Purification and characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from the basidiomycete Phanerochaete chrysosporium.

1,2,4-Trihydroxybenzene (THB) is an intermediate in the Phanerochaete chrysosporium degradation of vanillate and aromatic pollutants. A P. chrysosporium intracellular enzyme able to oxidatively cleave the aromatic ring of THB was purified by ammonium sulfate precipitation, hydrophobic and ion-exchange chromatographies, and native gel electrophoresis. The native protein has a molecular mass of 90 kDa and a subunit mass of 45 kDa. The enzyme catalyzes an intradiol cleavage of the substrate aromatic ring to produce maleylacetate. 18O2 incorporation studies demonstrate that molecular oxygen is a cosubstrate in the reaction. The enzyme exhibits high substrate specificity for THB; however, catechol cleavage occurs at approximately 20% of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product of THB cleavage, is reduced to beta-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts.     

Spectroscopic characterization of secondary amine mono-oxygenase. Comparison to cytochrome P-450 and myoglobin

Secondary amine mono-oxygenase from Pseudomonas aminovorans catalyzes the NAD(P)H- and dioxygen-dependent N-dealkylation of secondary amines to yield a primary amine and an aldehyde. Heme iron, flavin, and non-heme iron prosthetic groups are known to be present in the oligomeric enzyme. The N-dealkylation reaction is also catalyzed by the only other heme-containing mono-oxygenase, cytochrome P-450. In order to identify the heme iron axial ligands of secondary amine mono-oxygenase so as to better define the structural requirements for oxygen activation by heme enzymes, we have investigated the spectroscopic properties of the enzyme. The application of three different spectroscopic techniques, UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance, to study eight separate enzyme derivatives has provided extensive and convincing evidence for the presence of a proximal histidine ligand. This conclusion is based primarily on comparisons of the spectral properties of the enzyme with those of parallel derivatives of myoglobin (histidine proximal ligand) and P-450 (cysteinate proximal ligand). Spectral studies of ferric secondary amine mono-oxygenase as a function of pH have led to the proposal that the distal ligand is water. Deprotonation of the distal water ligand occurs upon either raising the pH to 9.0 or substrate (dimethylamine) binding. In contrast, the deoxyferrous enzyme appears to have a weakly bound nitrogen donor distal ligand. Initial spectroscopic studies of the iron-sulfur units in the enzyme are interpreted in terms of a pair of Fe2S2 clusters. Secondary amine mono-oxygenase is unique in its ability to function as cytochrome P-450 in activating molecular oxygen but to do so with a myoglobin-like active site. As such, it provides an important system with which to probe structure-function relations in heme-containing oxygenases.     

Purification and characterization of a monooxygenase involved in the biosynthetic pathway of the antitumor drug mithramycin

A monooxygenase encoded by the mtmOIV gene from the mithramycin gene cluster of Streptomyces argillaceus was purified 21-fold by a three-step purification procedure. This monooxygenase catalyzes the oxidative cleavage of the fourth ring of premithramycin B. The enzyme was dependent on NADPH and flavin adenine dinucleotide for activity with optimal pH at 9.5, and the K(m) values for NADPH and premithramycin B were 269.22 and 23.35 micro M, respectively. The reaction catalyzed by MtmOIV yields two possible isomers of the same basic shortened aliphatic chain molecule. One of the reaction products showed important biological activity, thus highlighting the importance of the cleavage of the fourth ring of the aglycon for biological activity.     

Formation of benzoquinol moiety in cornoside by salidroside mono-oxygenase,a cytochrome P450 enzyme,from <Emphasis Type="Italic">Abeliophyllum distichum </Emphasis>cell suspension cultures

A microsomal fraction prepared from Abeliophyllum distichumNakai (Oleaceae) cell suspension cultures oxidized salidroside, a glucoside of 4-hydroxyphenylethyl alcohol, to cornoside possessing a unique benzoquinol ring. The enzyme named salidroside mono-oxygenase required NADPH as the only cofactor, and molecular oxygen. The reaction was strongly inhibited by CO as well as several cytochrome P450 inhibitors, such as cytochrome c and miconazole, indicating the involvement of a cytochrome P450 enzyme. Salidroside mono-oxygenase accepted salidroside as the only substrate, but did not oxidize 4-hydroxyphenylethyl alcohol, the salidroside aglucone, and 4-hydroxybenzoic acid. The optimum pH of the reaction was 7.5, and apparent K(m) values for salidroside and NADPH were 44 micro M and 33 micro M, respectively. The benzoquinol ring formation mechanism is discussed in comparison to the mechanism for ipso substitution of 4-hydroxybenzoate by active oxygen species followed by elimination leading to hydroquinone.     

Pyrrolidine ring conformations in prolyl peptides from 13C spin-lattice relaxation times

A method was developed in the framework of a bistable jump model to obtain the pyrrolidine ring conformations in proline peptides from 13C spin-lattice relaxation times. Equations are presented expressing the ring torsions in terms of the 13C spin-lattice relaxation times of the ring carbons. This method was applied to 26 pyrrolidine ring systems and acceptable conformations were obtained.     

Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ ¹H nuclear magnetic resonance (¹H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and ¹H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. ¹H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring.     

Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes

The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.     

Discovery of new diphenyloxazole derivatives containing a pyrrolidine ring: orally active prostacyclin mimetics. Part 2

Synthetic and biological evaluation of novel diphenyloxazole derivatives containing a pyrrolidine ring, as a prostacyclin mimetic without the PG skeleton, are described. Asymmetric reduction of a ketone using a chiral Ru complex and reductive amination by NaBH(4) produces four isomers of the tetrahydronaphthalene ring and the pyrrolidine ring with high stereoselectivity. FR193262 (4), (R,R)-diphenyloxazolyl pyrrolidine derivative, displays high potency and agonist efficacy at the IP receptor and has good bioavailability in rats and dogs.     

Recombinant lysine:N(6)-hydroxylase: effect of cysteine-->alanine replacements on structural integrity and catalytic competence

Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed.     

9-Mercaptodethiobiotin is formed as a competent catalytic intermediate by Escherichia coli biotin synthase

Biotin synthase (BS) catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This enzyme is an S-adenosylmethionine (AdoMet) radical enzyme that catalyzes the reductive cleavage of AdoMet, generating methionine and a transient 5'-deoxyadenosyl radical. In our working mechanism, the 5'-deoxyadenosyl radical oxidizes DTB by abstracting a hydrogen from C6 or C9, generating a dethiobiotinyl carbon radical that is quenched by a sulfide from a [2Fe-2S] (2+) cluster. A similar reaction sequence directed at the other position generates the second C-S bond in the thiophane ring. Since the BS active site holds only one AdoMet and one DTB, it follows that dissociation of methionine and 5'-deoxyadenosine and binding of a second equivalent of AdoMet must be intermediate steps in the formation of biotin. During these dissociation-association steps, a discrete DTB-derived intermediate must remain bound to the enzyme. In this work, we confirm that the conversion of DTB to biotin is accompanied by the reductive cleavage of 2 equiv of AdoMet. A discrepancy between DTB consumption and biotin formation suggests the presence of an intermediate, and we use liquid chromatography and mass spectrometry to demonstrate that this intermediate is indeed 9-mercaptodethiobiotin, generated at approximately 10% of the total enzyme concentration. The amount of intermediate observed is increased when the reaction is run with substoichiometric levels of AdoMet or with the defective enzyme containing the Asn153Ser mutation. The retention of 9-mercaptodethiobiotin as a tightly bound intermediate is consistent with a mechanism involving the stepwise radical-mediated oxidative abstraction of sulfide from an iron-sulfur cluster.     

Isolation and characterization of alkene-utilizing Xanthobacter spp.

Yellow-pigmented bacteria showing typical characteristics of Xanthobacter spp. were isolated from enrichments with propene and 1-butene, using classical techniques. The generation time for growth on propene and 1-butene of these bacteria ranged from 5 to 7h. A NADH-dependent mono-oxygenase was identified in cell-free extract of Xanthobacter Py2. This mono-oxygenase was not influenced by potential inhibitors tested indicating that propene mono-oxygenase is different from other hydrocarbon mono-oxygenases described until now. Nitrogenase activity could be measured using the acetylene reduction assay with propene as energy source, because acetylene did not inhibit the mono-oxygenase activity.     

New scaffolds in the development of mu opioid-receptor ligands

A new class of mu selective receptor antagonists has been developed using a combinatorial approach based on previously reported Dmt-Tic dipeptide ligands. Modified tetrahydroisoquinoline (Tiq) residues were reacted with different electrophiles in order to create novel molecules that would mimic the original dipeptide. A specific class of thioureas bearing basic pyrrolidine residues were shown to give good binding affinities. Further alkylation of the pyrrolidine ring with benzyl derivatives also proved to increase the mu binding affinity. In addition, it was demonstrated that mu binding was enhanced by the presence of polar groups around the benzyl ring having hydrogen-bonding character (donor/acceptor). This new class of ligands represents a novel scaffold in the development of opioid analogues.     

Biosynthesis of the pyrrolidine ring of nicotine in Nicotiana glutinosa

The biosynthesis of the pyrrolidine ring of nicotine has been studied using short-term steady-state exposures of Nicotiana glutinosa seedlings to ¹⁴CO₂. The pyrrolidine ring of the labeled nicotine has been degraded in a systematic manner to ascertain the radioactivity at each carbon, and a new method has been developed for obtaining C-2′ with complete radiochemical integrity. Some of the labeling patterns obtained were symmetrical while others were clearly unsymmetrical. The duality of the labeling patterns found in these ¹⁴CO₂ biosyntheses, together with other data on pyrrolidine ring biosynthesis which are critically examined, is best rationalized by postulating two biosynthetic pathways for formation of the pyrrolidine ring, one involving a symmetrical precursor and the other an unsymmetrical one.     

Substrate specificity of Sphingobium chlorophenolicum 2,6-dichlorohydroquinone 1,2-dioxygenase

PcpA is an aromatic ring-cleaving dioxygenase that is homologous to the well-characterized Fe(II)-dependent catechol extradiol dioxygenases. This enzyme catalyzes the oxidative cleavage of 2,6-dichlorohydroquinone in the catabolism of pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. (1)H NMR and steady-state kinetics were used to determine the regiospecificity of ring cleavage and the substrate specificity of the enzyme. PcpA exhibits a high degree of substrate specificity for 2,6-disubstituted hydroquinones, with halogens greatly preferred at those positions. Notably, the k(cat)(app)/K(mA)(app) of 2,6-dichlorohydroquinone is ~40-fold higher than that of 2,6-dimethylhydroquinone. The asymmetric substrate 2-chloro-6-methylhydroquinone yields a mixture of 1,2- and 1,6-cleavage products. These two modes of cleavage have different K(mO(2))(app) values (21 and 260 μM, respectively), consistent with a mechanism in which the substrate binds in two catalytically productive orientations. In contrast, monosubstituted hydroquinones show a limited amount of ring cleavage but rapidly inactivate the enzyme in an O(2)-dependent fashion, suggesting that oxidation of the Fe(II) may be the cause. Potent inhibitors of PcpA include ortho-disubstituted phenols and 3-bromocatechol. 2,6-Dibromophenol is the strongest competitive inhibitor, consistent with PcpA's substrate specificity. Several factors that could yield this specificity for halogen substituents are discussed. Interestingly, 3-bromocatechol also inactivates the enzyme, while 2,6-dihalophenols do not, indicating a requirement for two hydroxyl groups for ring cleavage and for enzyme inactivation. These results provide mechanistic insights into the hydroquinone dioxygenases.     

Crystal structure of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E, a key enzyme involved in polychlorinated aromatics biodegradation

Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail.     

Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi

The car gene cluster of the ascomycete Fusarium fujikuroi encodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid in F. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion of carX did not impede neurosporaxanthin biosynthesis. On the contrary, DeltacarX mutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in beta-carotene-accumulating Escherichia coli cells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its beta-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as gamma-carotene, torulene, and beta-apo-8'-carotenal. Our data indicate that the occurrence of at least one beta-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the beta-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.     

Substrate specificity and reaction kinetics of an X-motif ribozyme

The X-motif is an in vitro-selected ribozyme that catalyzes RNA cleavage by an internal phosphoester transfer reaction. This ribozyme class is distinguished by the fact that it emerged as the dominant clone among at least 12 different classes of ribozymes when in vitro selection was conducted to favor the isolation of high-speed catalysts. We have examined the structural and kinetic properties of the X-motif in order to provide a framework for its application as an RNA-cleaving agent and to explore how this ribozyme catalyzes phosphoester transfer with a predicted rate constant that is similar to those exhibited by the four natural self-cleaving ribozymes. The secondary structure of the X-motif includes four stem elements that form a central unpaired junction. In a bimolecular format, two of these base-paired arms define the substrate specificity of the ribozyme and can be changed to target different RNAs for cleavage. The requirements for nucleotide identity at the cleavage site are GD, where D = G, A, or U and cleavage occurs between the two nucleotides. The ribozyme has an absolute requirement for a divalent cation cofactor and exhibits kinetic behavior that is consistent with the obligate binding of at least two metal ions.     

Reaction of 1-amino-2-methylenecyclopropane-1-carboxylate with 1-aminocyclopropane-1-carboxylate deaminase: analysis and mechanistic implications

1-aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme which catalyzes the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia. In an early study of this unusual C(alpha)-C(beta) ring cleavage reaction, 1-amino-2-methylenecyclopropane-1-carboxylic acid (2-methylene-ACC) was shown to be an irreversible inhibitor of ACC deaminase. The sole turnover product was identified as 3-methyl-2-oxobutenoic acid. These results provided strong evidence supporting the ring cleavage of ACC via a nucleophilic addition initiated process, thus establishing an unprecedented mechanism of coenzyme B(6) dependent catalysis. To gain further insight into this inactivation, tritiated 2-methylene-ACC was prepared and used to trap the critical enzyme nucleophiles. Our results revealed that inactivation resulted in the modification of an active site residue, Ser-78. However, an additional 5 equiv of inhibitor was also found to be incorporated into the inactivated enzyme after prolonged incubation. In addition to Ser-78, other nucleophilic residues modified include Lys-26, Cys-41, Cys-162, and Lys-245. The location of the remaining unidentified nucleophile has been narrowed down to be one of the residues between 150 and 180. Labeling at sites outside of the active site is not enzyme catalyzed and may be a consequence of the inherent reactivity of 2-methylene-ACC. Further experiments showed that Ser-78 is responsible for abstracting the alpha-H from d-vinylglycine and may serve as the base to remove the beta-H in the catalysis of ACC. However, it is also likely that Ser-78 serves as the active site nucleophile that attacks the cyclopropane ring and initiates the fragmentation of ACC, while the conserved Lys-51 is the base required for beta-H abstraction. Clearly, the cleavage of ACC to alpha-ketobutyrate by ACC deaminase represents an intriguing conversion beyond the common scope entailed by coenzyme B(6) dependent catalysts.     

Cyclopropylglyoxylate as a mechanistic probe of thiamine pyrophosphate dependent pyruvate-metabolizing enzymes

Four pyruvate-decarboxylating enzymes with thiamine pyrophosphate (TPP) cofactors catalyze the decarboxylation of the cyclopropyl substrate analog cyclopropylglyoxylate. Pyruvate: ferredoxin oxidoreductase, an archaebacterial enzyme which catalyzes oxidation of the hydroxyethyl-TPP (HETPP) intermediate by two one-electron transfers to an iron-sulfur center, generates the coenzyme A thioester of cyclopropylcarboxylic acid. A long-lived free radical, HETPP is thought to be an intermediate in the pyruvate to acetyl-CoA conversion; however, cleavage of the cyclopropyl ring was not detected. Pyruvate decarboxylase, pyruvate oxidase, and pyruvate dehydrogenase also generate the corresponding cyclopropyl products. The applicability of cyclopropyl substrate analogs as indicators of free-radical enzyme mechanisms is discussed in light of these results.     

Substrate specificity and kinetics for VP14, a carotenoid cleavage dioxygenase in the ABA biosynthetic pathway

The plant growth regulator, abscisic acid (ABA), is synthesized via the oxidative cleavage of an epoxy-carotenoid. Specifically, a double bond is cleaved by molecular oxygen and an aldehyde is formed at the site of cleavage in both products. The Vp14 gene from maize encodes an oxidative cleavage enzyme for ABA biosynthesis and the recombinant VP14 protein catalyzes the cleavage reaction in vitro. The enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage (the 11-12 bond), but shows some plasticity in other features of carotenoids that are cleaved. A kinetic analysis with the 9-cis isomer of five carotenoids displays several substrate activity relationships. One of the carotenoids was not readily cleaved, but inhibited the cleavage of another substrate in mixed assays. Of the remaining four carotenoids used in this study, three of the substrates have similar V(max) values. The V(max) for the cleavage of one carotenoid substrate was significantly higher. Molecular modeling and several three-dimensional quantitative substrate-activity relationship programs were used to analyze these results. In addition to a 9-cis double bond, the presence and orientation of the ring hydroxyl affects substrate binding or the subsequent cleavage. Additional variations that affect substrate cleavage are proposed.