RESUMEN

UNLABELLED: Biphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400 in which a seven-residue segment, (335)TFNNIRI(341), has been replaced by the corresponding segment of BphAEB356, (333)GINTIRT(339), transforms a broader range of PCB congeners than does either BphAELB400 or BphAEB356, including 2,6-dichlorobiphenyl, 3,3'-dichlorobiphenyl, 4,4'-dichlorobiphenyl, and 2,3,4'-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400 reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9 that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9 transformed 2,3,4'-trichlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl more efficiently than did BphAELB400 and BphAEB356 BphAEII9 also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparent kcat/Km of 2.2 ± 0.5 mM(-1) s(-1), versus 0.9 ± 0.5 mM(-1) s(-1) for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes. IMPORTANCE: Biphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.

Asunto(s)

Compuestos de Bifenilo/metabolismo , Oxigenasas/química , Oxigenasas/genética , Ingeniería de Proteínas/métodos , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Simulación del Acoplamiento Molecular , Oxigenasas/metabolismo , Unión Proteica , Conformación Proteica , Especificidad por Sustrato

RESUMEN

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-ß-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.

Asunto(s)

Dominio Catalítico , Glucosiltransferasas/química , Oryza/enzimología , Membrana Celular/metabolismo , Pared Celular/metabolismo , Celulosa/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Modelos Moleculares , Conformación Molecular , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes , Especificidad por Sustrato

RESUMEN

The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (ßnuc ∼ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

Asunto(s)

Ácido Aspártico/química , Proteínas Bacterianas/química , Dipéptidos/química , Ácidos Grasos Insaturados/química , Hidrolasas/química , Sphingomonas/enzimología , Acilación , Ácido Aspártico/metabolismo , Proteínas Bacterianas/metabolismo , Catálisis , Cromatografía Liquida , Dipéptidos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Hidrolasas/metabolismo , Hidrólisis , Cinética , Modelos Químicos , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato

RESUMEN

DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ES(red), an intermediate possessing a bathochromically shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/ß-hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme's ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/ß-hydrolases.

Asunto(s)

Proteínas Bacterianas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Hidrolasas/metabolismo , Bifenilos Policlorados/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Burkholderia/enzimología , Burkholderia/genética , Cristalografía por Rayos X , Ácidos Grasos Insaturados/química , Hidrolasas/química , Hidrolasas/genética , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Bifenilos Policlorados/química , Multimerización de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrofotometría , Sphingomonas/enzimología , Sphingomonas/genética

RESUMEN

The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356)). BPDO(B356), a heterohexameric (αß)(3) Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDO(B356) with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDO(B356) and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2'-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments.

Asunto(s)

Compuestos de Bifenilo/metabolismo , Burkholderiaceae/enzimología , Dioxigenasas/química , Dioxigenasas/metabolismo , Bifenilos Policlorados/metabolismo , Burkholderiaceae/química , Burkholderiaceae/genética , Dominio Catalítico , Cristalografía por Rayos X , Dioxigenasas/genética , Modelos Moleculares , Mutagénesis , Filogenia , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Especificidad por Sustrato

RESUMEN

Members of the calcium/cation antiporter superfamily, including the cardiac sodium/calcium exchangers, are secondary active transporters that play an essential role in cellular Ca(2+) homeostasis. A notable feature of this group of transporters is the high levels of sequence similarity in relatively short sequences constituting the functionally important α-1 and α-2 regions in contrast to relatively lower degrees of similarity in the extended adjoining sequences. This suggests a similar structure and function of core transport machinery but possible differences in topology and/or oligomerization, a topic that has not been adequately addressed. Here we present the first example of purification of a bacterial member of this superfamily (CAX(CK31)) and analyze its quaternary structure. Purification of CAX(CK31) required the presence of a choline headgroup-containing detergent or lipid to yield stable preparations of the monomeric transporter. H(+)-driven Ca(2+) transport was demonstrated by reconstituting purified CAX(CK31) into liposomes. Dimeric CAX(CK31) could be isolated by manipulation of detergent micelles. Dimer formation was shown to be dependent on micelle composition as well as protein concentration. Furthermore, we establish that CAX(CK31) forms dimers in the membrane by analysis of cross-linked proteins. Using a dimeric homology model derived from the monomeric structure of the archaeal NCX homologue (Protein Data Bank entry 3V5U ), we introduced cysteine residues and through cross-linking experiments established the role of transmembrane helices 2 and 6 in the putative dimer interface.

Asunto(s)

Antiportadores/química , Proteínas de Transporte de Catión/química , Proteínas de Escherichia coli/química , Dicroismo Circular , Detergentes/química , Dimerización , Electroforesis en Gel de Poliacrilamida , Micelas , Modelos Moleculares

RESUMEN

The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) is a Rieske-type oxygenase that catalyzes the stereospecific oxygenation of many heterocyclic aromatics including dibenzofuran. In a previous work, we evolved BphAE(LB400) and obtained BphAE(RR41). This variant metabolizes dibenzofuran and 2-chlorodibenzofuran more efficiently than BphAE(LB400). However, the regiospecificity of BphAE(RR41) toward these substrates differs. Dibenzofuran is metabolized principally through a lateral dioxygenation whereas 2-chlorodibenzofuran is metabolized principally through an angular dioxygenation. In order to explain this difference, we examined the crystal structures of both substrate-bound forms of BphAE(RR41) obtained under anaerobic conditions. This structure analysis, in combination with biochemical data for a Ser283Gly mutant provided evidences that the substrate is compelled to move after oxygen-binding in BphAE(RR41):dibenzofuran. In BphAE(RR41):2-chlorodibenzofuran, the chlorine atom is close to the side chain of Ser283. This contact is missing in the BphAE(RR41):dibenzofuran, and strong enough in the BphAE(RR41):2-chlorodibenzofuran to help prevent substrate movement during the catalytic reaction.

Asunto(s)

Benzofuranos/metabolismo , Burkholderia/enzimología , Dioxigenasas/química , Catálisis , Cristalización , Dioxigenasas/genética , Glicina/química , Glicina/genética , Mutación , Conformación Proteica , Serina/química , Serina/genética

RESUMEN

Meta-cleavage product (MCP) hydrolases are members of the α/ß-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C-C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 Å resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme's catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of 'oxyanion hole' residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of (18)O into the benzoate produced during hydrolysis in H(2)(18)O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES(red), previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad.

Asunto(s)

Hidrolasas/química , Acilación , Biocatálisis , Enlace de Hidrógeno , Hidrólisis , Modelos Moleculares

RESUMEN

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE(p4), a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE(LB400) by the double substitution T335A/F336M, and BphAE(RR41), obtained by changing Asn(338), Ile(341), and Leu(409) of BphAE(p4) to Gln(338), Val(341), and Phe(409), metabolize dibenzofuran two and three times faster than BphAE(LB400), respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE(LB400) showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

Asunto(s)

Oxigenasas/metabolismo , Burkholderia/enzimología , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Cinética , Modelos Moleculares , Oxigenasas/química , Oxigenasas/genética , Especificidad por Sustrato

RESUMEN

Biphenyl 2,3-dioxygenase (BPDO; EC 1.14.12.18) catalyzes the initial step in the degradation of biphenyl and some polychlorinated biphenyls (PCBs). BPDOLB400, the terminal dioxygenase component from Burkholderia xenovorans LB400, a proteobacterial species that degrades a broad range of PCBs, has been crystallized under anaerobic conditions by sitting-drop vapour diffusion. Initial crystals obtained using various polyethylene glycols as precipitating agents diffracted to very low resolution (∼8 Å) and the recorded reflections were diffuse and poorly shaped. The quality of the crystals was significantly improved by the addition of 0.2% agarose to the crystallization cocktail. In the presence of agarose, wild-type BPDOLB400 crystals that diffracted to 2.4 Šresolution grew in space group P1. Crystals of the BPDOP4 and BPDORR41 variants of BPDOLB400 grew in space group P2(1).

Asunto(s)

Proteínas Bacterianas/química , Burkholderia/enzimología , Cristalización/métodos , Proteínas Hierro-Azufre/química , Oxigenasas/química , Sefarosa/química , Anaerobiosis , Cristalografía por Rayos X , Datos de Secuencia Molecular

RESUMEN

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE(LB400)) are largely unknown. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.

Asunto(s)

Burkholderiaceae/enzimología , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Oxigenasas/química , Oxigenasas/metabolismo , Bifenilos Policlorados/metabolismo , Sustitución de Aminoácidos , Evolución Biológica , Cristalografía por Rayos X , Cromatografía de Gases y Espectrometría de Masas , Proteínas Hierro-Azufre/genética , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Oxigenasas/genética , Estructura Terciaria de Proteína , Especificidad por Sustrato

RESUMEN

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H>F>Cl>Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A.3-Cl HOPDA and S112A.3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A.HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A.3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.

Asunto(s)

Proteínas Bacterianas/química , Comamonas testosteroni/enzimología , Contaminantes Ambientales/química , Ácidos Grasos Insaturados/química , Hidrolasas/química , Bifenilos Policlorados/química , Sustitución de Aminoácidos , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Comamonas testosteroni/genética , Hidrolasas/genética , Hidrólisis , Isomerismo , Cinética , Mutación Missense

RESUMEN

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO(B356) from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO(LB400) from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro approximately 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro approximately 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro approximately 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDO(B356) transformed each congener at a higher rate than BPDO(LB400). The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDO(B356) activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDO(LB400) had a greater apparent specificity for biphenyl than BPDO(B356) (k(cat)/K(m) = 2.4 x 10(6) +/- 0.7 x 10(6) M(-1) s(-1) versus k(cat)/K(m) = 0.21 x 10(6) +/- 0.04 x 10(6) M(-1) s(-1)). However, the latter transformed biphenyl at a higher maximal rate (k(cat) = 4.1 +/- 0.2 s(-1) versus k(cat) = 0.4 +/- 0.1 s(-1)). A variant of BPDO(LB400) containing four active site residues of BPDO(B356) transformed para-substituted congeners better than BPDO(LB400). Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O(2) consumption was approximately proportional to congener depletion. At 2.4-A resolution, the crystal structure of the BPDO(B356)-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.

Asunto(s)

Burkholderiaceae/química , Burkholderiaceae/enzimología , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Oxigenasas/química , Oxigenasas/metabolismo , Bifenilos Policlorados/metabolismo , Sustitución de Aminoácidos , Biotransformación , Cristalografía por Rayos X , Cromatografía de Gases y Espectrometría de Masas , Proteínas Hierro-Azufre/aislamiento & purificación , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oxigenasas/aislamiento & purificación , Estructura Terciaria de Proteína , Especificidad por Sustrato

RESUMEN

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (lambda(max) is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (lambda(max) is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (lambda(max) is 506 nm) with a similar rate, 1/tau approximately 500 s(-1). The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 A away.

Asunto(s)

Proteínas Bacterianas/química , Burkholderia/enzimología , Ácidos Grasos Insaturados/química , Hidrolasas/química , Modelos Moleculares , Proteínas Bacterianas/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Ácidos Grasos Insaturados/metabolismo , Histidina , Hidrolasas/metabolismo , Hidrólisis , Cinética , Estructura Cuaternaria de Proteína

RESUMEN

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.

Asunto(s)

Burkholderia/enzimología , Hidrolasas/química , Hidrolasas/metabolismo , Fenoles/metabolismo , Sitios de Unión , Ácidos Grasos Insaturados/metabolismo , Cinética , Modelos Moleculares , Conformación Proteica

RESUMEN

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.

Asunto(s)

Dioxigenasas/metabolismo , Hidrocarburos Aromáticos/metabolismo , Oxígeno/metabolismo , Oxigenasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Catálisis , Dioxigenasas/química , Activación Enzimática , Hidrocarburos Aromáticos/química , Modelos Moleculares , Oxidación-Reducción , Oxigenasas/química

RESUMEN

DoxG, an extradiol dioxygenase involved in the aerobic catabolism of naphthalene, possesses a weak ability to cleave 3,4-dihydroxybiphenyls (3,4-DHB), critical polychlorinated biphenyl metabolites. A directed evolution strategy combining error-prone PCR, saturation mutagenesis, and DNA shuffling was used to improve the polychlorinated biphenyl-degrading potential of DoxG. Screening was facilitated through analysis of filtered, digital imaging of plated colonies. A simple scheme, which is readily adaptable to other activities, enabled the screening of >10(5) colonies/h. The best variant, designated DoxGSMA2, cleaved 3,4-DHB with an apparent specificity constant of 2.0 +/- 0.3 x 10(6) m(-1) s(-1), which is 770 times that of wild-type (WT) DoxG. The specificities of DoxGSMA2 for 1,2-DHN and 2,3-DHB were increased by 6.7-fold and reduced by 2-fold, respectively, compared with the WT enzyme. DoxGSMA2 contained three substituted residues with respect to the WT enzyme: L190M, S191W, and L242S. Structural data indicate that the side chains of residues 190 and 242 occur on opposite walls of the substrate binding pocket and may interact directly with the distal ring of 3,4-DHB or influence contacts between this substrate and other residues. Thus, the introduction of two bulkier residues on one side of the substrate binding pocket and a smaller residue on the other may reshape the binding pocket and alter the catalytically relevant interactions of 3,4-DHB with the enzyme and dioxygen. Kinetic analyses reveal that the substitutions are anti-cooperative.

Asunto(s)

Dioxigenasas/metabolismo , Evolución Molecular Dirigida , Bifenilos Policlorados/metabolismo , Secuencia de Bases , Cartilla de ADN , Cinética , Modelos Moleculares

RESUMEN

The extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD, EC 1.13.11.39), has been studied using 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, which are analogous to methods used in earlier studies on the extradiol dioxygenase catechol 2,3-dioxygenase [Mabrouk et al. J. Am. Chem Soc. 1991, 113, 4053-4061]. For DHBD, the spectroscopic data can be correlated to the results of crystallography and with the results from density functional calculations to obtain detailed geometric and electronic structure descriptions of the resting and substrate (DHB) bound forms of the enzyme. The geometry of the active site of the resting enzyme, square pyramidal with a strong Fe-glutamate bond in the equatorial plane, localizes the redox active orbital in an orientation appropriate for O(2) binding. However, the O(2) reaction is not favorable, as it would produce a ferric superoxide intermediate with a weak Fe-O bond. Substrate binding leads to a new square pyramidal structure with the strong Fe-glutamate bond in the axial direction as indicated by a decrease in the (5)E(g) and increase in the (5)T(2g) splitting. Electronic structure calculations provide insight into the relative lack of dioxygen reactivity for the resting enzyme and its activation upon substrate binding.

Asunto(s)

Dioxigenasas/química , Dioxigenasas/metabolismo , Hierro/metabolismo , Oxígeno/metabolismo , Sitios de Unión , Modelos Moleculares , Conformación Proteica , Pseudomonas/enzimología , Análisis Espectral , Especificidad por Sustrato

RESUMEN

The microbial degradation of polychlorinated biphenyls (PCBs) provides the potential to destroy these widespread, toxic and persistent environmental pollutants. For example, the four-step upper bph pathway transforms some of the more than 100 different PCBs found in commercial mixtures and is being engineered for more effective PCB degradation. In the critical third step of this pathway, 2,3-dihydroxybiphenyl (DHB) 1,2-dioxygenase (DHBD; EC 1.13.11.39) catalyzes aromatic ring cleavage. Here we demonstrate that ortho-chlorinated PCB metabolites strongly inhibit DHBD, promote its suicide inactivation and interfere with the degradation of other compounds. For example, k(cat)(app) for 2',6'-diCl DHB was reduced by a factor of approximately 7,000 relative to DHB, and it bound with sufficient affinity to competitively inhibit DHB cleavage at nanomolar concentrations. Crystal structures of two complexes of DHBD with ortho-chlorinated metabolites at 1.7 A resolution reveal an explanation for these phenomena, which have important implications for bioremediation strategies.

Asunto(s)

Dioxigenasas , Contaminantes Ambientales/metabolismo , Modelos Moleculares , Oxigenasas/metabolismo , Bifenilos Policlorados/metabolismo , Sitios de Unión , Biodegradación Ambiental , Catálisis , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cinética , Sustancias Macromoleculares , Modelos Químicos , Oxigenasas/química , Bifenilos Policlorados/química

RESUMEN

Ultraviolet resonance Raman spectroscopy (UVRRS), electronic absorption spectroscopy, and X-ray crystallography were used to probe the nature of the binding of 2,3-dihydroxybiphenyl (DHB) to the extradiol ring-cleavage enzyme, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD; EC 1.13.11.39). The lowest lying transitions in the electronic absorption spectrum of DHBD-bound DHB occurred at 299 nm, compared to 305 nm for the monoanionic DHB species in buffer. In contrast, the corresponding transitions in neutral and dianionic DHB occurred at 283 and 348 nm, respectively, indicating that DHBD-bound DHB is monoanionic. These binding-induced spectral changes, and the use of custom-designed optical fiber probes, facilitated UVRR experiments. The strongest feature of the UVRR spectrum of DHB was a Y8a-like mode around 1600 cm(-1), whose position depended strongly on the protonation state of the DHB. In the spectrum of the DHBD-bound species, this feature occurred at 1603 cm(-1), as observed in the spectrum of monoanionic DHB. Raman band shifts were observed in deuterated solvent, ruling out dianionic binding of the substrate. Thus, the electronic absorption and UVRRS data demonstrate that DHBD binds its catecholic substrate as a monoanion, definitively establishing this feature of the proposed mechanism of extradiol dioxygenases. This conclusion is supported by a crystal structure of the DHBD:DHB complex at 2.0 A resolution, which suggests that the substrate's 2-hydroxyl substituent, and not the 3-hydroxyl group, deprotonates upon binding. The structural data also show that the aromatic rings of the enzyme-bound DHB are essentially orthogonal to each other. Thus, the 6 nm blue shift of the transition for bound DHB relative to the monoanion in solution could indicate a conformational change upon binding. Catalytic roles of active site residues are proposed based on the structural data and previously proposed mechanistic schemes.

Asunto(s)

Compuestos de Bifenilo/metabolismo , Catecoles/metabolismo , Dioxigenasas , Oxigenasas/metabolismo , Sitios de Unión , Compuestos de Bifenilo/química , Catecoles/química , Cristalografía por Rayos X , Modelos Moleculares , Oxigenasas/química , Unión Proteica , Espectrofotometría Ultravioleta/métodos , Espectrometría Raman/métodos