza sativa: comparison with the wild halophytic rice, Porteresia coarctata

“…However, the present study demonstrates that the »60 kDa subunit of the chloroplastic MIPS protein is not an evolutionary descendent of the cyanobacterial MIPS(s) as none of the experimentally determined peptide masses obtained from MALDI-TOF analysis of the chloroplastic MIPS matched the in silico trypsin digest of either sll1722 or sll1981, the two MIPS proteins in Synechocystis functionally identiWed earlier from this laboratory (Chatterjee et al , 2006. Further, a comprehensive search of the rice Fig.…”

“…The native protein from Spirulina showed similar enzymatic and immunological properties to other MIPS proteins. However, no unique structural gene coding for MIPS from Spirulina or any other cyanobacterium was reported until, two unidentiWed open reading frames (ORFs), sll1722 and sll1981 from Synechocystis sp PCC 6803 were reported as MIPS coding genes (Chatterjee et al , 2006.…”

Enhancing photosynthetic production of ethylene in genetically engineered Synechocystis sp. PCC 6803

“…PCC 6803 was putatively assigned as a bifunctional enzyme with conserved domains for both acetolactate synthase (ALS) activities 48 and L-myo-inositol 1-phosphate synthase (MIPS). 49 Nevertheless, our enzymatic analysis showed that Sll1981 and Slr0370 functioned as OGDC and SSADH, respectively. Meanwhile, our results were in line with the metabolic flux analysis observed by Knoop and coworkers.…”