Homoprotocatechuate 2,3-dioxygenase Research Articles

Nuclear Resonance Vibrational Spectroscopy Definition of Peroxy Intermediates in Catechol Dioxygenases: Factors that Determine Extra- versus Intradiol Cleavage.

The extradiol dioxygenases (EDOs) and intradiol dioxygenases (IDOs) are nonheme iron enzymes that catalyze the oxidative aromatic ring cleavage of catechol substrates, playing an essential role in the carbon cycle. The EDOs and IDOs utilize very different FeII and FeIII active sites to catalyze the regiospecificity in their catechol ring cleavage products. The factors governing this difference in cleavage have remained undefined. The EDO homoprotocatechuate 2,3-dioxygenase (HPCD) and IDO protocatechuate 3,4-dioxygenase (PCD) provide an opportunity to understand this selectivity, as key O2 intermediates have been trapped for both enzymes. Nuclear resonance vibrational spectroscopy (in conjunction with density functional theory calculations) is used to define the geometric and electronic structures of these intermediates as FeII-alkylhydroperoxo (HPCD) and FeIII-alkylperoxo (PCD) species. Critically, in both intermediates, the initial peroxo bond orientation is directed toward extradiol product formation. Reaction coordinate calculations were thus performed to evaluate both the extra- and intradiol O-O cleavage for the simple organic alkylhydroperoxo and for the FeII and FeIII metal catalyzed reactions. These results show the FeII-alkylhydroperoxo (EDO) intermediate undergoes facile extradiol O-O bond homolysis due to its extra e-, while for the FeIII-alkylperoxo (IDO) intermediate the extradiol cleavage involves a large barrier and would yield the incorrect extradiol product. This prompted our evaluation of a viable mechanism to rearrange the FeIII-alkylperoxo IDO intermediate for intradiol cleavage, revealing a key role in the rebinding of the displaced Tyr447 ligand in this rearrangement, driven by the proton delivery necessary for O-O bond cleavage.

Structures of an extradiol catechol dioxygenase – C23O64, from 3-nitrotoluene degrading Diaphorobacter sp. strain DS2 in substrate-free, substrate-bound and substrate analog-bound states

AbstractThis manuscript reports structure–function studies of Catechol 2,3-dioxygenase (C23O64), which is the second enzyme in the metabolic degradation pathway of 3-nitrotoluene by Diaphorobacter sp. strain DS2. The recombinant protein is a ring cleavage enzyme for 3-methylcatechol and 4-methylcatechol products formed after dioxygenation of the aromatic ring. Here we report the substrate-free, substrate-bound, and substrate-analog bound crystal structures of C23O64. The protein crystallizes in the P6(2)22 space-group. The structures were determined by molecular replacement and refined to resolutions of 2.4, 2.4, 2.2 Å, respectively. A comparison of the structures with related extradiol dioxygenases showed 22 conserved residues. A comparison of the active site pocket with catechol 2,3-dioxygenase (LapB) from Pseudomonas sp KL28 and homoprotocatechuate 2,3-dioxygenase (HPCD) from Brevibacterium fuscum shows significant similarities to suggest that the mechanism of enzyme action is similar to HPCD.

Enhancement of biocatalyst activity and protection against stressors using a microbial exoskeleton

Whole cell biocatalysts can perform numerous industrially-relevant chemical reactions. While they are less expensive than purified enzymes, whole cells suffer from inherent reaction rate limitations due to transport resistance imposed by the cell membrane. Furthermore, it is desirable to immobilize the biocatalysts to enable ease of separation from the reaction mixture. In this study, we used a layer-by-layer (LbL) self-assembly process to create a microbial exoskeleton which, simultaneously immobilized, protected, and enhanced the reactivity of a whole cell biocatalyst. As a proof of concept, we used Escherichia coli expressing homoprotocatechuate 2,3-dioxygenase (HPCD) as a model biocatalyst and coated it with up to ten alternating layers of poly(diallyldimethylammonium chloride) (PDADMAC) and silica. The microbial exoskeleton also protected the biocatalyst against a variety of external stressors including: desiccation, freeze/thaw, exposure to high temperatures, osmotic shock, as well as against enzymatic attack by lysozyme, and predation by protozoa. While we observed increased permeability of the outer membrane after exoskeleton deposition, this had a moderate effect on the reaction rate (up to two-fold enhancement). When the exoskeleton construction was followed by detergent treatment to permeabilize the cytoplasmic membrane, up to 15-fold enhancement in the reaction rate was reached. With the exoskeleton, we increased in the reaction rate constants as much as 21-fold by running the biocatalyst at elevated temperatures ranging from 40 °C to 60 °C, a supraphysiologic temperature range not accessible by unprotected bacteria.

Nuclear Resonance Vibrational Spectroscopy Definition of O2 Intermediates in an Extradiol Dioxygenase: Correlation to Crystallography and Reactivity.

The extradiol dioxygenases are a large subclass of mononuclear nonheme Fe enzymes that catalyze the oxidative cleavage of catechols distal to their OH groups. These enzymes are important in bioremediation, and there has been significant interest in understanding how they activate O2. The extradiol dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) provides an opportunity to study this process, as two O2 intermediates have been trapped and crystallographically defined using the slow substrate 4-nitrocatechol (4NC): a side-on Fe-O2-4NC species and a Fe-O2-4NC peroxy bridged species. Also with 4NC, two solution intermediates have been trapped in the H200N variant, where H200 provides a second-sphere hydrogen bond in the wild-type enzyme. While the electronic structure of these solution intermediates has been defined previously as FeIII-superoxo-catecholate and FeIII-peroxy-semiquinone, their geometric structures are unknown. Nuclear resonance vibrational spectroscopy (NRVS) is an important tool for structural definition of nonheme Fe-O2 intermediates, as all normal modes with Fe displacement have intensity in the NRVS spectrum. In this study, NRVS is used to define the geometric structure of the H200N-4NC solution intermediates in HPCD as an end-on FeIII-superoxo-catecholate and an end-on FeIII-hydroperoxo-semiquinone. Parallel calculations are performed to define the electronic structures and protonation states of the crystallographically defined wild-type HPCD-4NC intermediates, where the side-on intermediate is found to be a FeIII-hydroperoxo-semiquinone. The assignment of this crystallographic intermediate is validated by correlation to the NRVS data through computational removal of H200. While the side-on hydroperoxo semiquinone intermediate is computationally found to be nonreactive in peroxide bridge formation, it is isoenergetic with a superoxo catecholate species that is competent in performing this reaction. This study provides insight into the relative reactivities of FeIII-superoxo and FeIII-hydroperoxo intermediates in nonheme Fe enzymes and into the role H200 plays in facilitating extradiol catalysis.

Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies.

The extradiol, aromatic ring-cleaving enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes a complex chain of reactions that involve second sphere residues of the active site. The importance of the second-sphere residue His200 was demonstrated in studies of HPCD variants, such as His200Cys (H200C), which revealed significant retardations of certain steps in the catalytic process as a result of the substitution, allowing novel reaction cycle intermediates to be trapped for spectroscopic characterization. As the H200C variant largely retains the wild-type active site structure and produces the correct ring-cleaved product, this variant presents a valuable target for mechanistic HPCD studies. Here, the high-spin Fe(II) states of resting H200C and the H200C-homoprotocatechuate enzyme-substrate (ES) complex have been characterized with Mössbauer spectroscopy to assess the electronic structures of the active site in these states. The analysis reveals a high-spin Fe(II) center in a low symmetry environment that is reflected in the values of the zero-field splitting (ZFS) (D ≈ - 8 cm(-1), E/D ≈ 1/3 in ES), as well as the relative orientations of the principal axes of the (57)Fe magnetic hyperfine (A) and electric field gradient (EFG) tensors relative to the ZFS tensor axes. A spin Hamiltonian analysis of the spectra for the ES complex indicates that the magnetization axis of the integer-spin S = 2 Fe(II) system is nearly parallel to the symmetry axis, z, of the doubly occupied dxy ground orbital deduced from the EFG and A-values, an observation, which cannot be rationalized by DFT assisted crystal-field theory. In contrast, ORCA/CASSCF calculations for the ZFS tensor in combination with DFT calculations for the EFG- and A-tensors describe the experimental data remarkably well.

Unravelling the Molecular Origin of the Regiospecificity in Extradiol Catechol Dioxygenases.

Many factors have been suggested to control the selectivity for extradiol or intradiol cleavage in catechol dioxygenases. The varied selectivity of model complexes and the ability to force an extradiol enzyme to do intradiol cleavage indicate that the problem may be complex. In this paper we focus on the regiospecificity of the proximal extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase (HPCD), for which considerable advances have been made in our understanding of the mechanism from an experimental and computational standpoint. Two key steps in the reaction mechanism were investigated: (1) attack of the substrate by the superoxide moiety and (2) attack of the substrate by the oxyl radical generated by O-O bond cleavage. The selectivity at both steps was investigated through a systematic study of the role of the substrate and the first and second coordination spheres. For the isolated native substrate, intradiol cleavage is calculated to be both kinetically and thermodynamically favored, therefore nature must use the enzyme environment to reverse this preference. Two second sphere residues were found to play key roles in controlling the regiospecificity of the reaction: Tyr257 and His200. Tyr257 controls the selectivity by modulating the electronic structure of the substrate, while His200 controls selectivity through steric effects and by preventing alternative pathways to intradiol cleavage.

Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography

Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ∼16 min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.

A Long-Lived Fe(III)-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by Mössbauer, Electron Paramagnetic Resonance, and Density Functional Theory Methods.

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site Fe(II). Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both kcat and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 Fe(III) center coupled to an SR = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in Hexch = JŜ1·ŜR. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone(•))Fe(III)(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mössbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the (57)Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound (17)OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe(III)-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.

Thermodynamics of substrate binding to the metal site in homoprotocatechuate 2,3-dioxygenase: Using ITC under anaerobic conditions to study enzyme–substrate interactions

BackgroundExtradiol dioxygenases are a family of nonheme iron (and sometimes manganese) enzymes that catalyze an O2-dependent ring-opening reaction in a biodegradation pathway of aromatic compounds. Here we characterize the thermodynamics of two substrates binding in homoprotocatechuate 2,3-dioxygenase (HPCD) prior to the O2 activation step. MethodsThis study uses microcalorimetry under an inert atmosphere to measure thermodynamic parameters associated with catechol binding to nonheme metal centers in HPCD. Several stopped-flow rapid mixing experiments were used to support the calorimetry experiments. ResultsThe equilibria constant for 4-nitrocatechol and homoprotocatechuate binding to the iron(II) and manganese(II) forms of HPCD range from 2×104 to 1×106, suggesting there are distinctive differences in how the enzyme–substrate complexes are stabilized. Further experiments in multiple buffers allowed us to correct the experimental ΔH for substrate ionization and to fully derive the pH and buffer independent thermodynamic parameters for substrate binding to HPCD. Fewer protons are released from the iron(II) dependent processes than their manganese(II) counterparts. ConclusionsCondition independent thermodynamic parameters for 4-nitrocatechol and homoprotocatechuate binding to HPCD are highly consistent with each other, suggesting these enzyme–substrate complexes are more similar than once thought, and the ionization state of metal coordinated waters may be playing a role in tuning redox potential and in governing reactivity. General significanceSubstrate binding to HPCD is a complex set of equilibria that includes ionization of substrate and water release, yet it is also the key step in O2 activation. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

Reaction Mechanism of Homoprotocatechuate 2,3-Dioxygenase with 4-Nitrocatechol: Implications for the Role of Substrate

The reaction mechanism of the dioxygen activation by homoprotocatechuate 2,3-dioxygenase (HPCD) with the substrate 4-nitrocatechol was investigated by quantum mechanical/molecular mechanical calculations. Our results demonstrated that the experimentally determined side-on iron-oxygen complex in crystallo is a semiquinone substrate radical (SQ(•))-Fe(III)-hydroperoxo species, which could not act as the reactive species. In fact, the Fe(III)-superoxo species with a hydrogen bond between His200 and the proximal oxygen is the reactive oxygen species. The second-sphere His200 residue was found to play an important role in manipulating the orientation of the superoxide in the Fe-O2 adduct for the further reaction. The rate-limiting step is the attack of the superoxo group on the substrate with a barrier of 17.2 kcal/mol, in good agreement with the experimental value of 16.8 kcal/mol. The reaction mechanism was then compared with the one for HPCD with its native substrate homoprotocatechuate studied recently by the same methods, in which a hybrid SQ(•)-Fe(II)-O2(•-)/Fe(III)-O2(•-) was suggested to be the reactive species. Therefore, our studies suggested that the substrate plays important roles in the dioxygen activation by HPCD.

Oxygen activation by homoprotocatechuate 2,3-dioxygenase: a QM/MM study reveals the key intermediates in the activation cycle

Oxygen activation by homoprotocatechuate 2,3-dioxygenase (HPCD) was investigated by quantum mechanical/molecular mechanical (QM/MM) calculations. Our results demonstrated that the semiquinone substrate radical-FeII-superoxo (SQ•–FeII–O2•−) and the corresponding FeIII-superoxo species are both present within the protein environment. Moreover, we also located a species, which possesses a hybrid SQ•–FeII–O2•−/FeIII–O2•− character (so-called hybrid state) with a hydrogen bond between His200 and the proximal oxygen. His200 was found to play an important role in controlling the electronic configuration of the superoxide species. A mere reorientation of the hydrogen bonding donated by His200, from its interaction with the substrate's oxygen to interaction with the proximal oxygen of the dioxygen moiety, causes a fast rearrangement from FeIII-superoxo to the hybrid state with partial electron transfer from the substrate to the Fe center. Since the hybrid state reacts further with a low barrier, then during the oxidation process all the FeIII-superoxo species are converted to the hybrid state, which is consumed rapidly by the substrate oxidation process. This theoretical result agrees quite well with the mechanism proposed in previous experimental investigation by Lipscomb et al., Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 16788–16793, where the FeIII–O2•− was suggested to be able to convert to the true reactive species, the SQ•–FeII–O2•− species, rapidly with one-electron transfer from the substrate to iron.

Exploring substrate binding in homoprotocatechuate 2,3-dioxygenase using isothermal titration calorimetry

Homoprotocatechuate 2,3-dioxygenase (HPCD) is a member of the extradiol dioxygenase family of non-heme iron enzymes. These enzymes catalyze the ring-cleavage step in the aromatic degradation pathway commonly found in soil bacteria. In this study, isothermal titration calorimetry (ITC) is used to measure the equilibrium constant (K = 1.1 ± 0.6 × 10(6)) and enthalpy change (ΔH = -17.0 ± 1.7 kcal/mol) associated with homoprotocatechuate binding to HPCD. The ITC data are consistent with the release of approximately 2.6 protons upon binding of the substrate to HPCD. These results raise new questions regarding the relationships between substrate, protein, and the oxygen activation mechanism for this class of non-heme metalloenzymes.

Oxygen activation in extradiol catecholate dioxygenases – a density functional study

Recent trapping and spectroscopic characterization of an O2 adduct for the non-heme enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) demonstrates it to be a FeIII-superoxo species. This proposal is in direct opposition to the consensus mechanism (J. P. Emerson, E. G. Kovaleva, E. R. Farquhar, J. D. Lipscomb and L. Que, Jr., Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 7347–7352) in which the metal facilitates the transfer of electrons from the substrate to O2 to form the reactive species in the mechanism without changing oxidation state. In this study we performed a detailed analysis of the electronic structure of the O2 adduct for the mutant and native enzymes and the nature of oxygen activation in the reaction mechanism of HPCD using a combination of computational chemistry and theoretical Mossbauer spectroscopy. Our results are in agreement with the available experimental data and demonstrate that even for the native enzyme changes in the metal oxidation state are an important factor in oxygen activation.

On the observation of a gem diol intermediate after O–O bond cleavage by extradiol dioxygenases. A hybrid DFT study

Catalytic cycle intermediates of a representative extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase (HPCD), have recently been characterized in crystallo by Kovaleva and Lipscomb. The structures of the identified species indicate that the process of inserting oxygen into the catechol ring occurs stepwise, and involves an Fe(II)-alkylperoxo intermediate and its O-O cleavage product: a gem diol species. In general, these findings corroborate the results of our previous computational studies; however, the fact that the gem diol species is stable enough to be observed in the crystal form seems to be at odds with the computational mechanistic data, which suggest that this intermediate should very readily and spontaneously convert to the epoxide species. The key question then becomes what is actually observed in the X-ray experiments. Here we report additional computational studies undertaken with the hope of clarifying this issue. The results obtained for active site models hosting both the native and the alternative (4-sulfonylcatechol) substrate indicate that the stability of the gem diol species is substantially increased if an electron and a proton are added. If this occurs somehow, the lifetime of the intermediate should be sufficient to observe it.

Intermediate in the O−O Bond Cleavage Reaction of an Extradiol Dioxygenase,

The reactive oxy intermediate of the catalytic cycle of extradiol aromatic ring-cleaving dioxygenases is formed by binding the catecholic substrate and O2 in adjacent ligand positions of the active site metal [usually Fe(II)]. This intermediate and the following Fe(II)-alkylperoxo intermediate resulting from oxygen attack on the substrate have been previously characterized in a crystal of homoprotocatechuate 2,3-dioxygenase (HPCD). Here a subsequent intermediate in which the O-O bond is broken to yield a gem diol species is structurally characterized. This new intermediate is stabilized in the crystal by using the alternative substrate, 4-sulfonylcatechol, and the Glu323Leu variant of HPCD, which alters the crystal packing.

Cloning, Overexpression, and Mutagenesis of the Gene for Homoprotocatechuate 2,3-Dioxygenase fromBrevibacterium fuscum

Homoprotocatechuate (hpca, 3,4-dihydroxyphenylacetate) is a central intermediate for the bacterial degradation of aromatic compounds. Homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes the key ring cleavage step in the metabolism of hpca by the Gram (+) bacteriumBrevibacterium fuscumto yield α-hydroxy-δ-carboxymethylcis-muconic semialdehyde. A genomic DNA library ofB. fuscumwas constructed inEscherichia coliusing a cosmid vector and screened by spraying the cells with hpca. One clone was found to contain the gene for HPCD based on its ability to convert hpca into the yellow-colored product. This cosmid clone was further subcloned and the gene for HPCD was localized and sequenced. The open reading frame codes for a protein with 365 amino acids andMr= 41,699, in accord with the characteristics of the previously purified wild-type enzyme. The gene for HPCD was overexpressed inE. colito ∼30% of the total soluble protein, and purification of the recombinant enzyme to apparent homogeneity was achieved by a two-step procedure. Iron was the only abundant metal found in the purified recombinant enzyme, and the specific activity per iron was comparable to that observed for the wild-type enzyme. The deduced amino acid sequence of HPCD has a very high level of homology (78.6% identity in the 337-aa overlap) to the manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) fromArthrobacter globiformisCM-2. The basis for the difference in metal selection by HPCD and MndD was investigated by mutagenesis of a 50-base-pair region of the HPCD gene containing three frame shifts relative to the MndD gene. The purified triple mutant of HPCD did not exhibit a significant change in the metal content; therefore, other factors must contribute to the selection of the active site metal.