Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH(2))-utilizing monooxygenase, and FADH(2) was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH(2) and retarded its rapid oxidation by O(2). A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH(2)-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.     

Biodegradation of 2,4,6-tribromophenol by Ochrobactrum sp. strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 microM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Anaerobic/aerobic conditions and biostimulation for enhanced chlorophenols degradation in biocathode microbial fuel cells

Anaerobic/aerobic conditions affected bacterial community composition and the subsequent chlorophenols (CPs) degradation in biocathode microbial fuel cells (MFCs). Bacterial communities acclimated with either 4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP) under anaerobiosis can degrade the respective substrates more efficiently than the facultative aerobic bacterial communities. The anaerobic bacterial communities well developed with 2,4-DCP were then adapted to 2,4,6-trichlorophenol (2,4,6-TCP) and successfully stimulated for enhanced 2,4,6-TCP degradation and power generation. A 2,4,6-TCP degradation rate of 0.10 mol/m³/d and a maximum power density of 2.6 W/m³ (11.7 A/m³) were achieved, 138 and 13 % improvements, respectively compared to the controls with no stimulation. Bacterial communities developed with the specific CPs under anaerobic/aerobic conditions as well as the stimulated biofilm shared some dominant genera and also exhibited great differences. These results provide the most convincing evidence to date that anaerobic/aerobic conditions affected CPs degradation with power generation from the biocathode systems, and using deliberate substrates can stimulate the microbial consortia and be potentially feasible for the selection of an appropriate microbial community for the target substrate (e.g. 2,4,6-TCP) degradation in the biocathode MFCs.     

Specificity of reductive dehalogenation of substituted ortho-chlorophenols by Desulfitobacterium dehalogenans JW/IU-DC1.

Resting cells of Desulfitobacterium dehalogenans JW/IU-DC1 growth with pyruvate and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor and inducer of dehalogenation reductively ortho-dehalogenate pentachlorophenol (PCP); tetrachlorophenols (TeCPs); the trichlorophenols 2,3,4-TCP, 2,3,6-TCP, and 2,4,6-TCP; the dichlorophenols 2,3-DCP, 2,4-DCP, and 2,6-DCP; 2,6-dichloro-4-R-phenols (2,6-DCl-4-RPs, where R is -H, -F, -Cl, -NO2, -CO2, or -COOCH3; 2-chloro-4-R-phenols (2-Cl-4-RPs, where R is -H, -F, -Cl, -Br, -NO2, -CO2-, -CH2CO2, or -COOCH3); and the bromophenols 2-BrP, 2,6-DBrP, and 2-Br-4ClP [corrected]. Monochlorophenols, the dichlorophenols 2,5-DCP, 3,4-DCP, and 3,5-DCP, the trichlorophenols 2,3,5-TCP, 2,4,5-TCP, and 3,4,5-TCP, and the fluorinated analog of 3-Cl-4-OHPA, 3-F-4-OHPA ("2-F-4-CH2CO2- P"), are not dehalogenated. A chlorine substituent in position 3 (meta), 4 (para), or 6 (second ortho) of the phenolic moiety facilitates ortho dehalogenation in position 2. Chlorine in the 5 (second meta) position has a negative effect on the dehalogenation rate or even prevents dechlorination in the 2 position. In general, 2,6-DCl-4-RPs are dechlorinated faster than the corresponding 2-Cl-4-RPs with the same substituent R in the 4 position. The highest dechlorination rate, however, was found for dechlorination of 2,3-DCP, with a maximal observed first-order rate constant of 19.4 h-1 g (dry weight) of biomass-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriavidus necator JMP134

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH₂)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH₂, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpX_H) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX_H had no activity toward NADPH or riboflavin. Coupling of TcpX_H and TcpA demonstrated that TcpX_H provided FADH₂ for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX_H in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.     

Reaction of the soil microflora after contamination with chlorinated aromatic compounds and HCH

Abstract The effect of the pollution of an industrial land site with chlorinated benzenes, chlorinated phenols, hexachlorocyclohexane-isomers (HCH) on the soil microflora was investigated. Cell counts (microscopic and by plate count) as well as respiration rated did not correlate negatively with the concentration of the contaminants. Soil microorganisms grew in the presence of up to 750 μmol 1⁻¹ pf chlorinated compounds in liquid culture. Only 150 μmol l⁻¹ 2,4,5-trichlorophenol (2,4,5-TCP) inhibited growth totally. In enrichment cultures, bacteria used α- and γ-HCH, 3-chlorophenol (3-CP), 2,3-dichlorophenol (2,3-DCP), 2,6-DCP, 2,4,5-TCP, and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB) as a sole source of carbon and energy under aerobic conditions. No growth was observed with β-HCH. Under anaerobic conditions no growth was observed with any of the substances tested     

Anaerobic transformation and toxicity of trichlorophenols in a stable enrichment culture.

The transformation and toxicity of trichlorophenols (TCPs) were studied with a methanogenic enrichment culture derived from sewage sludge. Transformation of TCPs rapidly resumed after heating of the culture at *) degrees C for 1 h, suggesting that the dechlorinating bacteria are spore-forming anaerobes. 2,4,6-TCP was rapidly dechlorinated via 2,4-dichlorophenol to 4-chlorophenol. During the transformation of 2,4,6-TCP, the most probable number of dechlorinating bacteria increased by 4 orders of magnitude. The most extensive dechlorination was observed in media with complex carbon sources such as yeast extract, peptone, and Casamino Acids, but glucose, galactose, and lactose were also used by the consortium. Experiments using chloramphenicol indicated that the reductive dechlorination of 2,4,6-TCP was regulated by an inducible enzyme system. The highest initial concentration at which dechlorination of 2,4,6-TCP was observed was 400 microM. 2,4,5-TCP and 3,4,5-TCP were dechlorinated to, respectively, 3,4-dichlorophenol and 3-chlorophenol at initial concentrations of less than or equal to 40 microM. Toxicity for the acid-producing and methanogenic bacteria in the consortium was a function of chemical structure, as the inhibition of these activities increased from 2,4,6-TCP, via 2,4,5-TCP, to 3,4,5,-TCP.     

Anaerobic transformation and toxicity of trichlorophenols in a stable enrichment culture.

The transformation and toxicity of trichlorophenols (TCPs) were studied with a methanogenic enrichment culture derived from sewage sludge. Transformation of TCPs rapidly resumed after heating of the culture at *) degrees C for 1 h, suggesting that the dechlorinating bacteria are spore-forming anaerobes. 2,4,6-TCP was rapidly dechlorinated via 2,4-dichlorophenol to 4-chlorophenol. During the transformation of 2,4,6-TCP, the most probable number of dechlorinating bacteria increased by 4 orders of magnitude. The most extensive dechlorination was observed in media with complex carbon sources such as yeast extract, peptone, and Casamino Acids, but glucose, galactose, and lactose were also used by the consortium. Experiments using chloramphenicol indicated that the reductive dechlorination of 2,4,6-TCP was regulated by an inducible enzyme system. The highest initial concentration at which dechlorination of 2,4,6-TCP was observed was 400 microM. 2,4,5-TCP and 3,4,5-TCP were dechlorinated to, respectively, 3,4-dichlorophenol and 3-chlorophenol at initial concentrations of less than or equal to 40 microM. Toxicity for the acid-producing and methanogenic bacteria in the consortium was a function of chemical structure, as the inhibition of these activities increased from 2,4,6-TCP, via 2,4,5-TCP, to 3,4,5,-TCP.     

A monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions

Ralstonia eutropha JMP134 2,4,6-trichlorophenol (2,4,6-TCP) 4-monooxygenase catalyzes sequential dechlorinations of 2,4,6-TCP to 6-chlorohydroxyquinol. Although 2,6-dichlorohydroxyquinol is a logical metabolic intermediate, the enzyme hardly uses it as a substrate, implying it may not be a true intermediate. Evidence is provided to support the proposition that the monooxygenase oxidized 2,4,6-TCP to 2,6-dichloroquinone that remained with the enzyme and got hydrolyzed to 2-chlorohydroxyquinone, which was chemically reduced by ascorbate and NADH to 6-chlorohydroxyquinol. When the monooxygenase oxidized 2,6-dichlorophenol, the product was 2,6-dichloroquinol, which was not further converted to 6-chlorohydroxyquinol, implying that the enzyme only converts 2,6-dichloroquinone to 6-chlorohydroxyquinol. Stoichiometric analysis indicated the consumption of one O2 molecule per 2,4,6-TCP converted to 6-chlorohydroxyquinol, ruling out the possibility of two oxidative reactions. Experiments with 18O-labeling gave direct evidence for the incorporation of oxygen from both O2 and H2O into the produced 6-chlorohydroxyquinol. A monooxygenase that catalyzes hydroxylation by both oxidative and hydrolytic reactions has not been reported to date. The ability of the enzyme to perform two types of reactions is not due to the presence of a second functional domain but rather is due to catalytic promiscuity, as a homologous monooxygenase converts 2,4,6-TCP to only 2,6-dichloroquinol. Employing both conventional catalysis and catalytic promiscuity of a single enzyme in two consecutive steps of a metabolic pathway has been unknown previously.     

厌氧微生物菌群XH-1对2,4,6-三氯苯酚的降解特性

有机污染物2,4,6-三氯苯酚(2,4,6-TCP)普遍存在于地下水和河流底泥等厌氧环境中。为了探究厌氧微生物菌群XH-1对2,4,6-TCP的降解能力,本研究以2,4,6-TCP为底物,接种XH-1建立微宇宙培养体系,并以中间产物4-氯苯酚(4-CP)和苯酚为底物分别进行分段富集培养,利用高效液相色谱分析底物的降解转化,同时基于16S rRNA基因高通量测序分析微生物群落结构变化。结果表明: 2,4,6-TCP(122 μmol·L^-1)以0.15 μmol·d^-1的速率在80 d内被完全降解转化,降解中间产物分别为2,4-二氯苯酚(2,4-DCP)、4-氯苯酚和苯酚,所有中间产物最终在325 d被完全降解。高通量测序结果表明,脱卤杆菌和脱卤球菌可能驱动2,4,6-TCP还原脱氯,其中,脱卤球菌可能在4-CP的脱氯转化中发挥重要作用,并与丁酸互营菌和产甲烷菌联合作用彻底降解2,4,6-TCP。     

Environmental significance of O-demethylation of chloroanisoles by soil bacterial isolates as a mechanism that improves the overall biodegradation of chlorophenols

The biodegradation rate of chlorophenols in the environment seems to be limited by a competitive mechanism of O-methylation which produces chloroanisoles with a high potential of being bioconcentrated in living organisms. In this work we report for the first time the isolation of three soil bacterial strains able to efficiently degrade 2,4,6-trichloroanisole (2,4,6-TCA). These strains were identified as Xanthomonas retroflexus INBB4, Pseudomonas putida INBP1 and Acinetobacter radioresistens INBS1. In these isolates 2,4,6-TCA was efficiently metabolized in a minimal medium containing methanol and 2,4,6-TCA as the only carbon sources, with a concomitant release of 3 mol of chloride ion from 1 mol of 2,4,6-TCA, indicating complete dehalogenation of 2,4,6-TCA. 2,4,6-trichlorophenol (2,4,6-TCP) was identified as a degradative intermediate, indicating that 2,4,6-TCA underwent O-demethylation as the first step in the biodegradation process. 2,4,6-TCP was further transformed into 2,6-dichloro-para-hydroquinone (2,6-DCHQ) and subsequently mineralized. The degradation of chloroanisoles could improve the overall biodegradation of chlorophenols in the environment, because those chlorophenols previously biomethylated might also be later biodegraded. Xanthomonas retroflexus INBB4 has two O-demethylation systems: one is an oxygenase-type demethylase, and the other is a tetrahydrofolate (THF)-dependent O-demethylase. On the contrary O-demethylation of 2,4,6-TCA in P. putida INBP1 is just catalysed by an oxygenase-type NADH/NADPH-dependent O-demethylase, whereas in A. radioresistens INBS1 a THF-dependent O-demethylase activity was detected.     

Biodegradation of 2,4,6-Tribromophenol by Ochrobactrum sp. Strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 μM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1.

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.     

Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols.

Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP. These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP. Of these strains, P. pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs). Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol. The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.     

Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols.

Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP. These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP. Of these strains, P. pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs). Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol. The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.     

Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme

Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors.     

Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100.

Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH-dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself.     

Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --> FAD --> [2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.     

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.