RESUMO
Enzymes are natures' catalysts and will have a lasting impact on (organic) synthesis as they possess unchallenged regio- and stereo selectivity. On the downside, this high selectivity limits enzymes' substrate range and hampers their universal application. Therefore, substrate scope expansion of enzyme families by either modification of known biocatalysts or identification of new members is a key challenge in enzyme-driven catalysis. Here, we present a streamlined approach to rationally select enzymes with proposed functionalities from the ever-increasing amount of available sequence data. In a case study on 4-phenol oxidoreductases, eight enzymes of the oxidase branch were selected from 292 sequences on basis of the properties of first shell residues of the catalytic pocket, guided by the computational tool A2CA. Correlations between these residues and enzyme activity yielded robust sequence-function relations, which were exploited by site-saturation mutagenesis. Application of a peroxidase-independent oxidase screening resulted in 16 active enzyme variants which were up to 90-times more active than respective wildtype enzymes and up to 6-times more active than the best performing natural variants. The results were supported by kinetic experiments and structural models. The newly introduced amino acids confirmed the correlation studies which overall highlights the successful logic of the presented approach.
RESUMO
Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for É£-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.
Assuntos
Oxirredutases do Álcool , Ascomicetos , Biocatálise , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Ascomicetos/enzimologia , Fenóis/química , Fenóis/metabolismo , Especificidade por Substrato , Hidroxilação , Éteres/química , Éteres/metabolismoRESUMO
Flavoprotein monooxygenases are a versatile group of enzymes for biocatalytic transformations. Among these, group E monooxygenases (GEMs) catalyze enantioselective epoxidation and sulfoxidation reactions. Here, we describe the crystal structure of an indole monooxygenase from the bacterium Variovorax paradoxus EPS, a GEM designated as VpIndA1. Complex structures with substrates reveal productive binding modes that, in conjunction with force-field calculations and rapid mixing kinetics, reveal the structural basis of substrate and stereoselectivity. Structure-based redesign of the substrate cavity yielded variants with new substrate selectivity (for sulfoxidation of benzyl phenyl sulfide) or with greatly enhanced stereoselectivity (from 35.1 % to 99.8 % ee for production of (1S,2R)-indene oxide). This first determination of the substrate binding mode of GEMs combined with structure-function relationships opens the door for structure-based design of these powerful biocatalysts.
Assuntos
Oxigenases de Função Mista , Oxigenases , Biocatálise , Indóis , Oxigenases de Função Mista/metabolismo , Oxigenases/metabolismo , Especificidade por Substrato , Oxirredução , Enxofre/químicaRESUMO
Flavoprotein monooxygenases (FPMOs) are single- or two-component enzymes that catalyze a diverse set of chemo-, regio- and enantioselective oxyfunctionalization reactions. In this review, we describe how FPMOs have evolved from model enzymes in mechanistic flavoprotein research to biotechnologically relevant catalysts that can be applied for the sustainable production of valuable chemicals. After a historical account of the development of the FPMO field, we explain the FPMO classification system, which is primarily based on protein structural properties and electron donor specificities. We then summarize the most appealing reactions catalyzed by each group with a focus on the different types of oxygenation chemistries. Wherever relevant, we report engineering strategies that have been used to improve the robustness and applicability of FPMOs.
Assuntos
Flavoproteínas , Oxigenases de Função Mista , Biocatálise , Catálise , Flavoproteínas/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , OxirreduçãoRESUMO
Myxococcus xanthus kills other species to use their biomass as energy source. Its predation mechanisms allow feeding on a broad spectrum of bacteria, but the identity of predation effectors and their mode of action remains largely unknown. We initially focused on the role of hydrolytic enzymes for prey killing and compared the activity of secreted M. xanthus proteins against four prey strains. 72 secreted proteins were identified by mass spectrometry, and among them a family 19 glycoside hydrolase that displayed bacteriolytic activity in vivo and in vitro This enzyme, which we name LlpM (lectin/lysozyme-like protein of M. xanthus), was not essential for predation, indicating that additional secreted components are required to disintegrate prey. Furthermore, secreted proteins lysed only Gram-positive, but not Gram-negative species. We thus compared the killing of different preys by cell-associated mechanisms: Individual M. xanthus cells killed all four test strains in a cell-contact dependent manner, but were only able to disintegrate Gram-negative, not Gram-positive cell envelopes. Thus, our data indicate that M. xanthus uses different, multifactorial mechanisms for killing and degrading different preys. Besides secreted enzymes, cell-associated mechanisms that have not been characterized so far, appear to play a major role for prey killing.IMPORTANCEPredation is an important survival strategy of the widespread myxobacteria, but it remains poorly understood on the mechanistic level. Without a basic understanding of how prey cell killing and consumption is achieved, it also remains difficult to investigate the role of predation for the complex myxobacterial lifestyle, reciprocal predator-prey relationships or the impact of predation on complex bacterial soil communities.We study predation in the established model organism Myxococcus xanthus, aiming to dissect the molecular mechanisms of prey cell lysis. In this study, we addressed the role of secreted bacteriolytic proteins, as well as potential mechanistic differences in the predation of Gram-positive and Gram-negative bacteria. Our observation shows that secreted enzymes are sufficient for killing and degrading Gram-positive species, but that cell-associated mechanisms may play a major role for killing Gram-negative and Gram-positive prey on fast timescales.
RESUMO
Styrene and indole are naturally occurring compounds, which are also produced and processed by various chemical industries. Thus, it is not surprisingly that microorganisms evolved pathways to detoxify or even to utilize those compounds as carbon sources. Especially, among bacteria several routes are described specifically for the activation and degradation of styrene and indole. Respectively, the initial attack toward these compounds occurs via a flavin-dependent monooxygenase: styrene monooxygenase (SMO) or indole monooxygenase (IMO). In the first place, SMOs have been described to initiate a styrene specific degradation. These are in general two-component systems, whereas a small FAD-reductase (SMOB) delivers reduced FAD on the expense of NADH toward the monooxygenase (SMOA). Various modes of interaction are possible and for both mostly dimeric protein subunits structural data were reported. Thus, this flavoprotein monooxygenase-especially the one from Pseudomonas putida S12 can be seen as the prototype of this class of enzymes. In the course of describing related members of this enzyme family some remarkable findings were made. For example, self-sufficient fusion proteins have been reported as well as enzymes, which could not be assigned to a styrene metabolic activity, rather to indole conversion. Later it was found that this flavoprotein group can be separated at least into two subgroups: styrene and indole monooxygenases. And both enzymes rely on a FAD-reductase to obtain the reduced cofactor (FADred), which is employed to activate molecular oxygen toward hydroperoxy-FAD, which allows substrate epoxidation and the formation of hydroxy-FAD, which finally yields H2O and oxidized FAD.
Assuntos
Biocatálise , Biodegradação Ambiental , Flavoproteínas/química , Oxigenases de Função Mista/química , Oxigenases/química , Indóis/químicaRESUMO
Ene-reductases allow regio- and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene-reductase FOYE-1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel FOYE-1 to produce valuable compounds. A maximum activity of (319.7±3.2)â U mg-1 with NADPH or of (206.7±3.4)â U mg-1 with 1-benzyl-1,4-dihydronicotinamide (BNAH) for the reduction of N-methylmaleimide was observed at 30 °C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: FOYE-1 showed excellent performance in most systems with up to 20â vol% solvent and at temperatures up to 40 °C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200â mL biotransformation of 750â mg (R)-carvone afforded 495â mg of (2R,5R)-dihydrocarvone (>95 % ee), demonstrating the simplicity of handling and application of FOYE-1.