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PubMed1998

Acyl transfer activity of an amidase from Rhodococcus sp. strain R312: formation of a wide range of hydroxamic acids.

The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with alpha-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the Km(amide) values (e.g., Km(amide) = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (kcat) were obtained with linear aliphatic amides (e.g., kcat = 333 s-1 with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, alpha-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., KmNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be L-enantioselective towards alpha-hydroxy- and alpha-aminoamides.

Acylation↗

PubMed1998

Initial transformations in the biodegradation of benzothiazoles by Rhodococcus isolates.

Benzothiazole-2-sulfonate (BTSO3) is one of the side products occurring in 2-mercaptobenzothiazole (MBT) production wastewater. We are the first to isolate an axenic culture capable of BTSO3 degradation. The isolate was identified as a Rhodococcus erythropolis strain and also degraded 2-hydroxybenzothiazole (OBT) and benzothiazole (BT), but not MBT, which was found to inhibit the biodegradation of OBT, BT, and BTSO3. In anaerobic resting cell assays, BTSO3 was transformed into OBT in stoichiometric amounts. Under aerobic conditions, OBT was observed as an intermediate in BT breakdown and an unknown compound transiently accumulated in several assays. This product was identified as a dihydroxybenzothiazole. Benzothiazole degradation pathways seem to converge into OBT, which is then transformed further into the dihydroxy derivative.

Aerobiosis↗

PubMed1999

Microbial desulfurization of alkylated dibenzothiophenes from a hydrodesulfurized middle distillate by Rhodococcus erythropolis I-19.

Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 micromol g dry cell weight(-1) min(-1), more than 25-fold higher than that for wild-type bacteria. According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes. Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S. XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones. I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs. Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane. Apparent saturation rate constant (K(0)) and half-saturation rate constant (K(1)) values were calculated to be 2.8 micromol g dry cell weight(-1) min(-1) and 130 ppm, respectively. However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior. A competitive-substrate model suggested that the apparent K(1) values for each group of Cx-DBTs increased with increasing alkylation. Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure.

Alkylation↗

PubMed1999

Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and a Rhodococcus sp.

A single microorganism able to mineralize chloronitrobenzenes (CNBs) has not been reported, and degradation of CNBs by coculture of two microbial strains was attempted. Pseudomonas putida HS12 was first isolated by analogue enrichment culture using nitrobenzene (NB) as the substrate, and this strain was observed to possess a partial reductive pathway for the degradation of NB. From high-performance liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance analyses, NB-grown cells of P. putida HS12 were found to convert 3- and 4-CNBs to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For the degradation of CNBs, Rhodococcus sp. strain HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with P. putida HS12 to give a coculture. This coculture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. A degradation pathway for 3- and 4-CNBs by the two isolated strains was also proposed.

Acetylation↗

PubMed1999

Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene.

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.

Alcohol Oxidoreductases↗

PubMed1999

Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp. strain Q15.

We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.

Adaptation, Physiological↗

PubMed2000

Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitriles.

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein(-1)) and amidase activity (38.4 nkat mg of protein(-1)) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C. I. Basic Blue 9.

Acrylic Resins↗

PubMed2000

Targeted disruption of the kstD gene encoding a 3-ketosteroid delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1.

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by Delta(1)-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1)-dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta(1)-dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9alpha-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Delta(1)-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the Delta(1)-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

Cloning, Molecular↗

PubMed2000

Identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in Rhodococcus opacus 1cp.

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and (19)F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The (19)F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2, 3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.

Biotransformation↗

PubMed2001

Physical and metabolic interactions of Pseudomonas sp. strain JA5-B45 and Rhodococcus sp. strain F9-D79 during growth on crude oil and effect of a chemical surfactant on them.

Methods to enhance crude oil biodegradation by mixed bacterial cultures, for example, (bio)surfactant addition, are complicated by the diversity of microbial populations within a given culture. The physical and metabolic interactions between Rhodococcus sp. strain F9-D79 and Pseudomonas sp. strain JA5-B45 were examined during growth on Bow River crude oil. The effects of a nonionic chemical surfactant, Igepal CO-630 (nonylphenol ethoxylate), also were evaluated. Strain F9-D79 grew attached to the oil-water interface and produced a mycolic acid-containing capsule. Crude oil emulsification and surface activity were associated with the cellular fraction. Strain JA5-B45 grew in the aqueous phase and was unable to emulsify oil, but cell-free supernatants mediated kerosene-water emulsion formation. In coculture, stable emulsions were formed and strain JA5-B45 had an affinity for the capsule produced by strain F9-D79. Igepal CO-630 inhibited F9-D79 cells from adhering to the interface, and cells grew dispersed in the aqueous phase as 0.5-microm cocci rather than 2.5-microm rods. The surfactant increased total petroleum hydrocarbon removal by strain JA5-B45 from 4 to 22% and included both saturated compounds and aromatics. In coculture, TPH removal increased from 13 to 40% following surfactant addition. The culture pH normally increased from 7.0 to between 7.5 and 8.5, although addition of Igepal CO-630 to F9-D79 cultures resulted in a drop to pH 5.5. We suggest a dual role for the nonylphenol ethoxylate surfactant in the coculture: (i) to improve hydrocarbon uptake by strain JA5-B45 through emulsification and (ii) to prevent strain F9-D79 from adhering to the oil-water interface, indirectly increasing hydrocarbon availability. These varied effects on hydrocarbon biodegradation could explain some of the known diversity of surfactant effects.

Biodegradation, Environmental↗

PubMed2001

Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14.

A monoterpene epsilon-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30 degrees C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.

Amino Acid Sequence↗

PubMed2001

Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1.

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K(m) values for NADH and FMN were 208 and 10.8 microM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35 degrees C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80 degrees C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705-1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.

Escherichia coli↗

PubMed2001

Long-range (1)H-(15)N heteronuclear shift correlation at natural abundance: a tool to study benzothiazole biodegradation by two rhodococcus strains.

The biodegradation of benzothiazole and 2-hydroxybenzothiazole by two strains of Rhodococcus was monitored by reversed phase high-pressure liquid chromatography and by (1)H nuclear magnetic resonance (NMR). Both xenobiotics were biotransformed into a hydroxylated derivative of 2-hydroxybenzothiazole by these two strains. The chemical structure of this metabolite was determined by a new NMR methodology: long-range (1)H-(15)N heteronuclear shift correlation without any previous (15)N enrichment of the compound. This powerful NMR tool allowed us to assign the metabolite structure to 2,6-dihydroxybenzothiazole.

Benzothiazoles↗

PubMed2001

Deep desulfurization of extensively hydrodesulfurized middle distillate oil by Rhodococcus sp. strain ECRD-1.

Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by Rhodococcus sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.

Biodegradation, Environmental↗

PubMed2001

Characterization of the 450-kb linear plasmid in a polychlorinated biphenyl degrader, Rhodococcus sp. strain RHA1.

A strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). The diverse degradative genes are distributed mainly on the pRHL1 and pRHL2 plasmids. In this study, the structural and functional characteristics of pRHL2 were determined. We constructed a physical map of pRHL2, and the degradative enzyme genes, including bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4, were localized in three regions. Conjugal transfer of pRHL2 between RHA1 mutant derivatives was observed at a frequency of 7.5 x 10(-5) transconjugant per recipient. These results suggested that the linear plasmid is a possible determinant of propagation of the diverse degradative genes in rhodococci. The termini of pRHL2 were cloned and sequenced. The left and right termini of pRHL2 had 3-bp perfect terminal inverted repeats and were not as similar to each other (64% identity) as the known actinomycete linear replicons are. Southern hybridization analysis with pRHL2 terminal probes suggested that the right terminus of pRHL2 is similar to pRHL1 and pRHL3 termini. Retardation of both terminal fragments in the gel shift assay indicated that each terminus of pRHL2 is linked to a protein. We suggest that pRHL2 has invertron termini, as has been reported previously for Streptomyces linear replicons.

Base Sequence↗

PubMed2001

Epidemiology of Rhodococcus equi strains on Thoroughbred horse farms.

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of Rhodococcus equi, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of R. equi which carry the vapA gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.

Actinomycetales Infections↗

PubMed2002

Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp. strain DN22.

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 microM) when used as an N source for strain DN22. RDX was converted to nitrite (NO(2)(-)) (30%), nitrous oxide (N(2)O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [(15)N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N(2)O with two molecular mass ions: one at 44 Da, corresponding to (14)N(14)NO, and the second at 45 Da, corresponding to (15)N(14)NO. The nonlabeled N(2)O could be formed only from -NO(2), whereas the (15)N-labeled one was presumed to originate from a nitramine group ((15)N-(14)NO(2)) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C(2)H(5)N(3)O(3). When the experiment was repeated with ring-labeled [(15)N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-(14)C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in (14)CO(2) (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO(2) and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.

Aerobiosis↗

PubMed2002

Cloning, sequencing, and characterization of the hexahydro-1,3,5-Trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.

Amino Acid Sequence↗