Purification and properties of NADP‐isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803
Abstract
NADP‐dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive‐Red‐120‐agarose affinity chromatography and phenyl‐Sepharose chromatography as main steps. The enzyme was purified about 600‐fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 μM for Mg 2+, d,l‐isocitrate and NADP, respectively, using Mg 2+ as divalent cation and 4, 5.7 and 6 μM for Mn 2+, d,l‐isocitrate and NADP, respectively, using Mn 2+ as a cofactor. The enzyme was inhibited non‐competitively by ADP (Ki, 6.4 mM) and 2‐oxoglutarate, (Ki, 6mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular‐dichroism spectrum showed a protein with a secondary structure consisting of about 30%α‐helix and 36%β‐pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH 2‐terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well‐known E. coli enzyme.
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In cyanobacteria, reducing power is mainly generated during photosynthesis.
“Thus, in cyanobacteria, the reducing power is mainly generated during photosynthesis (Muro-Pastor and Florencio 1992).”
From Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering (2011) · cites this paper as Muro-Pastor and Florencio 1992
The production of 2-oxoglutarate by the GS-GOGAT mechanism is insignificant compared with IDH activity.
“However, the production of 2-OG by this mechanism is rather insignificant if compared with IDH activity (Muro-Pastor and Florencio 1992).”
From Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering (2011) · cites this paper as Muro-Pastor and Florencio 1992
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Renewable energy from Cyanobacteria: energy production optimization by metabolic pathway engineering
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Self-citation
- Authors
- MURO‐PASTOR, M. I., & FLORENCIO, F. J.
- Affiliations
- Consejo Superior de Investigaciones Científicas (ES)
- Corresponding author
- FLORENCIO, F. Javier
- Venue
- European Journal of Biochemistry
- Publisher
- Wiley
- Published
- 1992-01
- Volume
- 203
- Issue
- 1-2
- Pages
- 99-105
- ISSN
- 0014-2956 (ISSN-L)
- 1432-1033
- OpenAlex
- W2070207560
- Cited by
- 74
- References
- 35
- Citations by year
- 2026: 1 · 2024: 4 · 2023: 1 · 2022: 4 · 2021: 10 · 2020: 4 · 2019: 3 · 2018: 2 · 2017: 2 · 2014: 2 · 2012: 1
- FWCI
- 2.16
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- European Journal of Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Biochemical Acid Research Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
- Keywords
- NADP-isocitrate dehydrogenase, Synechocystis sp. PCC 6803, cyanobacteria, enzyme purification, enzyme kinetics, ADP inhibition, protein secondary structure, isoelectric point, NH2-terminal sequence
- MeSH terms
- Cyanobacteria, Amino Acid Sequence, Cations, Divalent, Chromatography, Gel, Circular Dichroism, Electrophoresis, Polyacrylamide Gel, Isocitrate Dehydrogenase, Isoelectric Point, Kinetics, Magnesium, Manganese, Molecular Sequence Data, Precipitin Tests, Sequence Homology, Nucleic Acid, Blotting, Western
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