RESUMEN
Light-dependent or light-stimulated catalysis provides a multitude of perspectives for implementation in technological or biomedical applications. Despite substantial progress made in the field of photobiocatalysis, the number of usable light-responsive enzymes is still very limited. Flavoproteins have exceptional potential for photocatalytic applications because the name-giving cofactor intrinsically features light-dependent reactivity, undergoing photoreduction with a variety of organic electron donors. However, in the vast majority of these enzymes, photoreactivity of the enzyme-bound flavin is limited or even suppressed. Here, we present a flavoprotein monooxygenase in which catalytic activity is controllable by blue light illumination. The reaction depends on the presence of nicotinamide nucleotide-type electron donors, which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrate that catalysis depends on a protein-mediated photoreduction of the flavin cofactor, which proceeds via a radical mechanism and a transient semiquinone intermediate.
Asunto(s)
Proteínas Bacterianas/metabolismo , Transporte de Electrón , Flavina-Adenina Dinucleótido/metabolismo , Oxigenasas de Función Mixta/metabolismo , NAD/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Cristalografía por Rayos X , Flavoproteínas Transportadoras de Electrones/química , Flavoproteínas Transportadoras de Electrones/genética , Flavoproteínas Transportadoras de Electrones/metabolismo , Luz , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Modelos Moleculares , NADP/metabolismo , Oxidación-Reducción , Procesos Fotoquímicos , Pseudomonas aeruginosa/genéticaRESUMEN
Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
Asunto(s)
Alcohol Deshidrogenasa/química , Escherichia coli/genética , Mutación , Ingeniería de Proteínas/métodos , Temperatura de Transición , Alcohol Deshidrogenasa/genética , Computadores Moleculares , Cristalización , Estabilidad de Enzimas , Biblioteca de Genes , Cinética , Conformación Proteica , Saccharomycetales/enzimologíaRESUMEN
Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these challenges, but known BVMOs have shortcomings in either stability or substrate selectivity. We characterized a novel BVMO from the thermophilic fungus Thermothelomyces thermophila, determined its three-dimensional structure, and demonstrated its use as a promising biocatalyst. This fungal enzyme displays excellent enantioselectivity, acts on various ketones, and is particularly active on polycyclic molecules. Most notably we observed that the enzyme can perform oxidations on both the A and D ring when converting steroids. These functional properties can be linked to unique structural features, which identify enzymes acting on bulky substrates as a distinct subgroup of the BVMO class.
Asunto(s)
Hongos/enzimología , Cetonas/química , Oxigenasas de Función Mixta/química , Ciclización , EstereoisomerismoRESUMEN
A novel series of substituted chalcones were designed and synthesized to be evaluated as selective human MAO-B inhibitors. A combination of either methylsulfonyl or trifluoromethyl substituents on the aromatic ketone moiety with a benzodioxol ring on the other end of the chalcone scaffold was investigated. The compounds were tested for their inhibitory activities on both human MAO-A and B. All compounds appeared to be selective MAO-B inhibitors with Ki values in the micromolar to submicromolar range. Molecular modeling studies have been performed to get insight into the binding mode of the synthesized compounds to human MAO-B active site.
Asunto(s)
Chalconas/síntesis química , Chalconas/farmacología , Diseño de Fármacos , Inhibidores de la Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Chalconas/química , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de la Monoaminooxidasa/síntesis química , Relación Estructura-ActividadRESUMEN
Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7ß-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the ß-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Actinobacteria/enzimología , Hidroxiesteroide Deshidrogenasas/química , Hidroxiesteroide Deshidrogenasas/metabolismo , Actinobacteria/química , Actinobacteria/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Modelos Moleculares , NADP/metabolismo , Conformación Proteica , Estereoisomerismo , Especificidad por SustratoRESUMEN
The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å(3) volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å(3) (entrance cavity) and ~400 Å(3) (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala-Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared with wild-type enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100-103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine K(M) but unaltered k(cat) values. The altered K(M) values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to wild-type enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate that the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provide insights into specific reversible inhibitor design for these membrane-bound enzymes.
Asunto(s)
Inhibidores de la Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bencilaminas/metabolismo , Dominio Catalítico , Humanos , Isoleucina/metabolismo , Cinética , Monoaminooxidasa/química , Monoaminooxidasa/genética , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
Putrescine oxidase (PuO) from Rhodococcus erythropolis is a soluble homodimeric flavoprotein, which oxidizes small aliphatic diamines. In this study, we report the crystal structures and cofactor binding properties of wild-type and mutant enzymes. From a structural viewpoint, PuO closely resembles the sequence-related human monoamine oxidases A and B. This similarity is striking in the flavin-binding site even if PuO does not covalently bind the cofactor as do the monoamine oxidases. A remarkable conserved feature is the cis peptide conformation of the Tyr residue whose conformation is important for substrate recognition in the active site cavity. The structure of PuO in complex with the reaction product reveals that Glu324 is crucial in recognizing the terminal amino group of the diamine substrate and explains the narrow substrate specificity of the enzyme. The structural analysis also provides clues for identification of residues that are responsible for the competitive binding of ADP versus FAD (~50% of wild-type PuO monomers isolated are occupied by ADP instead of FAD). By replacing Pro15, which is part of the dinucleotide-binding domain, enzyme preparations were obtained that are almost 100% in the FAD-bound form. Furthermore, mutants have been designed and prepared that form a covalent 8α-S-cysteinyl-FAD linkage. These data provide new insights into the molecular basis for substrate recognition in amine oxidases and demonstrate that engineering of flavoenzymes to introduce covalent linkage with the cofactor is a possible route to develop more stable protein molecules, better suited for biocatalytic purposes.
Asunto(s)
Coenzimas/química , Coenzimas/metabolismo , Flavina-Adenina Dinucleótido/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Rhodococcus/enzimología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Biocatálisis , Coenzimas/genética , Cristalografía por Rayos X , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Modelos Moleculares , Monoaminooxidasa/química , Monoaminooxidasa/metabolismo , Mutagénesis Sitio-Dirigida , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Unión Proteica/genética , Conformación Proteica , Multimerización de Proteína/genética , Rhodococcus/genética , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-ActividadRESUMEN
Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substrate-reactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity.
Asunto(s)
Oxidorreductasas de Alcohol/química , Streptomyces coelicolor/enzimología , Oxidorreductasas de Alcohol/genética , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Manitol/química , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Sorbitol/química , Estereoisomerismo , Sulfitos/química , Xilitol/químicaRESUMEN
Alditol oxidase is a 45 kDa enzyme containing a covalently bound FAD cofactor. This oxidase efficiently oxidizes a range of alditols to the corresponding aldoses. Owing to its substrate range and regioselectivity, this enzyme is an interesting candidate for biotechnological applications. Crystals of alditol oxidase from Streptomyces coelicolor A3(2) were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.1 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 107, b = 68, c = 58 A, beta = 94 degrees. Crystals of seleno-L-methionine-labelled alditol oxidase were obtained after seeding the crystallization drops with native microcrystals and showed a diffraction limit of 2.4 A.
Asunto(s)
Oxidorreductasas de Alcohol/química , Streptomyces coelicolor/enzimología , Oxidorreductasas de Alcohol/aislamiento & purificación , Cristalización , Cristalografía por Rayos XRESUMEN
The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (kcat/Km) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Benzaldehídos/metabolismo , Oxidorreductasas de Alcohol/genética , Benzaldehídos/química , Catálisis , Cresoles/metabolismo , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Flavina-Adenina Dinucleótido/metabolismo , Aromatizantes , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Estructura Molecular , Mutagénesis , Oxidación-Reducción , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols. We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 A), which is the first structure of a member of this flavin reductase family. We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 A). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group (K(d) = 10 nm) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking. This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide. The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate. In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis.