Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for oxidizing nitrobenzene to 3-nitrocatechol, 4-nitrocatechol, and nitrohydroquinone

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 was found to oxidize nitrobenzene (NB) to form m-nitrophenol (m-NP, 72%) and p-NP (28%) with an initial rate of 0.098 and 0.031 nmol/(min mg protein), respectively. It was also discovered that wild-type ToMO forms 4-nitrocatechol (4-NC) from m-NP and p-NP with an initial rate of 0.15 and 0.0082 nmol/(min mg protein), respectively, and 3-NC (12%) and nitrohydroquinone (NHQ, 88%) from o-NP with an initial rate of 0.11 and 0.8 nmol/(min mg protein), respectively. To increase the oxidation rate and alter the oxidation regiospecificity of nitro aromatics as well as to study the role of the active site residues I100, Q141, T201, and F205 of the alpha hydroxylase fragment of ToMO (TouA), DNA shuffling and saturation mutagenesis were used to generate random mutants. The mutants were initially identified by screening via a rapid agar plate assay and then were further examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC). Several mutants with higher rates of activities and with different regiospecificities were identified; for example, Escherichia coli TG1 cells expressing either TouA mutant M180T/E284G or E214G/D312N/M399V produce 4-NC 4.5- and 20-fold faster than wild-type ToMO (0.037 and 0.16 nmol/min mg protein from p-NP, respectively). TouA mutant A107T/E214A had the regiospecificity of NB changed significantly from 28% to 79% p-NP. From 200 microM NB, TouA variants A101T/M114T, A110T/E392D, M180T/E284G, and E214G/D312N/M399V produce 4-NC whereas wild-type ToMO does not. From m-NP, TouA mutant I100Q produces 4-NC (37%) and NHQ (63%), whereas wild-type ToMO produces only 4-NC (100%). Variant A107T/E214A acts like a para enzyme and forms p-cresol as the major product (93%) from toluene with enhanced activity (2.3-fold), whereas wild-type ToMO forms 32%, 21%, and 47% of o-, m-, and p-cresol, respectively. Hence, the non-specific ToMO was converted into a regiospecific enzyme, which rivals toluene 4-monooxygenase of P. mendocina KR1 and toluene o-monooxygenase of Burkholderia cepacia G4 in its specificity.     

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for synthesizing 4-methylresorcinol, methylhydroquinone, and pyrogallol

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.     

Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42.

Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion-exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase2NT) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin NAP from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin2NT. Experiments with 18O2 showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase.     

Protein Engineering of Toluene-o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.     

Construction of chimeric catechol 2,3-dioxygenase exhibiting improved activity against the suicide inhibitor 4-methylcatechol

Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.     

Construction of Chimeric Catechol 2,3-Dioxygenase Exhibiting Improved Activity against the Suicide Inhibitor 4-Methylcatechol

Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-toluate because 4-methylcatechol, a metabolite of p-toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-toluate.     

Catechol 2,3-oxygenase production by genetically engineered Escherichia coli and its application to catechol determination

Catechol 2,3-oxygenase was produced by Escherichia coli, harbouring the recombinant plasmid pBH100 which contained the pheB gene cloned from phenol-degrading Pseudomonas putida BH, and was applied for the determination of catechol in the liquor. E. coli JM103 (pBH100) and C600 (pBH100) showed, respectively, about 5 and 8.5 times higher activities than that of P. putida BH. Using the crude extract prepared from the culture broth of the recombinant, catechol between 0.1 and 3.0 g/ml could be determined quantitatively in phosphate buffer, synthetic sewage and in mixtures of phenol, benzoate and sallcylate, and also in sodium pyruvate solution. In addition to catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol could be determined. Oxygenase activity of the crude extract was maintained completely during the 100-day storage at –20°C after being freeze-dried with 10% acelone.M. Fujita, M. Ike, Y. Kawagoshi and N. Shinohara are with the Department of Environmental Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565, Japan. T. Kamiya is with the Central Research Laboratory of Mitsubishi Electric Co., Amagasaki, Hyogo 661, Japan.     

Oxidation of 4-methylcatechol: implications for the oxidation of catecholamines

At alkaline pH, 4-methylcatechol oxidizes more rapidly than the related catecholamines: dopamine, norepinephrine, and epinephrine. This oxidation is not inhibited by superoxide dismutase or catalase, indicating that O2 itself is the oxidant, but the reduction potential of O2/O2-* is too low for it to oxidize 4-methylcatechol directly. Instead, O2 oxidizes the 4-methylcatechol semiquinone, which is formed by comproportionation of 4-methylcatechol and its o-quinone. Aniline reacts very quickly with the o-quinone and thus stops the comproportionation reaction that oxidizes 4-methylcatechol to the semiquinone. Oxidation of 4-methylcatechol then requires superoxide, and in the presence of aniline, oxidation of 4-methylcatechol by O2 is inhibited by superoxide dismutase. When catecholamines oxidize, the side chain amine inserts into the catechol o-quinone, forming a bicyclic compound. By eliminating the quinone, this ring closure prevents comproportionation and the consequent oxidation of catecholamines by O2. It also prevents reaction of the quinone with other compounds and the formation of potentially toxic products.     

Critical intra-linker interactions of HCN1-encoded pacemaker channels revealed by interchange of S3-S4 determinants

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels contribute to the spontaneous rhythmic activities in cardiac and neuronal cells. Recently, we reported that the S3-S4 linker of HCN1 channels influences activation, and that part of the linker is helical with the determinants G231, M232, and E235 clustered on one side. Here we explored the undefined role of the G(231)E(235)M(232) triplet by systematic substitutions. Replacing G231 or M232 next to the "neighboring" E235 in the S3-S4 helix with an anionic residue (i.e., G231E, M232E) rendered channels non-functional although they were localized on the membrane surface. Interestingly, this loss of function could be readily rescued either by introducing a countercharge at position 235 (G231E/E235R, M232E/E235R) or by interchanging residues 231 or 232 and 235 (G231E/E235G, M232E/E235M). We conclude that residues 231, 232, and 235 are in close spatial proximity to each other, and uniquely interact with one another to shape the phenotypes of HCN channels.     

The metabolism of cresols by species of Pseudomonas

1. A comparison of rates of oxidation of various compounds by whole cells indicated that protocatechuate was a reaction intermediate when a non-fluorescent species of Pseudomonas oxidized p-cresol. In contrast, a fluorescent Pseudomonas oxidized 3-methylcatechol and 4-methylcatechol when grown with p-cresol, but did not oxidize protocatechuate. 2. Heat-treated extracts of the fluorescent Pseudomonas oxidized catechol, 3-methylcatechol and 4-methylcatechol to ring-fission products, the spectroscopic properties of which were recorded. Rates of enzymic degradation of these products were also measured. 3. Acetic acid and formic acid were obtained by the action of a Sephadex-treated extract on 3-methylcatechol and 4-methylcatechol respectively. In each case 0.8mol. of the carboxylic acid was formed from 1.0mol. of substrate. 4. Dialysed extracts converted 3-methylcatechol into acetaldehyde and pyruvate, with 4-hydroxy-2-oxovalerate as a reaction intermediate. 4-Methylcatechol was converted first into 4-hydroxy-2-oxohexanoate and then into propionaldehyde and pyruvate. 5. The ring-fission product of catechol was formed from phenol by a fluorescent Pseudomonas, that of 3-methylcatechol was formed from o-cresol and m-cresol, and the ring-fission product of 4-methylcatechol was given from p-cresol. Propionate was readily oxidized by these cells after growth with p-cresol, but this compound was not attacked when phenol, o-cresol or m-cresol served as source of carbon. 6. Cell extracts appeared to attack only one enantiomer of synthetic 4-hydroxy-2-oxohexanoate.     

Naphthalene metabolism by pseudomonads: purification and properties of 1,2-dihydroxynaphthalene oxygenase.

1,2-Dihydroxynaphthalene oxygenase was purified from Pseudomonas putida NCIB 9816 grown on naphthalene as the sole source of carbon and energy. The enzyme had a subunit molecular weight of 19,000 and in a medium containing phosphate buffer, 1 mM mercaptoethanol, and 10% (vol/vol) ethanol had a native molecular weight greater than 275,000. The enzyme required Fe2+ for activity. It was inactivated slowly on standing, and inactivation was accelerated by dilution with aerated buffers and by H2O2. Bathophenanthroline sulfonate, o-phenanthroline, 8-hydroxyquinoline, and 2,2'-dipyridyl also inhibited the enzyme. The inactive enzyme was reactivated by anaerobic incubation with ferrous sulfate and ferrous ammonium sulfate. Thiol reagents and acetone, ethanol, or glycerol decreased the rate of loss of activity. The enzyme was most active with 1,2-dihydroxynaphthalene, for which the Km was 2.8 X 10(-4) M. 3-Methyl- and 4-methylcatechols were oxidized at 3 and 1.5%, respectively, of the rate of 1,2-dihydroxynaphthalene, and the Km for 3-methylcatechol was 1.5 X 10(-4) M. Purified 1,2-dihydroxynaphthalene oxygenase catalyzed the oxidation of 1,2-dihydroxynaphthalene, leading to the appearance of 2-hydroxychromene-2-carboxylic acid, but 3-methylcatechol was oxidized by this enzyme to 2-hydroxy-6-oxoheptadienoic acid. Thus, a product structurally analogous to 2-hydroxychromene-2-carboxylic acid was not observed when 3-methylcatechol was oxidized. This may indicate that 2-hydroxychromene-2-carboxylic acid results from cyclization of a ring fission product before release from the enzyme.     

AurF from Streptomyces thioluteus and a possible new family of manganese/iron oxygenases

We recently reported that the R2 subunit of class Ic ribonucleotide reductase from Chlamydia trachomatis contains a heterodinuclear Mn/Fe redox cofactor [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The N-oxygenase, AurF, from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate to p-nitrobenzoate and contains the EX2HX60-180EX2H sequence motif previously used to identify proteins with non-heme diiron clusters. Two research groups independently obtained evidence for the presence of iron and manganese in preparations of AurF. The electron paramagnetic resonance (EPR) spectrum of purified, resting AurF presented in one of these studies is markedly similar to the spectrum of the MnIII/FeIII form of C. trachomatis R2. We propose that S. thioluteus AurF also may harbor a heterodinuclear Mn/Fe cofactor, which it may use to activate O2 for oxidation of the aryl amine to the nitro compound. Hypothetical proteins encoded in the genomes of several other bacteria have similar sequences and may also be members of this nascent family of oxygen-activating Mn/Fe proteins.     

Active site mutations of recombinant deacetoxycephalosporin C synthase

Site-directed mutagenesis of active site residues of deacetoxycephalosporin C synthase active site residues was carried out to investigate their role in catalysis. The following mutations were made and their effects on the conversion of 2-oxoglutarate and the oxidation of penicillin N or G were assessed: M180F, G299N, G300N, Y302S, Y302F/G300A, Y302E, Y302H, and N304A. The Y302S, Y302E, and Y302H mutations reduced 2-oxoglutarate conversions and abolished (<2%) penicillin G oxidation. The Y302F/G300A mutation caused partial uncoupling of penicillin G oxidation from 2-oxoglutarate conversion, but did not uncouple penicillin N oxidation from 2-oxoglutarate conversion. Met-180 is involved in binding 2-oxoglutarate, and the M180F mutation caused uncoupling of 2-oxoglutarate from penicillin oxidation. The N304A mutation apparently enhanced in vitro conversion of penicillin N but had little effect on the oxidation of penicillin G, under standard assay conditions.     

Extradiol Cleavage of 3-Methylcatechol by Catechol 1,2-Dioxygenase from Various Microorganisms

The isofunctional enzymes of catechol 1,2-dioxygenase from species of Acinetobacter, Pseudomonas, Nocardia, Alcaligenes, and Corynebacterium oxidize 3-methylcatechol according to both the intradiol and extradiol cleavage patterns. However, the enzyme preparations from Brevibacterium and Arthrobacter have only the intradiol cleavage activity. Comparison of substrate specificity among these isofunctional dioxygenases shows striking differences in the oxidation of 3-methylcatechol, 4-methylcatechol and pyrogallol.     

In vitro oxidation of alpha-tocopherol (vitamin E) in human platelets upon incubation with unsaturated fatty acids, diamide and superoxide

Incubation of human blood platelets in vitro in Tyrode solution with unsaturated fatty acids, diamide or superoxide (generated in situ) resulted in the oxidation of tocopherol in the platelets. Arachidonate concentrations of (3-5).10(-4) M caused a 50% decrease in platelet alpha-tocopherol. The addition of saturated fatty acids or platelet-active substances such as ADP, dibutyryl cyclic AMP, and some prostaglandins, or peroxidizing agents such as hydrogen peroxide and tert-butylhydroperoxide to the incubation medium did not cause any change in platelet tocopherol content. During incubations of platelets with arachidonate, malonaldehyde as well as alpha-tocopherolquinone were produced. The latter was also produced during incubations with diamide or superoxide. The oxidation of tocopherol induced by unsaturated fatty acids may be one factor responsible for the well-known increase in dietary vitamin E requirements induced by polyunsaturated fatty acids. The oxidative consumption of tocopherol in the membranes could be expected to take place during localized release of oxidants such as superoxide and polyunsaturated fatty acids during normal biological function (e.g., phagocytosis) or pathological processes (e.g., ischemia). Tocopherol utilization is kept low probably by the regeneration of the compound by vitamin C and/or the preferential utilization of the other biological antioxidants.     

Kinetic characterization of the normal and detergent-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump. Rate-limiting step(s) under different conditions

We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and M?ller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.     

Biodegradation of 2-nitrotoluene by Pseudomonas sp. strain JS42.

A strain of Pseudomonas sp. was isolated from nitrobenzene-contaminated soil and groundwater on 2-nitrotoluene as the sole source of carbon, energy, and nitrogen. Bacterial cells growing on 2-nitrotoluene released nitrite into the growth medium. The isolate also grew on 3-methylcatechol, 4-methylcatechol, and catechol. 2-Nitrotoluene, 3-methylcatechol, and catechol stimulated oxygen consumption by intact cells regardless of the growth substrate. Crude extracts from the isolate contained catechol 2,3-dioxygenase and 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase activity. The results suggest that 2-nitrotoluene is subject to initial attack by a dioxygenase enzyme that forms 3-methylcatechol with concomitant release of nitrite. The 3-methylcatechol is subsequently degraded via the meta ring fission pathway.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. I. Biochemical, spectral, and kinetic characterization of surface mutants in subunit ii of Rhodobacter sphaeroides cytochrome aa(3)

To determine the interaction site for cytochrome c (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp(143), was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc(2), showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K(m) values, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp(143) severely inhibit activity without changing the K(m) for Cc or disturbing the adjacent Cu(A) center. From these data, we identify a Cc binding area on CcO with Trp(143) and Asp(214) close to the site of electron transfer and Glu(148), Glu(157), and Asp(195) providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem. 274, 38042-38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

M?ssbauer study of Clostridium pasteurianum hydrogenase II. Evidence for a novel three-iron cluster

Hydrogenase II contains two iron-sulfur clusters, one of the [4Fe-4S] type and one of unknown structure with unusual spectral properties (H-cluster). Using M?ssbauer spectroscopy we have studied the H-cluster under a variety of conditions. In the reduced state the cluster exhibits, in zero magnetic field, spectra with the typical 2:1 quadrupole pattern of reduced [3Fe-4S] clusters. However, whereas the latter are paramagnetic (S = 2) the H-cluster is diamagnetic (S = 0). Upon oxidation and exposure to CO the H-cluster exhibits an S = 1/2 EPR spectrum with g values at 2.03, 2.02, and 2.00. In this state, the M?ssbauer spectra reveal two cluster subsites with magnetic hyperfine coupling constants AI = +26.5 MHz and AII = -30 MHz. ENDOR data obtained by Hoffman and co-workers (Telser, J., Benecky, M. J., Adams, M. W. W., Mortenson, L. E., and Hoffman, B. M. (1986) J. Biol. Chem. 261, 13536-13541) show a 57Fe resonance at AIII approximately equal to 9.5 MHz. Analysis of the M?ssbauer spectra shows that this resonance represents one iron site. Our studies of the reduced and CO-bound oxidized states of hydrogenase II suggest that the H-cluster contains three iron atoms. The data obtained for the oxidized H-cluster suggest a novel type of 3-Fe cluster and bear little resemblance to those reported for oxidized [3Fe-4S] clusters with g = 2.01 EPR signals. In the reduced sample the [4Fe-4S]1+ cluster appears to occur in a mixture of two distinct electronic states.     

Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant human pseudocathepsin D

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).