Subfunctionality of hydride transferases of the old yellow enzyme family of flavoproteins of Pseudomonas putida.
To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.
van Dillewijn
Pieter
P
Departamento de Protección Ambiental, Estación del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Wittich
Rolf-Michael
RM
Caballero
Antonio
A
Ramos
Juan-Luis
JL
D003001:Cloning, Molecular; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D053858:Metabolic Networks and Pathways; D009252:NADPH Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010802:Phylogeny; D016958:Pseudomonas putida / Q000201:enzymology*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014303:Trinitrotoluene / Q000378:metabolism
D005420:Flavoproteins; D009573:Nitrites; D000644:Quaternary Ammonium Compounds; D014303:Trinitrotoluene; D009252:NADPH Dehydrogenase; D014166:Transferases
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