Crystal structures of two nitroreductases from hypervirulent Clostridium difficile and functionally related interactions with the antibiotic metronidazole.
Nitroreductases (NRs) are flavin mononucleotide (FMN)-dependent enzymes that catalyze the biotransformation of organic nitro compounds (RNO2; R = alkyl, aryl) to the nitroso RN=O, hydroxylamino RNHOH, or amine RNH2derivatives. Metronidazole (Mtz) is a nitro-containing antibiotic that is commonly prescribed for lower-gut infections caused by the anaerobic bacterium Clostridium difficile. C. difficile infections rank number one among hospital acquired infections, and can result in diarrhea, severe colitis, or even death. Although NRs have been implicated in Mtz resistance of C. difficile, no NRs have been characterized from the hypervirulent R20291 strain of C. difficile. We report the first expression, purification, and three-dimensional X-ray crystal structures of two NRs from the C. difficile R20291 strain. The X-ray crystal structures of the two NRs were solved to 2.1 Å resolution. Their homodimeric structures exhibit the classic NR α+β fold, with each protomer binding one FMN cofactor near the dimer interface. Functional assays demonstrate that these two NRs metabolize Mtz with associated re-oxidation of the proteins. Importantly, these results represent the first isolation and characterization of NRs from the hypervirulent R20291 strain of relevance to organic RNO2(e.g., Mtz) metabolism.
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, United States.
Bing
B
Wang
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, United States.
Samantha M
SM
Powell
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, United States.
Neda
N
Hessami
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, United States.
Fares Z
FZ
Najar
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, United States.
Leonard M
LM
Thomas
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Microbiology and Plant Biology, University of Oklahoma, Norman 73019, United States.
Elizabeth A
EA
Karr
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, United States.
Ann H
AH
West
Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman 73019, United States; Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, United States. Electronic address: grichteraddo@ou.edu.
George B
GB
Richter-Addo
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D016360:Clostridioides difficile / Q000201:enzymology*; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D008795:Metronidazole / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009601:Nitroreductases / Q000737:chemistry / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008795:Metronidazole; D005486:Flavin Mononucleotide; D009601:Nitroreductases
FMN cofactor; Metronidazole; Nitroorganic; Nitroreductase; UV–vis; X-ray structure