Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei. 2017

Xinying Wang, and Daniel Ken Inaoka, and Tomoo Shiba, and Emmanuel Oluwadare Balogun, and Stefan Allmann, and Yoh-Ichi Watanabe, and Michael Boshart, and Kiyoshi Kita, and Shigeharu Harada
Department of Biomedical Chemistry, School of International Health, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8523, Japan.

Isocitrate dehydrogenases (IDHs) are metabolic enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate. Depending on the electron acceptor and subcellular localization, these enzymes are classified as NADP+-dependent IDH1 in the cytosol or peroxisomes, NADP+-dependent IDH2 and NAD+-dependent IDH3 in mitochondria. Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and Nagana disease in animals. Here, for the first time, a putative glycosomal T. brucei type 1 IDH (TbIDH1) was expressed in Escherichia coli and purified for crystallographic study. Surprisingly, the putative NADP+-dependent TbIDH1 has higher activity with NAD+ compared with NADP+ as electron acceptor, a unique characteristic among known eukaryotic IDHs which encouraged us to crystallize TbIDH1 for future biochemical and structural studies. Methods of expression and purification of large amounts of recombinant TbIDH1 with improved solubility to facilitate protein crystallization are presented.

UI MeSH Term Description Entries
D007521 Isocitrate Dehydrogenase An enzyme of the oxidoreductase class that catalyzes the conversion of isocitrate and NAD+ to yield 2-ketoglutarate, carbon dioxide, and NADH. It occurs in cell mitochondria. The enzyme requires Mg2+, Mn2+; it is activated by ADP, citrate, and Ca2+, and inhibited by NADH, NADPH, and ATP. The reaction is the key rate-limiting step of the citric acid (tricarboxylic) cycle. (From Dorland, 27th ed) (The NADP+ enzyme is EC 1.1.1.42.) EC 1.1.1.41. NAD Isocitrate Dehydrogenase,Isocitrate Dehydrogenase (NAD+),Isocitrate Dehydrogenase-I,Dehydrogenase, Isocitrate,Dehydrogenase, NAD Isocitrate,Isocitrate Dehydrogenase I,Isocitrate Dehydrogenase, NAD
D007523 Isocitrates Derivatives of isocitric acid, a structural isomer of CITRIC ACID, including its salts and esters.
D007656 Ketoglutaric Acids A family of compounds containing an oxo group with the general structure of 1,5-pentanedioic acid. (From Lehninger, Principles of Biochemistry, 1982, p442) Oxoglutarates,2-Ketoglutarate,2-Ketoglutaric Acid,2-Oxoglutarate,2-Oxoglutaric Acid,Calcium Ketoglutarate,Calcium alpha-Ketoglutarate,Ketoglutaric Acid,Oxogluric Acid,alpha-Ketoglutarate,alpha-Ketoglutaric Acid,alpha-Ketoglutaric Acid, Calcium Salt (2:1),alpha-Ketoglutaric Acid, Diammonium Salt,alpha-Ketoglutaric Acid, Dipotassium Salt,alpha-Ketoglutaric Acid, Disodium Salt,alpha-Ketoglutaric Acid, Monopotassium Salt,alpha-Ketoglutaric Acid, Monosodium Salt,alpha-Ketoglutaric Acid, Potassium Salt,alpha-Ketoglutaric Acid, Sodium Salt,alpha-Oxoglutarate,2 Ketoglutarate,2 Ketoglutaric Acid,2 Oxoglutarate,2 Oxoglutaric Acid,Calcium alpha Ketoglutarate,alpha Ketoglutarate,alpha Ketoglutaric Acid,alpha Ketoglutaric Acid, Diammonium Salt,alpha Ketoglutaric Acid, Dipotassium Salt,alpha Ketoglutaric Acid, Disodium Salt,alpha Ketoglutaric Acid, Monopotassium Salt,alpha Ketoglutaric Acid, Monosodium Salt,alpha Ketoglutaric Acid, Potassium Salt,alpha Ketoglutaric Acid, Sodium Salt,alpha Oxoglutarate,alpha-Ketoglutarate, Calcium
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D009243 NAD A coenzyme composed of ribosylnicotinamide 5'-diphosphate coupled to adenosine 5'-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). (Dorland, 27th ed) Coenzyme I,DPN,Diphosphopyridine Nucleotide,Nadide,Nicotinamide-Adenine Dinucleotide,Dihydronicotinamide Adenine Dinucleotide,NADH,Adenine Dinucleotide, Dihydronicotinamide,Dinucleotide, Dihydronicotinamide Adenine,Dinucleotide, Nicotinamide-Adenine,Nicotinamide Adenine Dinucleotide,Nucleotide, Diphosphopyridine
D009249 NADP Nicotinamide adenine dinucleotide phosphate. A coenzyme composed of ribosylnicotinamide 5'-phosphate (NMN) coupled by pyrophosphate linkage to the 5'-phosphate adenosine 2',5'-bisphosphate. It serves as an electron carrier in a number of reactions, being alternately oxidized (NADP+) and reduced (NADPH). (Dorland, 27th ed) Coenzyme II,Nicotinamide-Adenine Dinucleotide Phosphate,Triphosphopyridine Nucleotide,NADPH,Dinucleotide Phosphate, Nicotinamide-Adenine,Nicotinamide Adenine Dinucleotide Phosphate,Nucleotide, Triphosphopyridine,Phosphate, Nicotinamide-Adenine Dinucleotide
D011993 Recombinant Fusion Proteins Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes. Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D003460 Crystallization The formation of crystalline substances from solutions or melts. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Crystalline Polymorphs,Polymorphism, Crystallization,Crystal Growth,Polymorphic Crystals,Crystal, Polymorphic,Crystalline Polymorph,Crystallization Polymorphism,Crystallization Polymorphisms,Crystals, Polymorphic,Growth, Crystal,Polymorph, Crystalline,Polymorphic Crystal,Polymorphisms, Crystallization,Polymorphs, Crystalline
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli

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