Abstract
The intestinal diplomonadid Giardia lamblia is a causative agent of persistent diarrhea. Current treatments are based on nitro drugs, especially metronidazole. Nitro compounds are activated by reduction, yielding toxic intermediates. The enzymatic systems responsible for this activation are not completely understood. By fractionating cell free crude extracts by size exclusion chromatography followed by mass spectrometry, enzymes with nitroreductase (NR) activities are identified. The protein encoded by ORF 17150 found in two pools with NR activities is overexpressed and characterized. In pools of fractions with main NR activities, previously-known NRs are identified, as well as a previously uncharacterized protein encoded by ORF 17150. Recombinant protein 17150 is a flavoprotein with NADPH-dependent quinone reductase and NR activities. Besides a set of previously identified NRs, we have identified a novel enzyme with NR activity.
Highlights
The intestinal diplomonadid Giardia lamblia is a causative agent of persistent diarrhea in humans and various animal species [1,2,3,4]
A useful system to further characterize these proteins is the overexpression in Escherichia coli BL21, a strain that is susceptible to metronidazole and various other antibiotics
NR activities were measured in the fractions obtained as described above using either NADH or NADPH as electron donors and MTT as a final electron acceptor
Summary
The intestinal diplomonadid Giardia lamblia is a causative agent of persistent diarrhea in humans and various animal species [1,2,3,4]. We have characterized two nitroreductases (NRs) with N-terminal ferredoxin and C-terminal flavine-NAD(P)H reductase domains, namely the nitazoxanide-binding protein NR1 [12,13] and a homologous protein referred to as NR2 [14]. NR1, NR2, and NR3 are good quinone reductases, but are modest NRs with only residual activities on nitro drugs [15,16]. 7-nitrocoumarin is a suitable substrate for assaying the nitroreductase activity of the previously characterized NR1 and NR2 [16], as well as for assaying NR activities of crude extracts [29]. The previously uncharacterized protein encoded by ORF17150 identified by this approach is characterized
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