Delineation of the caffeine C-8 oxidation pathway in Pseudomonas sp. strain CBB1 via characterization of a new trimethyluric acid monooxygenase and genes involved in trimethyluric acid metabolism

The molecular basis of the ability of bacteria to live on caffeine via the C-8 oxidation pathway is unknown. The first step of this pathway, caffeine to trimethyluric acid (TMU), has been attributed to poorly characterized caffeine oxidases and a novel quinone-dependent caffeine dehydrogenase. Here, we report the detailed characterization of the second enzyme, a novel NADH-dependent trimethyluric acid monooxygenase (TmuM), a flavoprotein that catalyzes the conversion of TMU to 1,3,7-trimethyl-5-hydroxyisourate (TM-HIU). This product spontaneously decomposes to racemic 3,6,8-trimethylallantoin (TMA). TmuM prefers trimethyluric acids and, to a lesser extent, dimethyluric acids as substrates, but it exhibits no activity on uric acid. Homology models of TmuM against uric acid oxidase HpxO (which catalyzes uric acid to 5-hydroxyisourate) reveal a much bigger and hydrophobic cavity to accommodate the larger substrates. Genes involved in the caffeine C-8 oxidation pathway are located in a 25.2-kb genomic DNA fragment of CBB1, including cdhABC (coding for caffeine dehydrogenase) and tmuM (coding for TmuM). Comparison of this gene cluster to the uric acid-metabolizing gene cluster and pathway of Klebsiella pneumoniae revealed two major open reading frames coding for the conversion of TM-HIU to S-(+)-trimethylallantoin [S-(+)-TMA]. The first one, designated tmuH, codes for a putative TM-HIU hydrolase, which catalyzes the conversion of TM-HIU to 3,6,8-trimethyl-2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (TM-OHCU). The second one, designated tmuD, codes for a putative TM-OHCU decarboxylase which catalyzes the conversion of TM-OHCU to S-(+)-TMA. Based on a combination of enzymology and gene-analysis, a new degradative pathway for caffeine has been proposed via TMU, TM-HIU, TM-OHCU to S-(+)-TMA.     

A novel EDTA-degrading Pseudomonas sp.

A bacterial strain LPM-410 capable of utilizing ethylenediaminetetraacetate (EDTA) as the sole source of energy, carbon, and nitrogen was isolated from sewage sludge and identified as a Pseudomonas sp. on the basis of its phenotypic characteristics. Suspensions of exponential-phase cells degraded EDTA, Mg–, Ca–, Ba–, and Mn–EDTA at constant specific rates ranging from 0.363 to 0.525 mmol EDTA/(g cells h). The more stable chelate, Zn–EDTA, was degraded at a lower rate (0.195 ± 0.030 mmol EDTA/(g cells h)), and here was no degradation of Co–, Cu–, Pb–, and Fe(III)–EDTA.     

Degradation of and sensitivity to cholate in Pseudomonas sp. strain Chol1

A facultative anaerobic bacterium, Pseudomonas sp. strain Chol1, degrading cholate and other bile acids was isolated from soil. We investigated how strain Chol1 grew with cholate and whether growth was affected by the toxicity of this compound. Under anoxic conditions with nitrate as electron acceptor, strain Chol1 grew by transformation of cholate to 7,12-dihydroxy-1,4-androstadiene-3,17-dione (DHADD) as end product. Under oxic conditions, strain Chol1 grew by transformation of cholate to 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (THSATD), which accumulated in the culture supernatant before its further oxidation to CO₂. Strain Chol1 converted DHADD into THSATD by an oxygenase-dependent reaction. Addition of cholate (≥10 mM) to cell suspensions of strain Chol1 caused a decrease of optical density and viable counts but aerobic growth with these toxic cholate concentrations was possible. Addition of CCCP or EDTA strongly increased the sensitivity of the cells to 10 mM cholate. EDTA also increased the sensitivity of the cells to DHADD and THSATD (≤1.7 mM). The toxicity of cholate and its degradation intermediates with a steroid structure indicates that strain Chol1 requires a strategy to minimize these toxic effects during growth with cholate. Apparently, the proton motive force and the outer membrane are necessary for protection against these toxic effects.     

A novel inducible formaldehyde dehydrogenase ofPseudomonas sp. (RJ1)

A novel, pyridine-nucleotide-inducible formaldehyde dehydrogenase activity was detected in cells ofPseudomonas sp. (RJ) propagated on methylamine and oxalate. The pH optimum of the dehydrogenase was 7.0. Dichlorophenol-indophenol or potassium ferricyanide served as an electron acceptor. The rate of reduction of these electron acceptors was shown to be stimulated by phenazine methosulfate. The dehydrogenase was inhibited by parahydroxymercuric benzoate and iodoacetamide. This inhibition suggests that the enzyme contains sulfhydryl groups. The stoichiometry of the reaction in terms of oxygen uptake to formate formation was 0.5, which agrees with the theoretical value.     

Biphenyl uptake by psychrotolerant Pseudomonas sp. strain Cam-1 and mesophilic Burkholderia sp. strain LB400

We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400.     

Monooxygenase-mediated 1,2-dichloroethane degradation by Pseudomonas sp. strain DCA1.

A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a Km value below the detection limit of 0.5 microM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.     

Constitutive NADP-dependent alcohol dehydrogenase ofAcinetobacter sp. strain HO1-N

An NADP-dependent alcohol dehydrogenase was purified to homogeneity fromAcinetobacter sp. strain HO1-N. The enzyme appears to be a tetramer of sub-unit M_r 40,600, and it has kinetic and other properties almost identical to those of an enzyme previously isolated fromAcinetobacter calcoaceticus strain NCIB 8250. The alcohol dehydrogenases from both of these strains ofAcinetobacter oxidized primary alcohols. The highestk _cat(app) values were with alcohols containing from four to eight carbon atoms; there was activity up to tetradecan-l-ol, although it was a poor substrate, but there was not measurable activity with hexadecan-l-ol. The highest specificity constant was found with hexan-l-ol as substrate when the messurements were made in the absence of dioxan, and with decan-l-ol as substrate when assayed in the presence of dioxan. It seems unlikely that this enzyme is involved in the metabolism of wax esters or of long-chain alkanes.     

A new NAD+-dependent opine dehydrogenase from Arthrobacter sp. strain 1C.

A new NAD+-dependent opine dehydrogenase was purified to homogeneity from Arthrobacter sp. strain 1C isolated from soil by an enrichment culture technique. The enzyme has a molecular weight of about 70,000 and consists of two identical subunits with molecular weights of about 36,000. The enzyme catalyzed a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids. In the oxidative deamination reaction, the enzyme was active toward unusual opines, such as N-[1-R-(carboxyl)ethyl]-S-methionine and N-[1-R-(carboxyl)ethyl]-S-phenylalanine. In the reductive secondary amine-forming reaction with NADH as a cofactor, the enzyme utilized L-amino acids such as L-methionine, L-isoleucine, L-valine, L-phenylalanine, L-leucine, L-alanine, and L-threonine as amino donors and alpha-keto acids such as pyruvate, oxaloacetate, glyoxylate, and alpha-ketobutyrate as amino acceptors. The product enzymatically synthesized from L-phenylalanine and pyruvate in the presence of NADH was identified as N-[1-R-(carboxyl)ethyl]-S-phenylalanine.     

Toxicity of chlorobenzene on Pseudomonas sp. strain RHO1, a chlorobenzene-degrading strain

Pseudomonas sp. strain RHO1 able to use chloro- and 1,4-dichlorobenzene as growth substrates was tested towards sensitivity against chlorobenzene. Concentrations of chlorobenzene higher than 3.5 mM were found to be toxic to cells independent of pregrowth with chlorobenzene or nutrient broth. Below this concentration, sensitivity towards chlorobenzene depended on the precultivation of the cells, i.e. type of growth substrate (chlorobenzene or nutrient broth) and the concentration of chlorobenzene as the growth substrate. Cells grown in continuous culture were especially sensitive with a threshold concentration of 2.5 mM chlorobenzene. In addition to chlorobenzene, metabolites also seem to function as toxic compounds. 2-Chlorophenol and 3-chlorocatechol were isolated from cell extracts. Cleavage of 3-chlorocatechol by catechol 1,2-dioxygenase seems to be the critical step in the metabolism of chlorobenzene.     

Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp. strain AT3.

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy. Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid. Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable. The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography. It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation. The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step. By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions. Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.     

NAD-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. I. Amino acid sequence

The primary structure of NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 is determined. The enzyme is composed of two identical subunits, each comprising 393 amino acid residues, and has a molecular weight of 43.1 kD. To elucidate the protein's amino acid sequence, four types of digestion were used: cyanogen bromide cleavage at methionine residues, endoproteinase Lys-C digestion at lysine residues, endoproteinase Glu-C cleavage at glutamic acid residues, and tryptic digestion. The peptides obtained were purified to homogeneity and characterized.     

Metabolism of glyphosate in Pseudomonas sp. strain LBr

Metabolism of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. strain LBr, a bacterium isolated from a glyphosate process waste stream, was examined by a combination of solid-state 13C nuclear magnetic resonance experiments and analysis of the phosphonate composition of the growth medium. Pseudomonas sp. strain LBr was capable of eliminating 20 mM glyphosate from the growth medium, an amount approximately 20-fold greater than that reported for any other microorganism to date. The bacterium degraded high levels of glyphosate, primarily by converting it to aminomethylphosphonate, followed by release into the growth medium. Only a small amount of aminomethylphosphonate (about 0.5 to 0.7 mM), which is needed to supply phosphorus for growth, could be metabolized by the microorganism. Solid-state 13C nuclear magnetic resonance analysis of strain LBr grown on 1 mM [2-13C,15N]glyphosate showed that about 5% of the glyphosate was degraded by a separate pathway involving breakdown of glyphosate to glycine, a pathway first observed in Pseudomonas sp. strain PG2982. Thus, Pseudomonas sp. strain LBr appears to possess two distinct routes for glyphosate detoxification.     

Uptake of methylamine and methanol by Pseudomonas sp. strain AM1.

The uptake of methylamine and of methanol by the facultative methylotroph Pseudomonas sp. strain AM1 was investigated. It was found that this organism possesses two uptake systems for methylamine. One of these operates when methylamine is the sole source of carbon, nitrogen, and energy. It has a Km of 1.33 X 10(-4) M and a Vmax of 67 nmol/min per mg of cells (dry weight). The other system, found when methylamine is the sole nitrogen source only, has a Km of 1.2 X 10(-5) M and a Vmax of 8.9 nmol/min per mg of cells (dry weight). Both uptake systems were severely inhibited by azide, cyanide, carbonyl cyanide-m-chlorophenyl hydrazone, and N-ethylmaleimide, but only the high-affinity system was inhibited by ammonium ions with a Ki of 7.7 mM. Both systems were susceptible to osmotic shock treatment, competitively inhibited by ethylamine, and unaffected by most amino acids. Methanol uptake showed a Km of 4.8 microM and a Vmax of 60.6 nmol/min per mg of cells (dry weight) and was not inhibited by osmotic shock treatment. Azide, cyanide, and N-ethylmaleimide curtailed uptake, but carbonyl cyanide-m-chlorophenyl hydrazone merely reduced the rate of uptake. A methanol dehydrogenase mutant, M15A, was unable to take up methanol. It is proposed that methanol diffuses into the cell where it is rapidly oxidized by methanol dehydrogenase.     

Co-metabolic degradation of chlorinated hydrocarbons by Pseudomonas sp. strain DCA1

Pseudomonas sp. strain DCA1, which is capable of utilizing 1,2-dichloroethane (DCA) as sole carbon and energy source, was used to oxidize chlorinated methanes, ethanes, propanes, and ethenes. Chloroacetic acid, an intermediate in the DCA degradation pathway of strain DCA1, was used as a co-substrate since it was readily oxidized by DCA-grown cells of strain DCAI and did not compete for the monooxygenase. All of the tested compounds except tetrachloroethylene (PER) were oxidized by cells expressing DCA monooxygenase. Strain DCAI could not utilize any of these compounds as a growth substrate. Co-metabolic oxidation during growth on DCA was studied with 1,2-dichloropropane. Although growth on this mixture occurred, 1,2-dichloropropane strongly inhibited growth of strain DCAI. This inhibition was not caused by competition for the monooxygenase. It was shown that the oxidation of 1,2dichloropropane resulted in the accumulation of 2,3-dichloro-1-propanol and 2-chloroethanol.     

Metabolism of glyphosate in Pseudomonas sp. strain LBr.

Metabolism of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. strain LBr, a bacterium isolated from a glyphosate process waste stream, was examined by a combination of solid-state 13C nuclear magnetic resonance experiments and analysis of the phosphonate composition of the growth medium. Pseudomonas sp. strain LBr was capable of eliminating 20 mM glyphosate from the growth medium, an amount approximately 20-fold greater than that reported for any other microorganism to date. The bacterium degraded high levels of glyphosate, primarily by converting it to aminomethylphosphonate, followed by release into the growth medium. Only a small amount of aminomethylphosphonate (about 0.5 to 0.7 mM), which is needed to supply phosphorus for growth, could be metabolized by the microorganism. Solid-state 13C nuclear magnetic resonance analysis of strain LBr grown on 1 mM [2-13C,15N]glyphosate showed that about 5% of the glyphosate was degraded by a separate pathway involving breakdown of glyphosate to glycine, a pathway first observed in Pseudomonas sp. strain PG2982. Thus, Pseudomonas sp. strain LBr appears to possess two distinct routes for glyphosate detoxification.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A

We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at -5 degrees C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35 degrees C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.     

Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6.

The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems. The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes. Pseudomonas sp. strain JS6 uses a similar system for growth on toluene or dichlorobenzenes. We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene. When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols. Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols. Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.     

A novel N-carbamoyl-L-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-L-cysteine amidohydrolase expressed in Escherichia coli

N-carbamoyl-l-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50°C, respectively. The enzyme required Mn²⁺ ion for activity expression and was inhibited by EDTA, Hg²⁺ and sulfhydryl reagents. The enzyme was strictly specific for the l-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-l-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel l-carbamoylase, different from the known l-carbamoylases.