Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E_mM for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of α-helix (56%) with lesser amounts of random coil (21%), β-turn (13%) and β-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% α-helix, 33.7% random coil and 10.5% β-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60°C for 5 min; thereafter by cooling at 1°C min⁻¹ to 45°C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60°C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330–350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.

Ammonium uptake by Chondrus crispus Stackhouse (Gigartinales,Rhodophyta) in culture

Cultivated Chondrus crispus was used in N-NH₄ uptake experiments in the laboratory. An elevation of temperature increased the apparent rate of uptake, especially up to 11 °C. Uptake in the dark was found to be 83 % of that in the light. The apparent uptake decreased with increasing internal N pool; rates were 26.5, 22.2 and 20.2 µg N g dry wt^–1 min^–1 for internal N pools of 2.7, 3.5 and 4.6%, respectively. Apparent uptake increased with the substrate N concentration. The resulting curve has two components: an active uptake and a diffusion component at high (> 5000 µg N L^–1) external N levels. Ks and V _max were calculated by deducting the diffusion component from the uptake curve: these were of 497 µg N L ^–1 and 14.4 µg N g dry wt^–1 min^–1. respectively, and reflect a low substrate affinity. This could be the result of 10 years of continuous culture of C. crispus. Uptake was similarly followed in the culture tanks and showed comparable results; nighttime would be the most appropriate time to supply nutrients.     

Dehalogenation of haloalkanes byRhodococcus erythropolis Y2

Phodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C₃ to C₆ 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C₇ to C₁₆ 1-haloalkanes andn-alkanes. The oxygenase-type activity dehalogenated C₄ to C₁₈ 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and ,-disubstituted haloalkanes and ,-substituted haloalcohols. In resting cell suspensions of hexadecane-grownR. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)^–1 towards 1-chlorotetradecane (3.67 mU mg^–1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)^–1 towards 1-chlorobutane.The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase     

Ecdysone 20-monooxygenase in a cricket,Gryllus bimaculatus (Ensifera,Gryllidae)—characterization of the microsomal midgut steroid hydroxylase in adult females

Summary Ecdysone 20-monooxygenase, the enzyme system which converts ecdysone into 20-hydroxyecdysone, was characterized in the midgut of 4-day-old female adult Gryllus bimaculatus using an in vitro radioassay. Differential centrifugation and sucrose gradient centrifugation revealed that ecdysone 20-monooxygenase activity is associated with the microsomal fractions. The 20-monooxygenase was found to be most active in potassium phosphate buffer, pH 7.8, at an osmolarity of 100 mOsm and at 39 °C assay temperature. The conversion of ecdysone into 20-hydroxyecdysone was linear over an incubation period of 12 min and with respect to a protein concentration of 3 mg·ml^–1. K⁺ and Na⁺ (10^–3–10^–1 M), Ca²⁺ (2.3 mM), and EDTA (1–5 mM) did not affect monooxygenase activity, whereas Mg²⁺ (2.3–10 mM) slightly inhibited enzyme activity. The enzyme complex has an apparent K_m for ecdysone of 3.7·10^–7 M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent K_i of 4·10^–6 M. The cytochrome P-450 nature of the steroid hydroxylase was shown by its obligate requirement for NADPH and its inhibition by carbon monoxide, metyrapone, and p-chloromercuribenzoate, but not by cyanide. The insect systemic growth disruptor, azadirachtin, was found to inhibit ecdysone 20-monooxygenase activity with a I₅₀ of 8·10^–4 M. From the CO-difference spectrum, a cytochrome P-450 content of 285 pmol·mg protein^–1 was calculated for midgut microsomes of 4-day-old females.Abbreviations GO carbon monoxide - EDTA ethylenediamine tetraacetic acid - HPLC high performance liquid chromatography - I ₅₀ concentration for 50% inhibition - KCN potassium cyanide - K ₁ inhibition constant - K _m Michaelis-Menten constant - MOPS 3-morpholinopropanesulfonic acid - NADH/NAD ⁺ nicotinamide adenine dinucleotide reduced/oxidized - NADPH/NADP ⁺ nicotinamide adenine dinucleotide phosphate reduced/oxidized - Na ₂ S ₂ O ₄ sodium dithionite - SEM Standard error of mean - TLC thin-layer chromatography - TRIS 2-amino 2-hydroxymethyl-1,3-propanediol (trishydroxymethyl aminomethane) - V _max maximal reaction velocity     

Physiological and Biochemical Analysis of the Transformants of Aerobic Methylobacteria Expressing the dcmA Gene of Dichloromethane Dehalogenase

Transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr^–/pME 8220 and DM4-2cr^–/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl^– ions, CH₂O, or HCOOH, nor to the impairment of glutathione synthesis or to the disturbance of intracellular pH homeostasis. The (exo^–) Klenow fragment–mediated incorporation of [-³²P]dATP into the DNA of the transformants DM4-2cr^–/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH₂Cl₂ than when they were incubated with CH₃OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH₂Cl₂ showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH₂Cl₂ degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.     

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F₄₃₀ as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO₂ (apparent V_max at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N ⁵,N¹⁰-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming), 33 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin reductase (coenzyme F₄₂₀ dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F₄₂₀-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K_m values for these enzymes and the effect of salts on their activities were determined.The coenzyme F₄₂₀ present in M. kandleri was identified as coenzyme F₄₂₀-2 with 2 -glutamyl residues.Abbreviations H–S-CoM coenzyme M - CH₃–S-CoM methylcoenzyme M - H–S-HTP 7-mercaptoheptanoylthreonine phosphate - MFR methanofuran - CHO-MFR formyl-MFR - H₄MPT tetrahydromethanopterin - CHO–H₄MPT N ⁵-formyl-H₄MPT - CH=H₄MPT⁺ N ⁵,N¹⁰-methenyl-H₄MPT - CH₂=H₄MPT N ⁵,N¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - 1 U= 1 mol/min     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Isolation of <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 °C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K_m and V_max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml^–1 and 85 mmol min^–1 mg^–1, respectively, and for chondroitin sulfate C, 0.5 mg ml^–1 and 103 mmol min^–1 mg^–1, respectively.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence     

Dual pathways of glycerol assimilation in Klebsiella aerogenes NCIB 418

Klebsiella aerogenes NCIB 418 assimilates glycerol via alternative pathways: one involves a glycerol kinase with a high affinity for glycerol (apparent K _m=1–2×10^–6 M), and the second a glycerol dehydrogenase with a much lower affinity for its substrate (apparent K _m=2–4×10^–2 M).In variously-limited chemostat cultures, one or the other pathway predominated. Thus, aerobic carbonlimited organisms contained only the glycerol kinase pathway whereas aerobic sulphate-limited or ammonia-limited organisms (grown on glycerol) used only the glycerol dehydrogenase pathway. Anaerobic cultures invariably contained glycerol dehydrogenase, and glycerol kinase was absent.Washed suspensions of aerobically-grown organisms oxidized glycerol with kinetics similar to that of the particular enzyme (the primary enzyme of the assimilatory pathway) which they possessed, thus indicating a close association between these two enzymes and the uptake process. But a supply of exogenous glycerol was not a prerequisite for the synthesis of either glycerol kinase or glycerol dehydrogenase, and nor was molecular oxygen the key factor in effecting modulation between the alternative pathways of glycerol metabolism, as had been previously suggested.The physiological significance of dual pathways of glycerol assimilation is discussed.     

Kinetics of Microbial Dehalogenation of Haloaromatic Substrates in Methanogenic Environments

The kinetic parameters associated with the microbial dehalogenation of 3-chlorobenzoate, 3,5-dichlorobenzoate, and 4-amino-3,5-dichlorobenzoate were measured in anoxic sediment slurries and in an enriched methanogenic culture grown on 3-chlorobenzoate. The initial dehalogenation of the substrates exhibited Michaelis-Menten kinetics. The apparent K_m values for the above substrates ranged from 30 to 67 μM. The pattern of degradation, however, was unusual. The enrichment culture accumulated partially dehalogenated intermediates to 72 and 98% of that possible when incubated with either 3,5-dichloro- or 4-amino-3,5-dichlorobenzoate, respectively, but did not accumulate significant amounts of benzoate when 3-chlorobenzoate was the sole carbon and energy source. The accumulated intermediates were rapidly metabolized only after the parent substrate concentrations were nearly depleted (<5 μM). A sequential Michaelis-Menten model was developed to account for the observed pattern of biodegradation. Using this model, we found that relative differences in the K_m and V_max parameters for substrate and intermediate dehalogenations alone were insufficient to explain the transitory accumulation of intermediates. However, by inserting a competitive inhibition term, with the primary substrate as the inhibitor, the observed pattern of degradation was simulated. Apparently, the dichlorinated substrates competitively inhibit the dehalogenation of the monochlorinated substrates. Similar kinetic patterns were noted for sediments, although the rates were slower than in the enrichment culture.     

Differential Photosynthetic Acclimation Pattern to Limiting Growth-Irradiance in Two Types of C4 Plants

Photosynthetic acclimation to reduced growth irradiances (650 and 200 µmol m^–2 s^–1) in Eleusine coracana (L.) Garten, a nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) C₄ species and Gomphrena globosa L., a nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) C₄ species were investigated. E. coracana plants acclimated in 4 and 8 d to 650 and 200 µmol m^–2 s^–1, respectively, whereas G. globosa plants took 8 and 10 d, respectively, to acclimate to the same irradiances. The acclimation to reduced irradiance was achieved in both species by greater partitioning of chlorophyll towards the light-harvesting antennae at the expense of functional components. However, magnitude of increase in the light-harvesting antenna was higher in E. coracana as compared to G. globosa. Superior photosynthetic acclimation to reduced irradiance in G. globosa was due to the smaller change in functions of the cytochrome b ₆/f complex, photosystem (PS) 1 and PS2 leading to the higher carbon fixation rates compared to E. coracana.     

Nitrogen and phosphorus uptake by the Brazilian kelp Laminaria abyssalis (Phaeophyta) in culture

The uptake of ammonium, nitrate and phosphate by laboratory-grown young sporophytes of Laminaria abyssalis was measured in a perturbed system (batch mode) at 18 °C and 35 ± 5 µE m^–2 s^–1 photon flux density. Uptake of all appeared to follow saturation-type nutrient uptake kinetics. The NO _inf3 ^sup– (K _s = 14.0 µM, V _max = 5.0 µmol h^–1 g^–1 dry wt) and NH _inf4 ^sup+ (K _s = 4.6 µM, V _max= 2.0 µmol h^–1 g^–1 dry wt) were taken up simultaneously, although NH _inf4 ^sup+ was taken up more rapidly. Values of K ₃ and V _max for phosphate were, respectively, 2.21 µM and 0.83 µmol h^–1 g^–1 dry wt. Nitrate and phosphate were both consumed in similar rates (V _max /Ks 0.37) at low concentrations. NH _inf4 ^sup+ , thus, might be a more efficient form of N fertilizer if artificial enrichment of seawater is used.     

Coenzyme F420 dependent N5, N10-methylenetetrahydromethanopterin dehydrogenase in methanol grown Methanosarcina barkeri

The dehydrogenation of N ⁵,N ¹⁰-methylenetetrahydromethanopterin (CH₂=H₄MPT) to N ⁵,N ¹⁰-methenyltetrahydromethanopterin (CH≡H₄MPT⁺) is an intermediate step in the oxidation of methanol to CO₂ in Methanosarcina barkeri. The reaction is catalyzed by CH₂=H₄MPT dehydrogenase, which was found to be specific for coenzyme F₄₂₀ as electron acceptor; neither NAD, NADP nor viologen dyes could substitute for the 5-deazaflavin. The dehydrogenase was anaerobically purified almost 90-fold to apparent homogeneity in a 32% yield by anion exchange chromatography on DEAE Sepharose and Mono Q HR, and by affinity chromatography on Blue Sepharose. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band with an apparent mass of 31 kDa. The apparent molecular mass of the native enzyme determined by polyacrylamide gradient gel electrophoresis was 240 kDa. The ultraviolet/visible spectrum of the purified enzyme was almost identical to that of albumin suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentrations were linear: the apparent K _m for CH₂=H₄MPT and for coenzyme F₄₂₀ were found to be 6 μM and 25 μM, respectively. V_max was 4,000 μmol min^-1·mg^-1 protein (k_cat=2,066 s^-1) at pH 6 (the pH optimum) and 37°C. The Arrhenius activation energy was 40 kJ/mol. The N-terminal amino acid sequence was found to be 50% identical with that of the F₄₂₀-dependent CH₂=H₄MPT dehydrogenase isolated from H₂/CO₂ grown Methanobacterium thermoautotrophicum.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification

Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O₂ with H₂ at a maximal specific rate of 0.4 U (mol/min) per mg protein with an apparent K _m for O₂ of 30 M. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F₄₂₀-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F₄₂₀H₂ with 1 O₂ to 2 F₄₂₀ and 2 H₂O. The apparent K _m for F₄₂₀ was 30 M and that for O₂ was 2 M with a V _max of 240 U/mg at 37°C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H₂O₂ as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H–X–E–X–D–X₆₂–H–X₁₈–D–X₆₀–H. Sequence comparisons revealed that the F₄₂₀H₂ oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30–40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F₄₂₀H₂ oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F₄₂₀H₂ oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.Dedicated to Hans Schlegel on the occasion of his 80th birthday.     

N 5-Methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein

An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N ⁵-methyltetrahydromethanopterin (CH₃–H₄MPT) to coenzyme M (H–S–CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H₂/CO₂-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH₃–S–CoM formation from CH₃–H₄MPT (apparent K _m=50 M) and H–S–CoM (apparent K _m=250 M) of approximately 0.6 mol·min^-1·mg protein^-1. For activity the presence of Ti(III) citrate (apparent K _m=15 M) and of ATP (apparent K _m=30 M) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH₃–H₄MPT in the absence of H–S–CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH₃–H₄MPT to H–S–CoM in the absence of ATP and Ti(III). Demethylation in the presence of H–S–CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.Abbreviations H₄MPT tetrahydromethanopterin - CH₃–H₄MPT N ⁵-methyl-H₄MPT - H–S–CoM 2-mercaptoethanesulfonate or coenzyme M - CH₃–S–CoM 2(methylthio)ethanesulfonate or methylcoenzyme M - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - DTT dithiothreitol - MOPS morpholinopropanesulfonate - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate - 1 U = 1 mol/min     

Nitrite inhibition of nitrogenase from soybean bacteroids

Nitrogenase from soybean bacteroids was purified and used to study NO ₂ ^– effects either as unfractionated enzyme or as reconstituted enzyme from separated nitrogenase components I and II. Partially purified enzyme was strongly inhibited by nitrite at concentrations less than 0.1 mM. This inhibition was typically referred to as competitive with an inhibition constant (K _i) for NO ₂ ^– which was 5.2 mM. Kinetics studies showed an abnormally low apparent constant of association between enzyme and NO ₂ ^– (k _a=60 M^-1·s^-1). Nitrite appeared to bind to the MoFe protein, without any effect on Fe component, giving a completely reversible inhibition. Nitrite was found not to be an alternative substrate for nitrogenase.Abbreviations TES N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid - PPG Polypropylene glycol