Homologous npdGI genes in 2,4-dinitrophenol- and 4-nitrophenol-degrading Rhodococcus spp.
ABSTRACT: Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F(420) reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F(420). Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F(420)-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F(420)-dependent glucose-6-phosphate dehydrogenases and F(420)-dependent N(5),N(10)-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F(420)-dependent enzymes involved in catabolism.
Project description:Two hydrogenation reactions in the initial steps of degradation of 2,4,6-trinitrophenol produce the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. The npdH gene (contained in the npd gene cluster of the 2,4,6-trinitrophenol-degrading strain Rhodococcus opacus HL PM-1) was shown here to encode a tautomerase, catalyzing a proton shift between the aci-nitro and the nitro forms of the dihydride Meisenheimer complex of 2,4,6-trinitrophenol. An enzyme (which eliminated nitrite from the aci-nitro form but not the nitro form of the dihydride complex of 2,4,6-trinitrophenol) was purified from the 2,4,6-trinitrophenol-degrading strain Nocardioides simplex FJ2-1A. The product of nitrite release was the hydride Meisenheimer complex of 2,4-dinitrophenol, which was hydrogenated to the dihydride Meisenheimer complex of 2,4-dinitrophenol by the hydride transferase I and the NADPH-dependent F(420) reductase from strain HL PM-1. At pH 7.5, the dihydride complex of 2,4-dinitrophenol is protonated to 2,4-dinitrocyclohexanone. A hydrolase was purified from strain FJ2-1A and shown to cleave 2,4-dinitrocyclohexanone hydrolytically to 4,6-dinitrohexanoate.
Project description:Rhodococcus opacus HL PM-1 utilizes 2,4,6-trinitrophenol (picric acid) as a sole nitrogen source. The initial attack on picric acid occurs through two hydrogenation reactions. Hydride transferase II (encoded by npdI) and hydride transferase I (encoded by npdC) are responsible for the hydride transfers. Database searches with the npd genes have indicated the presence of a putative transcriptional regulator, npdR. Here, the npdR gene was expressed in Escherichia coli, and the protein was purified and shown to form a complex with intergenic regions between open reading frames A and B and between npdH and npdI within the npd gene cluster. A change in DNA-NpdR complex formation occurred in the presence of 2,4-dinitrophenol, picric acid, 2-chloro-4,6-dinitrophenol, and 2-methyl-4,6-dinitrophenol. By constructing a promoter-probe vector, we demonstrated that both intergenic regions caused the expression of reporter gene xylE. Hence, both of these regions contain promoters. A deletion mutant of R. opacus HL PM-1 was constructed in which part of npdR was deleted. The expression of npdI and npdC was induced by 2,4-dinitrophenol in the wild-type strain, while in the mutant these genes were constitutively expressed. Hence, NpdR is a repressor involved in picric acid degradation.
Project description:Aldo-keto reductases (AKRs) are a major superfamily of monomeric NADPH-dependent carbonyl oxidoreductases. They are characterized by an (alpha/beta)(8)-barrel structure, which at its base contains a conserved catalytic tetrad of Tyr, Lys, His and Asp. Two AKR subfamilies contain other residues substituted for the catalytic His and perform different functions. First, the steroid 5beta-reductase (AKR1D1), which reduces CC double bonds instead of carbonyl groups, has a Glu substituted for His. Second, the Kvbeta subunits (AKR6A3, AKR6A5 and AKR6A9) which modulate opening of the voltage-gated potassium channel (Kv1) by oxidizing NADPH, have an Asn substituted for the His. Previously, we noted that conserved catalytic residues in AKRs perform similar functions in the short-chain dehydrogenases (SDRs). With the availability of crystal structures of AKR1D1 and two SDRs that catalyze double-bond reduction reactions, Digitalis steroid 5beta-reductase and 2,4-dienoyl-CoA reductase, we have compared their active sites to outline the features that govern whether 1,2-, 1,4- or 1,6-hydride transfer occurs.
Project description:2,4-Dinitroanisole (DNAN) is an insensitive munition ingredient used in explosive formulations as a replacement for 2,4,6-trinitrotoluene (TNT). Little is known about the environmental behavior of DNAN. There are reports of microbial transformation to dead-end products, but no bacteria with complete biodegradation capability have been reported. Nocardioides sp. strain JS1661 was isolated from activated sludge based on its ability to grow on DNAN as the sole source of carbon and energy. Enzyme assays indicated that the first reaction involves hydrolytic release of methanol to form 2,4-dinitrophenol (2,4-DNP). Growth yield and enzyme assays indicated that 2,4-DNP underwent subsequent degradation by a previously established pathway involving formation of a hydride-Meisenheimer complex and release of nitrite. Identification of the genes encoding the key enzymes suggested recent evolution of the pathway by recruitment of a novel hydrolase to extend the well-characterized 2,4-DNP pathway.
Project description:Most ene-reductases belong to the Old Yellow Enzyme (OYE) family of flavin-dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron-withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X-ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual-specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a ?-hairpin motif in an active site loop of both these enzymes were studied using stopped-flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the ?-hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15-fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55-fold) increase in efficiency in the reductive half-reaction, when compared to the essentially unreactive wild-type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs.
Project description:Glucuronides and mercapturates were examined as possible high-affinity substrates for a low-affinity ATP-dependent transport system for 2,4-dinitrophenyl S-glutathione (DNP-SG) in mouse L1210 cells. Initial inhibitor studies with inside-out vesicles revealed that the low-affinity transport of [3H]DNP-SG (Km 450 microM) exhibits a high sensitivity to N-acetyl 2,4-dinitrophenyl cysteine (NAc-DNP-Cys) (Ki 5.0 microM) and alpha-naphthyl beta-D-glucuronide (naphthyl glucuronide) (Ki 8.5 microM). Direct transport measurements showed the presence of ATP-dependent uptake activities for NAc-DNP-[35S]Cys and naphthyl [14C] glucuronide, and Km values for half-maximal transport were comparable to the Ki values of these compounds for inhibition of [3H]DNP-SG transport. Transport of [3H]DNP-SG, NAc-DNP-[35S]Cys and naphthyl [14C]glucuronide each showed the same sensitivity to various anions and anion conjugates. Inhibition was competitive and was most potent for bilirubin ditaurate, indoprofen, 4-biphenylacetic acid, 4-acridine 4 beta-D-glucuronide, N-acetyl leukotriene E4, 17 beta-oestradiol 3 beta-D-glucuronide and taurolithocholate 3-sulphate. Inside-out vesicles from human erythrocytes contain a comparable ATP-dependent transport system. These results show that NAc-DNP-Cys and naphthyl glucuronide are high-affinity substrates for a single system identified previously as a low-affinity transporter of DNP-SG. Substrate and inhibitor studies identify this system as a novel multispecific organic-anion transport system (MOAT4) that accommodates glucuronides and mercapturates and is distinct from other MOAT transporters. Human erythrocytes contain an additional ATP-dependent system for NAc-DNP-Cys (Km 33 microM) that does not transport monoglucuronides.
Project description:Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.
Project description:Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO(2). Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis.
Project description:Background:2,4-Dinitrophenol (2,4-DNP) is an effective but highly dangerous fat burner, not licensed for human consumption. Death cases reported for 2,4-DNP overdose, particularly among young adults, have raised concerns about the ineffective regulatory control, lack of education and risks associated with impurity, and the unknown concentration of 2,4-DNP purchased on the Internet. Methods:Using a sequential mixed method design and based on a hypothetical scenario as if 2,4-DNP was a licensed pharmaceutical drug, first we conducted a qualitative study to explore what product attributes people consider when buying a weight-loss aid. Focus group interviews with six females and three males (mean age?=?21.6?±?1.8?years) were audiorecorded, transcribed verbatim, and subjected to thematic analysis. Sixteen attributes were identified for the Best-Worst Scale (BWS) in the quantitative survey with 106 participants (64% female, mean age?=?27.1?±?11.9?years), focusing on 2,4-DNP. Demographics, weight satisfaction, and risk for eating disorder data were collected. Results:In contrast to experienced users such as bodybuilders, our study participants approached 2,4-DNP cautiously. Attributes of 2,4-DNP as a hypothetical weight-loss drug comprised a range of desirable and avoidable features. Of the 16 selected attributes, BWS suggested that long-term side effects were the most and branding was the least important attribute. Effectiveness and short-term side effects were also essential. Those in the?>25?year group showed least concerns for legality. Neutral BWS scores for cost, treatment, degree of lifestyle changes required, and specificity required for the hypothetical weight-loss drug to be effective were likely caused by disagreement about their importance among the participants, not indifference. Conclusion:With advances in research, 2,4-DNP as a pharmaceutical drug in the future for treating neurodegenerative diseases and potentially for weight loss is not inconceivable. Caution is warranted for interpreting the BWS scores. Owing to the difference in what data represent at individual vs. population levels, with pooled data, the method correctly identifies attributes by which most people are satisfied but misrepresents attributes that are individually very important but not universally agreed. Whilst this may be an advantage in marketing applications, it limits the utility of BWS as a research tool.
Project description:Structures of the iridoid synthase nepetalactol synthase in the presence of NAD(+) , NADPH or NAD(+) /10-oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 O? is positioned 2.5?Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4-C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5?-reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for ?-face hydride attack in progesterone 5?-reductases and is of general interest in the context of asymmetric synthesis.