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John D Lipscomb - One of the best experts on this subject based on the ideXlab platform.
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roles of the equatorial tyrosyl iron ligand of Protocatechuate 3 4 dioxygenase in catalysis
Biochemistry, 2005Co-Authors: Michael P Valley, Douglas H Ohlendorf, M W Vetting, Kent C Brown, David L Burk, John D LipscombAbstract:The active site Fe(III) of Protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH - . Tyr447 and OH - are displaced as Protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O 2 becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes. Protocatechuate 3,4-dioxygenase (3,4-PCD) 1 (E.C. 1.13.11.3) catalyzes the ring cleavage of Protocatechuate (PCA, 3,4- dihydroxybenzoate) with the incorporation of both atoms of molecular oxygen to form ‚-carboxy-cis,cis-muconate (1, 2).
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spectroscopic and electronic structure studies of Protocatechuate 3 4 dioxygenase nature of tyrosinate fe iii bonds and their contribution to reactivity
Journal of the American Chemical Society, 2002Co-Authors: Mindy I Davis, John D Lipscomb, Allen M Orville, Jeffrey M Zaleski, Frank Neese, Edward I SolomonAbstract:The geometric and electronic structure of the high-spin ferric active site of Protocatechuate 3,4-dioxygenase (3,4-PCD) has been examined by absorption (Abs), circular dichroism (CD), magnetic CD (MCD), and variable-temperature−variable-field (VTVH) MCD spectroscopies. Density functional (DFT) and INDO/S-CI molecular orbital calculations provide complementary insight into the electronic structure of 3,4-PCD and allow an experimentally calibrated bonding scheme to be developed. Abs, CD, and MCD indicate that there are at least seven transitions below 35 000 cm-1 which arise from tyrosinate ligand-to-metal-charge transfer (LMCT) transitions. VTVH MCD spectroscopy gives the polarizations of these LMCT bands in the principal axis system of the D-tensor, which is oriented relative to the molecular structure from the INDO/S-CI calculations. Three transitions are associated with the equatorial tyrosinate and four with the axial tyrosinate. This large number of transitions per tyrosinate is due to the π and impor...
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spectroscopic investigation of reduced Protocatechuate 3 4 dioxygenase charge induced alterations in the active site iron coordination environment
Inorganic Chemistry, 1999Co-Authors: Mindy I Davis, John D Lipscomb, Allen M Orville, Erik C Wasinger, Tami E Westre, Jeffrey M Zaleski, Britt Hedman, Keith O Hodgson, Edward I SolomonAbstract:Chemical reduction of the mononuclear ferric active site in the bacterial intradiol cleaving catecholic dioxygenase Protocatechuate 3,4-dioxygenase (3,4-PCD, Brevibacterium fuscum) produces a high-spin ferrous center. We have applied circular dichroism (CD), magnetic circular dichroism (MCD), variable-temperature-variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies to investigate the geometric and electronic structure of the reduced iron center. Excited-state ligand field CD and MCD data indicate that the site is six-coordinate where the 5Eg excited-state splitting is 2033 cm-1. VTVH MCD analysis of the ground state indicates that the site has negative zero-field splitting with a small rhombic splitting of the lowest doublet (δ = 1.6 ± 0.3 cm-1). XAS pre-edge analysis also indicates a six-coordinate site while EXAFS analysis provides accurate bond lengths. Since previous spectroscopic analysis and the crystal structure of oxidized ...
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the axial tyrosinate fe3 ligand in Protocatechuate 3 4 dioxygenase influences substrate binding and product release evidence for new reaction cycle intermediates
Biochemistry, 1998Co-Authors: Richard W Frazee, Douglas H Ohlendorf, Allen M Orville, Kevin B Dolbeare, John D LipscombAbstract:The essential active site Fe3+ of Protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with Protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.
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probing the reaction mechanism of Protocatechuate 3 4 dioxygenase with x ray crystallography
The Keio Journal of Medicine, 1998Co-Authors: Allen M Orville, John D Lipscomb, Douglas H OhlendorfAbstract:Protocatechuate 3,4-dioxygenase (3,4-PCD, E) catalyzes the oxidative ring cleavage of 3,4-dihydroxybenzoate (PCA) to produce β-carboxy-cis,cis-muconate. Previous results suggest that several short-lived intermediates and a conformational change are encountered during formation of the E • substrate complex followed by ternary E • PCA • O2 intermediates and product formation. In this review, the crystal structures of 3,4-PCD as isolated and complexed with 3-I,4-OH-benzoate (IHB), 3-F,4-OH-benzoate (FHB), PCA, or analogs of the ketonized PCA isomers [2-OH-isonicotinate N-oxide (INO) and 6-OH-nicotinate N-oxide (NNO)] plus CN− are compared as structural probes of the reaction pathway. The crystal structures of E • IHB and E • FHB appear to mimic the intermediate stages of PCA binding and suggest that the Fe3+-OH facilitates C4-OH proton abstraction. In contrast to the monodentate iron complexes of E • IHB and E • FHB, PCA asymmetrically chelates the iron in the anaerobic E • PCA complex. Concurrent dissociation of the axial tyrosinate (Tyr-447) generates an active site base (pK a ~ 10) capable of abstracting the PCA C3-OH proton to yield the dianionic Fe3+ • PCA complex. After its release from the iron, Tyr-447 forms the top of a small cavity adjacent to the C3-C4 bond of PCA. The ternary E • INO • CN complex mimics an E • PCA • O2 complex, and the structure shows that CN− binds to the Fe3+ in the adjacent cavity. This suggests that 3,4-PCD sequesters PCA and O2 as well as ensuing intermediates during catalysis.
Douglas H Ohlendorf - One of the best experts on this subject based on the ideXlab platform.
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Protocatechuate 3 4 dioxygenase
Encyclopedia of Inorganic and Bioinorganic Chemistry, 2006Co-Authors: Douglas H Ohlendorf, M W VettingAbstract:A key step in the degradation of aromatic compounds in the biosphere is the ring-opening step. Intradiol dioxygenases typically use a nonheme ferric iron to activate the substrate for an electrophilic attack by molecular oxygen to cleave catechol derivatives between the vicinal hydroxyls. Protocatechuate 3,4-dioxygenase (PCD) has been the most thoroughly studied of the intradiol dioxygenases because of the presence of optical and electron paramagnetic resonance (EPR) spectroscopic signals. The structures of PCD from Pseudomonas putida and Acinetobacter calcoaceticus alone and in complexes with more than a dozen substrates and inhibitors have been used to visualize steps in substrate binding and ligand dissociation. 3D Structure Keywords: iron; non-Heme Proteins; mononuclear Iron Proteins; enzyme; Protocatechuate 3; 4-dioxygenase; EC 1.13.11.13; non-heme iron oxidoreductase
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roles of the equatorial tyrosyl iron ligand of Protocatechuate 3 4 dioxygenase in catalysis
Biochemistry, 2005Co-Authors: Michael P Valley, Douglas H Ohlendorf, M W Vetting, Kent C Brown, David L Burk, John D LipscombAbstract:The active site Fe(III) of Protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH - . Tyr447 and OH - are displaced as Protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O 2 becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes. Protocatechuate 3,4-dioxygenase (3,4-PCD) 1 (E.C. 1.13.11.3) catalyzes the ring cleavage of Protocatechuate (PCA, 3,4- dihydroxybenzoate) with the incorporation of both atoms of molecular oxygen to form ‚-carboxy-cis,cis-muconate (1, 2).
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biophysical analyses of designed and selected mutants of Protocatechuate 3 4 dioxygenase
Annual Review of Microbiology, 2004Co-Authors: Kent C Brown, M W Vetting, Cathleen A Earhart, Douglas H OhlendorfAbstract:▪ Abstract The catechol dioxygenases allow a wide variety of bacteria to use aromatic compounds as carbon sources by catalyzing the key ring-opening step. These enzymes use specifically either catechol or Protocatechuate (2,3-dihydroxybenozate) as their substrates; they use a bare metal ion as the sole cofactor. To learn how this family of metalloenzymes functions, a structural analysis of designed and selected mutants of these enzymes has been undertaken. Here we review the results of this analysis on the nonheme ferric iron intradiol dioxygenase Protocatechuate 3,4-dioxygenase.
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structure of acinetobacter strain adp1 Protocatechuate 3 4 dioxygenase at 2 2 a resolution implications for the mechanism of an intradiol dioxygenase
Biochemistry, 2000Co-Authors: M W Vetting, L N Ornston, D A Dargenio, Douglas H OhlendorfAbstract:The crystal structures of Protocatechuate 3,4-dioxygenase from the soil bacteria Acinetobacterstrain ADP1 (Ac 3,4-PCD) have been determined in space group I23 at pH 8.5 and 5.75. In addition, the structures of Ac 3,4-PCD complexed with its substrate 3, 4-dihydroxybenzoic acid (PCA), the inhibitor 4-nitrocatechol (4-NC), or cyanide (CN(-)) have been solved using native phases. The overall tertiary and quaternary structures of Ac 3,4-PCD are similar to those of the same enzyme from Pseudomonas putida[Ohlendorf et al. (1994) J. Mol. Biol. 244, 586-608]. At pH 8.5, the catalytic non-heme Fe(3+) is coordinated by two axial ligands, Tyr447(OH) (147beta) and His460(N)(epsilon)(2) (160beta), and three equatorial ligands, Tyr408(OH) (108beta), His462(N)(epsilon)(2) (162beta), and a hydroxide ion (d(Fe-OH) = 1.91 A) in a distorted bipyramidal geometry. At pH 5.75, difference maps suggest a sulfate binds to the Fe(3+) in an equatorial position and the hydroxide is shifted [d(Fe-OH) = 2.3 A] yielding octahedral geometry for the active site Fe(3+). This change in ligation geometry is concomitant with a shift in the optical absorbance spectrum of the enzyme from lambda(max) = 450 nm to lambda(max) = 520 nm. Binding of substrate or 4-NC to the Fe(3+) is bidentate with the axial ligand Tyr447(OH) (147beta) dissociating. The structure of the 4-NC complex supports the view that resonance delocalization of the positive character of the nitrogen prevents substrate activation. The cyanide complex confirms previous work that Protocatechuate 3,4-dioxygenases have three coordination sites available for binding by exogenous substrates. A significant conformational change extending away from the active site is seen in all structures when compared to the native enzyme at pH 8.5. This conformational change is discussed in its relevance to enhancing catalysis in Protocatechuate 3,4-dioxygenases.
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substitution insertion deletion suppression and altered substrate specificity in functional Protocatechuate 3 4 dioxygenases
Journal of Bacteriology, 1999Co-Authors: Douglas H Ohlendorf, David A Dargenio, Mathew W Vetting, Nicholas L OrnstonAbstract:Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for Acinetobacter strains containing spontaneous mutations blocking expression of pcaH or -G, genes encoding the a and b subunits of Protocatechuate 3,4-dioxygenase. The crystal structure of the Acinetobacter oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivating Acinetobacter Protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate Acinetobacter strains in which mutations within pcaH or -G cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in pcaH or -G were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into the Acinetobacter chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.
Allen M Orville - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic and electronic structure studies of Protocatechuate 3 4 dioxygenase nature of tyrosinate fe iii bonds and their contribution to reactivity
Journal of the American Chemical Society, 2002Co-Authors: Mindy I Davis, John D Lipscomb, Allen M Orville, Jeffrey M Zaleski, Frank Neese, Edward I SolomonAbstract:The geometric and electronic structure of the high-spin ferric active site of Protocatechuate 3,4-dioxygenase (3,4-PCD) has been examined by absorption (Abs), circular dichroism (CD), magnetic CD (MCD), and variable-temperature−variable-field (VTVH) MCD spectroscopies. Density functional (DFT) and INDO/S-CI molecular orbital calculations provide complementary insight into the electronic structure of 3,4-PCD and allow an experimentally calibrated bonding scheme to be developed. Abs, CD, and MCD indicate that there are at least seven transitions below 35 000 cm-1 which arise from tyrosinate ligand-to-metal-charge transfer (LMCT) transitions. VTVH MCD spectroscopy gives the polarizations of these LMCT bands in the principal axis system of the D-tensor, which is oriented relative to the molecular structure from the INDO/S-CI calculations. Three transitions are associated with the equatorial tyrosinate and four with the axial tyrosinate. This large number of transitions per tyrosinate is due to the π and impor...
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spectroscopic investigation of reduced Protocatechuate 3 4 dioxygenase charge induced alterations in the active site iron coordination environment
Inorganic Chemistry, 1999Co-Authors: Mindy I Davis, John D Lipscomb, Allen M Orville, Erik C Wasinger, Tami E Westre, Jeffrey M Zaleski, Britt Hedman, Keith O Hodgson, Edward I SolomonAbstract:Chemical reduction of the mononuclear ferric active site in the bacterial intradiol cleaving catecholic dioxygenase Protocatechuate 3,4-dioxygenase (3,4-PCD, Brevibacterium fuscum) produces a high-spin ferrous center. We have applied circular dichroism (CD), magnetic circular dichroism (MCD), variable-temperature-variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies to investigate the geometric and electronic structure of the reduced iron center. Excited-state ligand field CD and MCD data indicate that the site is six-coordinate where the 5Eg excited-state splitting is 2033 cm-1. VTVH MCD analysis of the ground state indicates that the site has negative zero-field splitting with a small rhombic splitting of the lowest doublet (δ = 1.6 ± 0.3 cm-1). XAS pre-edge analysis also indicates a six-coordinate site while EXAFS analysis provides accurate bond lengths. Since previous spectroscopic analysis and the crystal structure of oxidized ...
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the axial tyrosinate fe3 ligand in Protocatechuate 3 4 dioxygenase influences substrate binding and product release evidence for new reaction cycle intermediates
Biochemistry, 1998Co-Authors: Richard W Frazee, Douglas H Ohlendorf, Allen M Orville, Kevin B Dolbeare, John D LipscombAbstract:The essential active site Fe3+ of Protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with Protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.
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probing the reaction mechanism of Protocatechuate 3 4 dioxygenase with x ray crystallography
The Keio Journal of Medicine, 1998Co-Authors: Allen M Orville, John D Lipscomb, Douglas H OhlendorfAbstract:Protocatechuate 3,4-dioxygenase (3,4-PCD, E) catalyzes the oxidative ring cleavage of 3,4-dihydroxybenzoate (PCA) to produce β-carboxy-cis,cis-muconate. Previous results suggest that several short-lived intermediates and a conformational change are encountered during formation of the E • substrate complex followed by ternary E • PCA • O2 intermediates and product formation. In this review, the crystal structures of 3,4-PCD as isolated and complexed with 3-I,4-OH-benzoate (IHB), 3-F,4-OH-benzoate (FHB), PCA, or analogs of the ketonized PCA isomers [2-OH-isonicotinate N-oxide (INO) and 6-OH-nicotinate N-oxide (NNO)] plus CN− are compared as structural probes of the reaction pathway. The crystal structures of E • IHB and E • FHB appear to mimic the intermediate stages of PCA binding and suggest that the Fe3+-OH facilitates C4-OH proton abstraction. In contrast to the monodentate iron complexes of E • IHB and E • FHB, PCA asymmetrically chelates the iron in the anaerobic E • PCA complex. Concurrent dissociation of the axial tyrosinate (Tyr-447) generates an active site base (pK a ~ 10) capable of abstracting the PCA C3-OH proton to yield the dianionic Fe3+ • PCA complex. After its release from the iron, Tyr-447 forms the top of a small cavity adjacent to the C3-C4 bond of PCA. The ternary E • INO • CN complex mimics an E • PCA • O2 complex, and the structure shows that CN− binds to the Fe3+ in the adjacent cavity. This suggests that 3,4-PCD sequesters PCA and O2 as well as ensuing intermediates during catalysis.
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cyanide and nitric oxide binding to reduced Protocatechuate 3 4 dioxygenase insight into the basis for order dependent ligand binding by intradiol catecholic dioxygenases
Biochemistry, 1997Co-Authors: Allen M Orville, John D LipscombAbstract:EPR-silent, chemically reduced Protocatechuate 3,4-dioxygenase (Er) binds NO at the active site Fe2+ to yield an EPR-active, S = 3/2 species that blocks subsequent binding of all other exogenous ligands. In contrast, addition of NO to a preformed Er.CN- complex yields an EPR-active, S = 1/2 species [Er.(CN)x.NO] that exhibits resolved superhyperfine splitting from 13CN-, 15/14NO, and a protein-derived 14N. Simulations of the EPR spectra observed for the Er.(CN)x.NO complex formed with 12CN- and 13CN- (1:1) show that CN- binds in two iron ligand sites (x >/= 2). The two cyanides exhibit similar, but distinguishable, hyperfine coupling constants. This demonstrates unambiguously that at least three exogenous ligands (two cyanides and NO) can bind to the Fe2+ simultaneously and strongly suggests that at least one histidine ligand is retained in the complex. The Er.(CN)>/=2.NO complex readily exchanges both of the bound cyanides for the substrate analog, 2-hydroxyisonicotinic acid N-oxide (INO), to form a Er.INO.NO complex exhibiting the same S = 3/2 type EPR spectrum that is observed for this complex in the absence of CN-. Because the dead-end Er.NO complex does not accumulate during the exchange, the results suggest that Er.(CN)>/=2. NO and Er.INO.NO are in conformational states that allow facile exchange of INO and CN- but not NO. The results are interpreted in the context of the known X-ray crystal structures for the ferric form of the resting enzyme (Eox) and numerous Eox.substrate, inhibitor, and CN- complexes, all of which have a charge neutral iron center. It is proposed that the binding of one CN- causes dissociation of an anionic endogenous ligand which begins a series of conformational changes analogous to those initiated by anionic substrate binding to Eox. This results in a new unique coordination site for NO, and a new second site for CN-; both cyanide sites are utilized when the enzyme subsequently binds substrates or INO.
Lawrence Que - One of the best experts on this subject based on the ideXlab platform.
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crystal structure and resonance raman studies of Protocatechuate 3 4 dioxygenase complexed with 3 4 dihydroxyphenylacetate
Biochemistry, 1997Co-Authors: Timothy E Elgren, Douglas H Ohlendorf, John D Lipscomb, Allen M Orville, Kimberly A Kelly, Lawrence QueAbstract:The crystal structure of the anaerobic complex of Pseudomonas putida Protocatechuate 3,4-dioxygenase (3,4-PCD) bound with the alternative substrate, 3,4-dihydroxyphenylacetate (HPCA), is reported at 2.4 A resolution and refined to an R factor of 0.17. Formation of the active site Fe(III)·HPCA chelated complex causes the endogenous axial tyrosinate, Tyr447 (147β), to dissociate from the iron and rotate into an alternative orientation analogous to that previously observed in the anaerobic 3,4-PCD·3,4-dihydroxybenzoate complex (3,4-PCD·PCA) [Orville, A. M., Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10052−10066]. Two orientations of the aromatic ring of HPCA related by an approximate 180° rotation within the active site are consistent with the electron density. Resonance Raman (rR) spectroscopic data from Brevibacterium fuscum 3,4-PCD·HPCA complex in solution reveals low frequency rR vibrational bands between 500 and 650 cm-1 as well as a band at ∼1320 cm-1 which are diagnostic of a HPCA·Fe(...
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resonance raman studies of the Protocatechuate 3 4 dioxygenase from brevibacterium fuscum
Biochemistry, 1992Co-Authors: Daniel C T Siu, Douglas H Ohlendorf, John D Lipscomb, Allen M Orville, Lawrence QueAbstract:Resonance Raman studies of the Protocatechuate 3,4-dioxygenase (PCD) from Brevibacterium fuscum have been carried out to take advantage of the high iron-site homogeneity of this enzyme. Native uncomplexed PCD exhibits individual resonance-enhanced nu CO and delta CH vibrations for the two tyrosinates coordinated to the active site iron center, which can be assigned to a particular residue by their excitation profiles. Of the two nu CO features observed at 1254 and 1266 cm-1, only the latter is upshifted (to 1272 cm-1) when H2O is replaced by D2O. Similarly the 1254-cm-1 feature is unaffected, while the 1266-cm-1 feature is shifted to approximately 1290 cm-1 when inhibitors such as phenolates or terephthalate bind to the active site. These observed shifts can be rationalized by the presence of hydrogen-bonding interactions with solvent in the active site cavity, which are modulated by D2O and eliminated upon inhibitor binding. Examination of the PCD crystal structure suggests that the axial tyrosine can be hydrogen bonded in the uncomplexed enzyme to water molecules present in the substrate binding pocket. The equatorial tyrosine may also be hydrogen bonded but to solvent molecules which are trapped in a pocket inaccessible to bulk solvent. These studies allow for the first time the association of particular Raman spectroscopic features, i.e., the nu CO's at 1254 and 1266 cm-1, with the equatorial and axial tyrosine residues in the PCD active site, respectively; they lay the groundwork for further Raman studies on catalytically important species to determine the roles these tyrosine residues may play in the PCD reaction cycle.
Richard W Frazee - One of the best experts on this subject based on the ideXlab platform.
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the axial tyrosinate fe3 ligand in Protocatechuate 3 4 dioxygenase influences substrate binding and product release evidence for new reaction cycle intermediates
Biochemistry, 1998Co-Authors: Richard W Frazee, Douglas H Ohlendorf, Allen M Orville, Kevin B Dolbeare, John D LipscombAbstract:The essential active site Fe3+ of Protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with Protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.
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cloning sequencing and expression of the pseudomonas putida Protocatechuate 3 4 dioxygenase genes
Journal of Bacteriology, 1993Co-Authors: Richard W Frazee, Dennis M Livingston, David C Laporte, John D LipscombAbstract:The genes that encode the alpha and beta subunits of Protocatechuate 3,4-dioxygenase (3,4-PCD [EC 1.13.11.3]) were cloned from a Pseudomonas putida (formerly P. aeruginosa) (ATCC 23975) genomic library prepared in lambda phage. Plaques were screened by hybridization with degenerate oligonucleotides designed using known amino acid sequences. A 1.5-kb SmaI fragment from a 15-kb primary clone was subcloned, sequenced, and shown to contain two successive open reading frames, designated pcaH and pcaG, corresponding to the beta and alpha subunits, respectively, of 3,4-PCD. The amino acid sequences deduced from pcaHG matched the chemically determined sequence of 3,4-PCD in all except three positions. Cloning of pcaHG into broad-host-range expression vector pKMY319 allowed high levels of expression in P. putida strains, as well as in Proteus mirabilis after specific induction of the plasmid-encoded nahG promoter with salicylate. The recombinant enzyme was purified and crystallized from P. mirabilis, which lacks an endogenous 3,4-PCD. The physical, spectroscopic, and kinetic properties of the recombinant enzyme were indistinguishable from those of the wild-type enzyme. Moreover, the same transient enzyme intermediates were formed during the catalytic cycle. These studies establish the methodology which will allow mechanistic investigations to be pursued through site-directed mutagenesis of P. putida 3,4-PCD, the only aromatic ring-cleaving dioxygenase for which the three-dimensional structure is known.
