Two atypical L-cysteine-regulated NADPH-dependent oxidoreductases involved in redox maintenance, L-cystine and iron reduction, and metronidazole activation in the enteric protozoan Entamoeba histolytica.
We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.
Jeelani
Ghulam
G
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; Center for Integrated Medical Research, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan.
Husain
Afzal
A
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Sato
Dan
D
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan.
Ali
Vahab
V
Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna-800007, India.
Suematsu
Makoto
M
Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Shinjuku, Tokyo 160-8582, Japan.
Soga
Tomoyoshi
T
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan.
Nozaki
Tomoyoshi
T
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Electronic address: nozaki@nih.go.jp.
D000595:Amino Acid Sequence; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000378:metabolism*; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008795:Metronidazole / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
D000981:Antiprotozoal Agents; D007506:Iron-Sulfur Proteins; D015800:Protozoan Proteins; D008795:Metronidazole; D005182:Flavin-Adenine Dinucleotide; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine