sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as L-myo-inositol 1-phosphate synthase
| Autor: | Klaus Eschrich, Susmita Maitra, Aparajita Das-Chatterjee, Krishnarup Ghosh Dastidar, Arun Lahiri Majumder, Hassan Dihazi, Anirban Chatterjee |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Cell Extracts
Protein subunit Auxotrophy Molecular Sequence Data Gene Expression Plant Science Saccharomyces cerevisiae Polymerase Chain Reaction chemistry.chemical_compound Open Reading Frames Schizosaccharomyces Genetics Inositol Phosphofructokinase 2 Amino Acid Sequence Acetolactate synthase ATP synthase biology Myo-Inositol-1-Phosphate Synthase Genetic Complementation Test Synechocystis biology.organism_classification Molecular biology Recombinant Proteins Molecular Weight Acetolactate Synthase chemistry Biochemistry Genes Bacterial Structural Homology Protein Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Schizosaccharomyces pombe biology.protein Peptides Sequence Alignment |
| Zdroj: | Planta. 224(2) |
| ISSN: | 0032-0935 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink