Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Abstract

Abstract:Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also docu mented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.Key words: nitroreductase, TNT, RDX, Enterobacter cloacae, Morganella morganii.Résumé : Différentes bactéries entériques produisent une nitroréductase de type I insensible à l'oxygène capable de ré duire les groupements nitro de divers composés nitroaromatiques dans des conditions anaérobies. La réduction enzyma tique des nitramines en anaérobiose est aussi connue chez les entérobactéries. La présente étude indique que la réduction des nitramines par les entérobactéries était catalysée par une nitroréductase de type I non sensible à l'oxygène plutôt que par une enzyme de type II. La souche B2 de Morganella morganii reconnue pour son activité hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroréductase, et la souche 96-3 d'Enterobacter cloacae reconnue pour son activité 2,4,6 trinitrotoluène (TNT) nitroréductase, ont toutes deux servi à démontrer que les deux explosifs TNT et RDX étaient réduits par une nitroréductase de type I. Les RDX et TNT nitroréductases de M. morganii et d'E. cloacae ont été observées lors d'essais avec des bactéries entières. La nitroréductase de type I, purifiée de l'E. cloacae, oxydait le NADPH en présence de TNT ou de RDX comme substrat. L'induction de l'expression du gène de la nitroréductase de type I de l'E. cloacae chez une souche d'Escherichia coli porteuse d'un plasmide a causé une augmentation spon tanée de l'activité des TNT et RDX nitroréductases. Par contre aucune activité TNT ou RDX nitroréductase n'a été ob servée chez des mutants de M. morganii résistants au nitrofurazone. La présente étude permet de conclure que la nitroréductase de type I ret...

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

“…Type I is oxygen insensitive (3,6,15,17,20,37,38,39), and type II is oxygen sensitive (21, 26). In type I, there are two nitroflavin reductase superfamiles: NfsA and NfsB (3,6,14,17,20,37,38,39).…”

Synthesis and evaluation of halogenated nitrophenoxazinones as nitroreductase substrates for the detection of pathogenic bacteria

“…[1][2][3][4] For example, the reduction of a nitroaromatic prodrug by a bacterial nitroreductase is central to the GDEPT anticancer therapeutic approach, 5,6 for which the mechanism of the Escherichia coli nitroreductase activation has been elucidated. 7 Nitroreductase enzymes have been subdivided into two different classes, 8,9 namely type I and type II nitroreductases, based on their relative sensitivity to oxygen. The type I nitroreductases are oxygen insensitive and can reduce nitro compounds to their corresponding amines via the nitroso and hydroxylamine intermediates, using a two electron transfer mechanism.…”

“…The type I nitroreductases are oxygen insensitive and can reduce nitro compounds to their corresponding amines via the nitroso and hydroxylamine intermediates, using a two electron transfer mechanism. [9][10][11] With the type II nitroreductases, which are oxygen sensitive, the reduction of nitro groups is initiated via a single electron transfer process, forming a nitro radical anion, which either 4 accepts a second electron to form the nitroso intermediate and then follows the two electron reduction process, or is rapidly reoxidised to a nitro group in the presence of oxygen. 8,9 The widespread distribution of nitroreductase enzymes in pathogenic bacteria and yeasts offers the potential to enhance the detection of such microorganisms in both clinical and food applications.…”

“…[9][10][11] With the type II nitroreductases, which are oxygen sensitive, the reduction of nitro groups is initiated via a single electron transfer process, forming a nitro radical anion, which either 4 accepts a second electron to form the nitroso intermediate and then follows the two electron reduction process, or is rapidly reoxidised to a nitro group in the presence of oxygen. 8,9 The widespread distribution of nitroreductase enzymes in pathogenic bacteria and yeasts offers the potential to enhance the detection of such microorganisms in both clinical and food applications. 11,12 The successful detection of pathogenic bacteria as a result of their nitroreductase action has been demonstrated in over thirty Gram negative and Gram positive microorganisms, 11 using the fluorogenic 7-nitrocoumarin-3-carboxylic acid 1a or 7-nitro-4-methylcoumarin 1b (Figure 1).…”

Identification of Metabolic Routes and Catabolic Enzymes Involved in Phytoremediation of the Nitro- Substituted Explosives TNT, RDX, and HMX

“…Cytochrome P-450 monooxygenases (CYPs) and glutathione S-transferases (GSTs) are known to be involved in plant metabolism of many xenobiotic pollutants in plants (Sandermann, 1994;Marrs, 1996;Ohkawa et al, 1999;Schwitzguebel and Vanek, 2003). On the other hand, cytochrome P-450, nitroreductases, hydrolases, peroxidases, and NADPH-dependent flavoenzymes have been shown, or are suspected, to play a role in RDX transformation by bacteria and fungi (Hawari, 2000;Kitts et al, 2000;Stahl et al, 2001;Bhushan et al, 2002;Hannink, Rosser, and Bruce, 2002;Seth-Smith et al, 2002;Bhushan et al, 2003).…”