Enhancement ofcis,cis-muconate productivity by overexpression of catechol 1,2-dioxygenase inPseudomonas putida BCM114

For enhancement ofcis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion tocis,cis-muconate) was cloned and expressed in recombinantPseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM),cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the case of wildtypeP. putida BM014, whereascis,cis-muconate productivity continuously increased and catechol was completely transformed tocis,cis-muconate forP. putida BCM114. Specific C12O activity ofP. putida BCM114 was about three times higher than that ofP. putida BM014, and productivity was enhanced more than two times.     

Production of cis,cis-muconate from benzoate and 2-fluoro-cis,cis-muconate from 3-fluorobenzoate by 3-chlorobenzoate degrading bacteria

Summary 3-Chlorobenzoate grown cells of Pseudomonas sp. strain B13 or Alcaligenes sp. strain A7-2 converted 3-fluorobenzoate to 2-fluoro-cis,cis-muconate with 87% yield. The latter strain produced 1.6 g/l. The type II muconate cycloisomerases of neither strain exhibit acitivity for 2-fluoro-cis,cis-muconate. Succinate grown cells of Pseudomonas sp. strain B13 converted benzoate to cis,cis-muconate (91% yield; 7.4 g/l). Enzyme tests confirmed that no muconate cycloisomerising enzyme was induced within 24 h.     

Molecular properties of cis,cis-muconate cycloisomerase from Pseudomonas putida

cis,cis-Muconate cycloisomerase (cis,cis-muconate lactonizing enzyme, EC 5.5.1.1.) was purified in crystalline form from Pseudomonas putida. Ultracentrifugation studies, as well as gel filtration chromatography and electrophoresis, indicate that the enzyme is an oligomeric protein of molecular weight 252,000 (s_20,w 12.20 × 10^?13 s), which is built of six homologous protomers of molecular weight 42,000. Studies of enzyme crystals and enzyme molecules in the electron microscope suggest that the cis,cis-muconate cycloisomerase is a hexamer in which the six protomers are arranged in a dihedral point-group symmetry 32 (D₃). Each protomer has a diameter of 42.5Åand six protomers are associated in a structure with a trigonal antiprismatic geometry (a hexamer D₃ octahedron). This model could account for the dimensions most frequently observed by negative staining of the enzyme in solution. A model for the three-dimensional structure of enzyme crystals in which each hexameric enzyme molecule is surrounded by eight neighbouring enzyme molecules, is described.     

Stereo- and regiospecific cis,cis-muconate cycloisomerization by Rhodococcus rhodochrous N75

cis,cis-Muconate cycloisomerase was purified to homogeneity from cells of Rhodococcus rhodochrous N75 grown at the expense of benzoate and p-toluate as the sole sources of carbon. A single cycloisomerase was found to be induced in this organism with no isoforms being detected when R. rhodochrous N75 was grown on either benzoate or p-toluate as the sole source of carbon. The enzyme is hexameric with a single subunit Mr of 40,000. cis,cis-Muconate cycloisomerase from R. rhodochrous N75 displayed strict regio- and stereospecificity whereby cis,cis-muconate is cycloisomerized to (4S)-muconolactone and 2-methyl- and 3-methyl-substituted muconates are cycloisomerized to 2-methyl- and 4-methyl-substituted muconolactones by 1,4- and 3,6-cycloisomerization, respectively.     

Biodegradation,biotransformation and the Belmont

Keynote Address at the Annual Meeting of the Society for Industrial Microbiology, San Diego, August 1992.     

The structure of Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme, a beta propeller cycloisomerase

Muconate lactonizing enzymes (MLEs) convert cis,cis-muconates to muconolactones in microbes as part of the beta-ketoadipate pathway; some also dehalogenate muconate derivatives of xenobiotic haloaromatics. There are three different MLE classes unrelated by evolution. We present the X-ray structure of a eukaryotic MLE, Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme (NcCMLE) at 2.5 A resolution, with a seven-bladed beta propeller fold. It is related neither to bacterial MLEs nor to other beta propeller enzymes, but is structurally similar to the G protein beta subunit. It reveals a novel metal-independent cycloisomerase motif unlike the bacterial metal cofactor MLEs. Together, the bacterial MLEs and NcCMLE structures comprise a striking structural example of functional convergence in enzymes for 1,2-addition-elimination of carboxylic acids. NcCMLE and bacterial MLEs may enhance the reaction rate differently: the former by electrophilic catalysis and the latter by electrostatic stabilization of the enolate.     

Formation of protoanemonin from 2-chloro-cis,cis-muconate by the combined action of muconate cycloisomerase and muconolactone isomerase

Muconate cycloisomerases are known to catalyze the reversible conversion of 2-chloro-cis,cis-muconate by 1,4- and 3,6-cycloisomerization into (4S)-(+)-2-chloro- and (4R/5S)-(+)-5-chloromuconolactone. 2-Chloromuconolactone is transformed by muconolactone isomerase with concomitant dechlorination and decarboxylation into the antibiotic protoanemonin. The low k(cat) for this compound compared to that for 5-chloromuconolactone suggests that protoanemonin formation is of minor importance. However, since 2-chloromuconolactone is the initially predominant product of 2-chloromuconate cycloisomerization, significant amounts of protoanemonin were formed in reaction mixtures containing large amounts of muconolactone isomerase and small amounts of muconate cycloisomerase. Such enzyme ratios resemble those observed in cell extracts of benzoate-grown cells of Ralstonia eutropha JMP134. In contrast, cis-dienelactone was the predominant product formed by enzyme preparations, in which muconolactone isomerase was in vitro rate limiting. In reaction mixtures containing chloromuconate cycloisomerase and muconolactone isomerase, only minute amounts of protoanemonin were detected, indicating that only small amounts of 2-chloromuconolactone were formed by cycloisomerization and that chloromuconate cycloisomerase actually preferentially catalyzes a 3,6-cycloisomerization.     

Inability of muconate cycloisomerases to cause dehalogenation during conversion of 2-chloro-cis,cis-muconate.

The conversion of 2-chloro-cis,cis-muconate by muconate cycloisomerase from Pseudomonas putida PRS2000 yielded two products which by nuclear magnetic resonance spectroscopy were identified as 2-chloro- and 5-chloromuconolactone. High-pressure liquid chromatography analyses showed the same compounds to be formed also by muconate cycloisomerases from Acinetobacter calcoaceticus ADP1 and Pseudomonas sp. strain B13. During 2-chloro-cis,cis-muconate turnover by the enzyme from P. putida, 2-chloromuconolactone initially was the major product. After prolonged incubation, however, 5-chloromuconolactone dominated in the resulting equilibrium. In contrast to previous assumptions, both chloromuconolactones were found to be stable at physiological pH. Since the chloromuconate cycloisomerases of Pseudomonas sp. strain B13 and Alcaligenes eutrophus JMP134 have been shown previously to produce the trans-dienelactone (trans-4-carboxymethylene-but-2-en-4-olide) from 2-chloro-cis,cis-muconate, they must have evolved the capability to cleave the carbon-chlorine bond during their divergence from normal muconate cycloisomerases.     

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: an alternate cycloisomerase motif.

3-Carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa catalyzes the reversible gamma-lactonization of 3-carboxy-cis,cis-muconate by a syn-1,2 addition-elimination reaction. The stereochemical and regiochemical course of the reaction is (i) opposite that of CMLE from Pseudomonas putida (EC 5.5.1.2) and (ii) identical to that of cis,cis-muconate lactonizing enzyme (MLE; EC 5.5.1.1) from P. putida. In order to determine the mechanistic and evolutionary relationships between N. crassa CMLE and the procaryotic cycloisomerases, we have purified CMLE from N. crassa to homogeneity and determined its nucleotide sequence from a cDNA clone isolated from a p-hydroxybenzoate-induced N. crassa cDNA library. The deduced amino acid sequence predicts a protein of 41.2 kDa (365 residues) which does not exhibit sequence similarity with any of the bacterial cycloisomerases. The cDNA encoding N. crassa CMLE was expressed in Escherichia coli, and the purified recombinant protein exhibits physical and kinetic properties equivalent to those found for the isolated N. crassa enzyme. We also report that N. crassa CMLE possesses substantially reduced yet significant levels of MLE activity with cis,cis-muconate and, furthermore, does not appear to be dependent on divalent metals for activity. These data suggest that the N. crassa CMLE may represent a novel eucaryotic motif in the cycloisomerase enzyme family.     

Insight into the reaction mechanism of cis,cis-muconate lactonizing enzymes: a DFT QM/MM study

MLEs derived from mycobacterium smegmatis and seudomonas fluorescens share ∼76% identity and have a very similar arrangement of catalytic residues in their active site configuration. However, while they catalyze the conversion of cis,cis-muconate to the same achiral product, muconolactone, studies in deuterated solvent surprisingly show that the cyclo-isomerization proceeds with the formation of a chiral product. In this paper we discuss the application of DFT QM/MM calculations on both MLEs, to our knowledge the first reported in the literature on this protein. We investigate the proposal that the base involved in the catalytic reaction is the lysine residue found at the end of the 2^nd strand given: (a) that the lysine residue at the end of the 6^th strand is in an apparently equally effective position to catalyze reaction and (b) that the structural related epimerase in-fact achieve their stereo-specific outcomes by relying on either the base from the 2^nd or 6^th strand.     

Synergism exerted by 4-methyl catechol, catechol, and their respective quinones on the rate of DL-DOPA oxidation by mushroom tyrosinase.

4-Methyl catechol and catechol, at concentrations ranging from 0.03 to 9 mM and 0.066 to 20 mM, respectively, have a synergistic effect on the rate of DL-DOPA oxidation by mushroom tyrosinase to material absorbing at 475 nm. The synergism results from the ability of 4-methyl catechol-o-quinone (4-methyl-o-benzoquinone) and of catechol-o-quinone (o-benzoquinone) to oxidize DL-DOPA non-enzymatically to dopaquinone, with the later being immediately converted to dopachrome (lambda max = 475 nm).     

Salsolinol,a catechol neurotoxin,induces oxidative modification of cytochrome c

Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), an endogenous neurotoxin, is known to perform a role in the pathogenesis of Parkinson’s disease (PD). In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with salsolinol. When cytochrome c was incubated with salsolinol, protein aggregation increased in a dosedependent manner. The formation of carbonyl compounds and the release of iron were obtained in salsolinol- treated cytochrome c. Salsolinol also led to the release of iron from cytochrome c. Reactive oxygen species (ROS) scavengers and iron specific chelator inhibited the salsolinol-mediated cytochrome c modification and carbonyl compound formation. It is suggested that oxidative damage of cytochrome c by salsolinol might induce the increase of iron content in cells, subsequently leading to the deleterious condition which was observed. This mechanism may, in part, provide an explanation for the deterioration of organs under neurodegenerative disorders such as PD. [BMB Reports 2013; 46(2): 119-123]     

Phytoalexins from crucifers: synthesis, biosynthesis, and biotransformation

Phytoalexins play a significant role in the defense response of plants. These secondary metabolites, which are synthesized de novo in response to diverse forms of stress, including fungal infection, are part of the plants' chemical and biochemical defense mechanisms. Phytoalexins from crucifers are structurally and biogenetically related, but display significantly different biological activities. Here, we review work reporting the chemical structures, synthesis, biosynthesis and metabolism of cruciferous phytoalexins, as well as their biological activity towards different microorganisms.     

Inducible Uptake System for β-Carboxy-cis, cis-muconate in a Permeability Mutant of Pseudomonas putida

A procedure for the large-scale enzymatic synthesis of beta-carboxymuconate is described. When used as a growth substrate, beta-carboxymuconate selected for mutant strains of Pseudomonas putida that were permeable to polycarboxylic acid intermediates of the beta-ketoadipate pathway. One mutant organism, strain PRS2110, was investigated in detail. It differed from the parental strain in that it possessed a beta-carboxymuconate uptake system that was formed when the compound was supplied exogenously to the cells. The uptake system was not induced by beta-carboxymuconate supplied endogenously during growth with p-hydroxybenzoate. These observations suggested that beta-carboxymuconate was contained within a physical compartment of enzymes during growth with p-hydroxybenzoate. Support for this hypothesis came from the demonstration that enzymes of the beta-ketoadipate pathway were held together by weak chemical interactions during the chromatography of crude extracts of benzoategrown P. putida on diethylaminoethyl-cellulose columns.