Anaerobic degradation of nitrilotriacetate (NTA) in a denitrifying bacterium: purification and characterization of the NTA dehydrogenase-nitrate reductase enzyme complex.
The initial step in the anoxic metabolism of nitrilotriacetate (NTA) was investigated in a denitrifying member of the gamma subgroup of the Proteobacteria. In membrane-free cell extracts, the first step of NTA oxidation was catalyzed by a protein complex consisting of two enzymes, NTA dehydrogenase (NTADH) and nitrate reductase (NtR). The products formed were iminodiacetate and glyoxylate. Electrons derived from the oxidation of NTA were transferred to nitrate only via the artificial dye phenazine methosulfate, and nitrate was stoichiometrically reduced to nitrite. NTADH activity could be measured only in the presence of NtrR and vice versa. The NTADH-NtrR enzyme complex was purified and characterized. NTADH and NtrR were both alpha 2 dimers and had molecular weights of 170,000 and 105,000, respectively. NTADH contained covalently bound flavin cofactor, and NtrR contained a type b cytochrome. Optimum NTA-oxidizing activity was achieved at a molar ratio of NTADH to NtrR of approximately 1:1. So far, NTA is the only known substrate for NTADH. This is the first report of a redox enzyme complex catalyzing the oxidation of a substrate and concomitantly reducing nitrate.
Jenal-Wanner
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Institute for Aquatic Sciences and Water Pollution Control, Swiss Federal Institute of Technology, Zurich ETHZ.
Egli
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D000693:Anaerobiosis; D001673:Biodegradation, Environmental; D003067:Coenzymes / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004579:Electron Transport; D004795:Enzyme Stability; D006090:Gram-Negative Bacteria / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000378:metabolism*; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D014871:Water Microbiology; D014874:Water Pollutants, Chemical / Q000378:metabolism
D003067:Coenzymes; D009097:Multienzyme Complexes; D014874:Water Pollutants, Chemical; D000587:Oxidoreductases Acting on CH-NH Group Donors; D009565:Nitrate Reductases; C084604:nitrilotriacetate dehydrogenase; D050901:Nitrate Reductase; D009571:Nitrilotriacetic Acid