Enantioselective oxidations catalyzed by diketocamphane monooxygenase from Pseudomonas putida with macromolecular NAD in a membrane reactor

Summary Several sulfides and bicyclo[3.2.0]hept-2-en-6-one were enantioselectively oxidized to the corresponding sulfoxides and oxa lactones by a crude preparation of the two diketocamphane monooxygenases from Pseudomonas putida. The reactions were carried out in a membrane reactor with the use of poly(ethylene glycol)-N⁶-(2-aminoethyl)-NAD and coenzyme regeneration by the formate/formate dehydrogenase system.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida

Toluene dioxygenase, a multicomponent enzyme system known to oxidize mononuclear aromatic hydrocarbons to cis-dihydrodiols, oxidized indene and indan to 1-indenol and 1-indanol, respectively. In addition, the enzyme catalyzed dioxygen addition to the nonaromatic double bond of indene to form cis-1,2-indandiol. The oxygen atoms in 1-indenol and cis-1,2-indandiol were shown to be derived from molecular oxygen, whereas 70% of the oxygen in 1-indanol was derived from water. All of the isolated products were optically active as demonstrated by 19F NMR and HPLC discrimination of diastereomeric esters and by chiroptic methods. The high optical purity of (-)-(1R)-indanol (84% enantiomeric excess) and the failure of scavengers of reactive oxygen species to inhibit the monooxygenation reaction supported the contention that the monooxygen insertion is mediated by an active-site process. Experiments with 3-[2H]indene indicated that equilibration between C-1 and C-3 occurred prior to the formation of the carbon-oxygen bond to yield 1-indenol. Naphthalene dioxygenase also oxidized indan to 1-indanol, which suggested that benzylic monoxygenation may be typical of this group of dioxygenases.     

Exchange reactions catalyzed by methioninase from Pseudomonas putida.

Highly purified methioninase from Pseudomonas putida, which catalyzes alpha, gamma-elimination reactions of homocysteine and its S-substituted derivatives as well as alpha, beta-elimination reactions of cysteine and its derivatives, was found to catalyze exchange reactions between the substituent at the gamma-carbon of homocysteine substrates and exogenously added alkanethiols, forming the corresponding S-alkylhomocysteines. It also catalyzed similar beta-exchange reactions between cysteine and alkanethiols. Thus, all the substrates for the methioninase-catalyzed elimination reactions also appear to be available for the exchange reactions.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.

Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol. Under similar conditions, P. putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P. putida F39/D that preferentially oxidizes (+)-(1S)-indanol.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate.

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

Degradation of L-djenkolate catalyzed by S-alkylcysteine alpha,beta-lyase from Pseudomonas putida

S-Alkylcysteine alpha, beta-lyase [EC 4.4.1.6] of Pseudomonas putida catalyzes alpha,beta-elimination of L-djenkolate [3,3'-methylenedithiobis(2-aminopropionic acid)] to produce pyruvate, ammonia, and S-(mercaptomethyl)cysteine initially. Secondly, S-(mercaptomethyl)-cysteine, which was identified in the form of S-(mercaptomethyl)cysteine thiolactone and S-(2-thia-3-carboxypropyl)cysteine in the absence and presence of iodoacetic acid, respectively, is decomposed enzymatically to pyruvate, ammonia, and bis(mercapto)methane, or spontaneously to cysteine, formaldehyde, and hydrogen sulfide. Balance studies showed that 1.3 mol each of pyruvate and ammonia and 0.2 mol each of formaldehyde and cysteine were produced with consumption of 1 mol of L-djenkolate. 1,2,4,5-Tetrathiane, 1,2,4-trithiolane, 1,2,4,6-tetrathiepane, and 1,2,3,5,6-pentathiepane, which are derivatives of bis(mercapto)methane, were also produced during the alpha,beta-elimination of L-djenkolate. In addition, a polymer with the general formula of -(CH2S)n- was produced as a white precipitate. When the alpha,beta-elimination of L-djenkolate was carried out in the presence of 20 mM iodoacetic acid, neither formaldehyde, cysteine, hydrogen sulfide, or the polymer were formed. Instead, the S-carboxymethyl derivatives of bis(mercapto)methane and S-(mercaptomethyl)cysteine were produced in addition to pyruvate and ammonia.     

Biotransformation of isoeugenol catalyzed by growing cells of Pseudomonas putida

Abstract

Growing cells of Pseudomonas putida transformed isoeugenol after 5 days of incubation to give mainly vanillin, eugenol, 4-(E)-(3-hydroxyprop-1-enyl)-2-methoxyphenol and the dimeric molecule (+)-4-[2,3-dihydro-7-methoxy-3-methyl-5-(E)-(1-propenyl)-2-benzofuranyl]-2-methoxyphenol (licarin A). The formation of the latter compound from isoeugenol by biotransformation with P. putida is reported here for the first time.     

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.     

Toxicity of Trichloroethylene to Pseudomonas putida F1 Is Mediated by Toluene Dioxygenase

Trichloroethylene was metabolically activated by toluene dioxygenase to produce toxic effects in Pseudomonas putida F1. Cytotoxicity was indicated by growth inhibition and by the covalent modification of cellular molecules in P. putida F1 exposed to [¹⁴C]trichloroethylene. With a toluene dioxygenase mutant, neither growth inhibition nor alkylation of intracellular molecules was observed.     

Plasmid control of the Pseudomonas aeruginosa and Pseudomonas putida phenotypes and of linalool and p-cymene oxidation.

Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P. aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P. aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observation implies that support of linalool oxidation effectively conceals the P. aeruginosa character.     

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.     

Monohydroxylation of Phenol and 2,5-Dichlorophenol by Toluene Dioxygenase in Pseudomonas putida F1

[This corrects the article on p. 2650 in vol. 55.].     

Enantioselective oxidations by the diketocamphane monooxygenase isozymes from Psevdomonas putida

Summary 2,5-Diketocamphane 1,2-monooxygenase and 3,6-diketocamphane 1,6-monooxygenase isolated from (±)-camphor grown Pseudomonas putida NCIMB 10007 catalysed two different types of Baeyer-Villiger oxidations. The former isozyme biotransformed various bicyclic [3.2.0] ketones and aryl alkyl sulfides with consistently greater regio- and/or enantioselectivity, yielding lactone and sulfoxide products with enantiomeric excesses > 55%.     

A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system.

A strain of Pseudomonas putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the O-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components: an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase. The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently. The absolute spectrum of the oxidized displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores. The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 415 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the NADH-dependent reductase. Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i.e., iron-containing and labile-sulfur-containing monooxygenases. From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed.     

Stereospecific Hydroxylation of Indan by Escherichia coli Containing the Cloned Toluene Dioxygenase Genes from Pseudomonas putida F1

[This corrects the article on p. 3407 in vol. 58.].