sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as L-myo-inositol 1-phosphate synthase.
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.
Chatterjee
Anirban
A
Plant Molecular Cellular Genetics, Bose Institute, P-1/12 CIT Scheme VII M, 700054 Kolkata, India.
Dastidar
Krishnarup Ghosh
KG
Maitra
Susmita
S
Das-Chatterjee
Aparajita
A
Dihazi
Hassan
H
Eschrich
Klaus
K
Majumder
Arun Lahiri
AL
D000095:Acetolactate Synthase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002457:Cell Extracts; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D007294:Inositol / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology; D012568:Schizosaccharomyces / Q000235:genetics; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D040681:Structural Homology, Protein; D046939:Synechocystis / Q000201:enzymology*
D002457:Cell Extracts; D010455:Peptides; D011994:Recombinant Proteins; D007294:Inositol; D000095:Acetolactate Synthase; D007296:Myo-Inositol-1-Phosphate Synthase