The ins and outs of ring-cleaving dioxygenases

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.     

The Ins and Outs of Ring-Cleaving Dioxygenases

ABSTRACT

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.     

Oxygenases without requirement for cofactors or metal ions

Mono- and dioxygenases usually depend on a transition metal or an organic cofactor to activate dioxygen, or their organic substrate, or both. This review points out that there are at least two separate families of oxygenases without any apparent requirement for cofactors or metal ions: the quinone-forming monooxygenases which are important 'tailoring enzymes' in the biosynthesis of several types of aromatic polyketide antibiotics, and the bacterial dioxygenases involved in the degradation of distinct quinoline derivatives, catalyzing the 2,4-dioxygenolytic cleavage of 3-hydroxy-4-quinolones with concomitant release of carbon monoxide. The quinone-forming monooxygenases might be useful for the modification of polyketide structures, either by using them as biocatalysts, or by employing combinatorial biosynthesis approaches. Cofactor-less oxygenases present the mechanistically intriguing problem of how dioxygen is activated for catalysis. However, the reactions catalyzed by these enzymes are poorly understood in mechanistic terms. Formation of a protein radical and a substrate-derived radical, or direct electron transfer from a deprotonated substrate to molecular oxygen to form a caged radical pair may be discussed as hypothetical mechanisms. The latter reaction route is expected for substrates that can easily donate an electron to dioxygen, and requires the ability of the enzyme to stabilize anionic intermediates. Histidine residues found to be catalytically relevant in both types of cofactor-less oxygenases might be involved in substrate deprotonation and/or electrostatic stabilization.     

Reactions involved in the lower pathway for degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1

In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds.     

An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Ppseudomonas putida mt-2.

BACKGROUND: Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. RESULTS: The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. CONCLUSIONS: The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.     

A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family.

Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.     

Bacterial aromatic ring-cleavage enzymes are classified into two different gene families

Dioxygenases that catalyze the cleavage of the aromatic ring are classified into two groups according to their mode of ring fission. Substrates of ring-cleavage dioxygenases usually contain hydroxyl groups on adjacent aromatic carbons, and intradiol enzymes cleave the ring between these two hydroxyl groups. Extradiol enzymes in contrast cleave the ring between one hydroxylated carbon and its adjacent nonhydroxylated carbon. In this study, we determined the complete nucleotide sequence of nahC, the structural gene for 1,2-dihydroxynaphthalene dioxygenase encoded in the NAH7 plasmid of Pseudomonas putida. This enzyme is an extradiol ring-cleavage enzyme that cleaves the first ring of 1,2-dihydroxynaphthalene. The amino acid sequence of the dioxygenase deduced from the DNA sequence demonstrated that the molecular weight of the enzyme is 33,882. This result was in agreement with those of maxicell analyses that showed that the nahC product was a 36-kDa protein. Interestingly, the amino acid sequence of 1,2-dihydroxynaphthalene dioxygenase was 50% homologous with that of 2,3-dihydroxybiphenyl dioxygenase, which catalyzes extradiol cleavage of the first ring of 2,3-dihydroxybiphenyl (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429). The amino acid sequence similarity of 1,2-dihydroxynaphthalene dioxygenase with catechol 2,3-dioxygenase, which is an authentic extradiol dioxygenase, was rather low (16%). However, a statistical analysis by the method of S. B. Needleman and C. D. Wunsch [1970) J. Mol. Biol. 48, 443-453) clearly showed that these two dioxygenases are evolutionarily related. Therefore, these extradiol enzymes are considered as products of the same gene superfamily. From the significant sequence similarity between intradiol enzymes, it has been shown (Neidle, E. L., Harnett, C., Bonitz, S., and Ornston, L. N. (1988) J. Bacteriol. 170, 4874-4880) that intradiol enzymes evolved from a common ancestor. Comparison of the amino acid sequence of extradiol enzymes with those of intradiol dioxygenases did not show any significant global or localized similarity.     

Catalytic mechanism of S-ribosylhomocysteinase: ionization state of active-site residues

S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether linkage in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial autoinducer (AI-2). The proposed catalytic mechanism involves two consecutive ribose carbonyl migration steps via an intramolecular redox reaction and a subsequent beta-elimination step, all catalyzed by a divalent metal ion (e.g., Fe(2+) or Co(2+)) and two general acids/bases in the active site. Absorption and EPR spectroscopic studies were performed with both wild-type and various mutant forms of LuxS under a wide range of pH conditions. The studies revealed a pK(a) of 10.4 for the metal-bound water. The pK(a) value of Cys-83 was determined to be <6 by (13)C-(1)H HSQC NMR experiments with [3-(13)C]cysteine-labeled Zn(2+)-substituted Escherichia coli LuxS. The active form of LuxS contains a metal-bound water and a thiolate ion at Cys-83, consistent with the proposed roles of the metal ion (Lewis acid) and Cys-83 (general acid/base) during catalysis. Finally, an invariant Arg-39 in the active site was demonstrated to be at least partially responsible for stabilizing the thiolate anion of Cys-83.     

Kinetic and spectroscopic studies on the quercetin 2,3-dioxygenase from Bacillus subtilis

Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin and molecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, the only known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe2+-dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme in Escherichia coli grown on minimal media in the presence of a number of divalent metals. The addition of Mn2+, Co2+, and Cu2+ generated active enzymes, but the addition of Zn2+, Fe2+, and Cd2+ did not increase quercetinase activity to any significant level over a control in which no divalent ions were added to the media. The Mn2+- and Co2+-containing QueDs were purified, characterized by metal analysis and EPR spectroscopy, and studied by steady-state kinetics. Mn2+ was found to be incorporated nearly stoichiometrically to the two cupin motifs. The hyperfine coupling constant of the g = 2 signal in the EPR spectra of the Mn2+-containing enzyme showed that the two Mn2+ ions are ligated in an octahedral coordination. The turnover number of this enzyme was found to be in the order of 25 s(-1), nearly 40-fold higher than that of the Fe2+-containing enzyme and similar in magnitude to that of the Cu2+-containing quercertin 2,3-dioxygenase from Aspergillus japonicus. In addition, kinetic and spectroscopic data suggest that the catalytic mechanism of QueD is different from that of the Aspergillus quercetinases but similar to that proposed for the extradiol catechol dioxygenases. This study provides evidence that Mn2+ might be the preferred cofactor for this enzyme and identifies QueD as a new member of the manganese dioxygenase family.     

Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)

The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.     

Single-turnover kinetics of homoprotocatechuate 2,3-dioxygenase

Homoprotocatechuate 2,3-dioxygenase isolated from Brevibacterium fuscum utilizes an active site Fe(II) and O(2) to catalyze proximal extradiol cleavage of the substrate aromatic ring. In contrast to other members of the ring cleaving dioxygenase family, the transient kinetics of the extradiol dioxygenase catalytic cycle have been difficult to study because the iron is nearly colorless and EPR silent. Here, it is shown that the reaction cycle kinetics can be monitored by utilizing the alternative substrate 4-nitrocatechol (4NC), which is also cleaved in the proximal extradiol position. Changes in the optical spectrum of 4NC occurring as a result of ionization, environmental changes, and ring cleavage allow both the substrate binding and product formation phases of the reaction to be studied. It is shown that substrate binding occurs in a four-step process probably involving binding to two ionization states of the enzyme at different rates. Following an initial rapid binding of the monoanionic 4NC in the active site, slower binding to the Fe(II) and conversion to the dianionic form occur. The bound dianionic 4NC reacts rapidly with O(2) in four additional steps, apparently occurring in sequence. On the basis of the optical properties of the intermediates, these steps are hypothesized to be O(2) binding to the iron, isomerization of the resulting complex, ring opening, and product release. The natural substrate appears to form the same intermediates but with much larger rate constants. These are the first transient intermediates to be reported for an extradiol dioxygenase reaction.     

3,4-Dihydroxyxanthone dioxygenase from Arthrobacter sp. strain GFB100.

Bacterial extradiol ring-fission dioxygenases play a critical role in the transformation of multiring aromatic compounds to more readily biodegradable aromatic or aliphatic intermediates. Arthrobacter sp. strain GFB100 utilizes an extradiol meta-fission dioxygenase, 3,4-dihydroxyxanthone dioxygenase (DHXD), in the catabolism of the three-ring oxygen heterocyclic compound xanthone. In this paper, we show that DHXD is a cytosolic enzyme, induced by growth on xanthone and maximally expressed during the stationary phase of growth. In addition, we characterize the DHXD activity in terms of its basic enzymological properties. 1,10-Phenanthroline and H2O2 treatments eliminated DHXD activity, indicating that the enzyme required Fe2+ ions for activity. Other divalent cations were either inhibitory or had no effect on activity. DHXD had a temperature optimum of 30 degrees C and a pH optimum of 7.0. DHXD followed typical saturation kinetics and had an apparent Km of 10 microM for 3,4-dihydroxyxanthone. The dye celestine blue served as a noncompetitive DHXD inhibitor (Ki, 5 microM). Several other structural analogs served neither as substrates nor inhibitors. DHXD was thermally labile at temperatures above 40 degrees C. The half-life for thermal DHXD inactivation was 5 min at 40 degrees C. DHXD activity was completely stable through one freeze-thaw cycle, and about 80% of the DHXD activity remained after 2 days of incubation at 0 degree C. The apparent tight binding of the Fe2+ cofactor to DHXD may be a factor contributing to the stability of this extradiol dioxygenase when it is stored.     

3,4-Dihydroxyxanthone dioxygenase from Arthrobacter sp. strain GFB100.

Bacterial extradiol ring-fission dioxygenases play a critical role in the transformation of multiring aromatic compounds to more readily biodegradable aromatic or aliphatic intermediates. Arthrobacter sp. strain GFB100 utilizes an extradiol meta-fission dioxygenase, 3,4-dihydroxyxanthone dioxygenase (DHXD), in the catabolism of the three-ring oxygen heterocyclic compound xanthone. In this paper, we show that DHXD is a cytosolic enzyme, induced by growth on xanthone and maximally expressed during the stationary phase of growth. In addition, we characterize the DHXD activity in terms of its basic enzymological properties. 1,10-Phenanthroline and H2O2 treatments eliminated DHXD activity, indicating that the enzyme required Fe2+ ions for activity. Other divalent cations were either inhibitory or had no effect on activity. DHXD had a temperature optimum of 30 degrees C and a pH optimum of 7.0. DHXD followed typical saturation kinetics and had an apparent Km of 10 microM for 3,4-dihydroxyxanthone. The dye celestine blue served as a noncompetitive DHXD inhibitor (Ki, 5 microM). Several other structural analogs served neither as substrates nor inhibitors. DHXD was thermally labile at temperatures above 40 degrees C. The half-life for thermal DHXD inactivation was 5 min at 40 degrees C. DHXD activity was completely stable through one freeze-thaw cycle, and about 80% of the DHXD activity remained after 2 days of incubation at 0 degree C. The apparent tight binding of the Fe2+ cofactor to DHXD may be a factor contributing to the stability of this extradiol dioxygenase when it is stored.     

Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1.

A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.     

Roles of exogenous divalent metals in the nucleolytic activity of Cu,Zn superoxide dismutase

It is well known that the wild type Cu,Zn superoxide dismutase (holo SOD) catalyzes the conversion of superoxide anion to peroxide hydrogen and dioxygen. However, a new function of holo SOD, i.e., nucleolytic activity has been found [W. Jiang, T. Shen, Y. Han, Q. Pan, C. Liu, J. Biol. Inorg. Chem. 11 (2006) 835-848], which is linked to the incorporation of exogenous divalent metals into the enzyme-DNA complex. In this study, the roles of exogenous divalent metals in the nucleolytic activity were explored in detail by a series of biochemical experiments. Based on a non-equivalent multi-site binding model, affinity of a divalent metal for the enzyme-DNA complex was determined by absorption titration, indicating that the complex can provide at least a high and a low affinity site for the metal ion. These mean that the holo SOD may use a "two exogenous metal ion pathway" as a mechanism in which both metal ions are directly involved in the catalytic process of DNA cleavage. In addition, the pH versus DNA cleavage rate profiles can be fitted to two ionizing-group models, indicating the presence of a general acid and a general base in catalysis. A model that requires histidine residues, metal-bound water molecules and two hydrated metal ions to operate in concert could be used to interpret the catalysis of DNA hydrolysis, supported by the dependences of loss of the nucleolytic activity on time and on the concentration of the specific chemical modifier to the histidine residues on the enzyme.     

Identification of an extradiol dioxygenase involved in tetralin biodegradation: gene sequence analysis and purification and characterization of the gene product

A genomic region involved in tetralin biodegradation was recently identified in Sphingomonas strain TFA. We have cloned and sequenced from this region a gene designated thnC, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to define a subfamily of 1, 2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7, 8-tetrahydronaphthalene was found to be the metabolite accumulated by a thnC insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (V(max), 40.5 U mg(-1); K(m), 18. 6 microM). The enzyme shows even higher activity with 1, 2-dihydroxynaphthalene and also significant activity toward 1, 2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1, 2-dihydroxynaphthalene dioxygenases.     

Subcloning and nucleotide sequence of the 3,4-dihydroxyphenylacetate (homoprotocatechuate) 2,3-dioxygenase gene from Escherichia coli C

A cloned gene encoding the Escherichia coli C homoprotocatechuate (HPC) dioxygenase, an aromatic ring cleavage enzyme, was used to produce large amounts of the protein. Preparations of E. coli C HPC dioxygenase, whether expressed from the cloned gene or produced by the bacterium, lost activity very rapidly. The pure protein showed one type of subunit of Mr 33,000. The first 21 N-terminal amino acids were sequenced and the data used to confirm that the open reading frame of 831 bp, identified from the nucleotide sequence, encoded HPC dioxygenase. Comparison of the derived amino acid sequence with those of other extradiol and intradiol dioxygenases showed no obvious similarity to any of them.     

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.     

Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease

On the basis of structural work, metal ions are proposed to play a catalytic role in reactions mediated by many phosphoryl transfer enzymes. To gain dynamic support for such mechanisms, the role of metal ion cofactors in phosphate diester hydrolysis catalysed by a flap endonuclease has been studied. The pH maximal rate profiles were measured in the presence of various metal ion cofactors; in each case, a single ionic form of the enzyme/cofactor accounts for the pH dependence. The kinetic pK(a)s display good correlation with the acidity of the corresponding hexahydrated metal ions, which strongly suggests a role for metal-bound hydroxide, or its equivalent ionic species, in the reaction. Comparing rates of reaction in the pH-independent regions, a small negative beta(nuc) value is observed. This suggests that expected trends in the nucleophilicity of the various metal-bound hydroxides are balanced by a second form of metal ion catalysis that is related to the acidity of the hexahydrated metal ion. This is likely to be either electrophilic catalysis or leaving group activation.     

Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by Mycobacterium Strain HL 4-NT-1

In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds.