Vanillyl alcohol oxidase from Diplodia corticola:Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives

Vanillyl alcohol oxidases 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 ɣ-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. </p

Vanillyl alcohol oxidase from Diplodia corticola:Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives

Vanillyl alcohol oxidases 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 ɣ-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. </p

Flavoprotein monooxygenases: Versatile biocatalysts

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.BT/Biocatalysi

Structural and Mechanistic Studies on Substrate and Stereoselectivity of the Indole Monooxygenase Vp IndA1: New Avenues for Biocatalytic Epoxidations and Sulfoxidations

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

Asymmetric Reduction of (R)-Carvone through a Thermostable and Organic-Solvent-Tolerant Ene-Reductase

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) U mg−1 with NADPH or of (206.7±3.4) 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 20 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 200 mL biotransformation of 750 mg (R)-carvone afforded 495 mg of (2R,5R)-dihydrocarvone (&gt;95 % ee), demonstrating the simplicity of handling and application of FOYE-1.BT/Biocatalysi

Vanillyl alcohol oxidase from Diplodia corticola\textit {Diplodia corticola}

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, $\it Dc$VAO, from $\textit {Diplodia corticola}$. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 $\pm$ 0.02 U $mg^{−1}$) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 $\pm$ 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 $\gamma$-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 $\pm$ 0.1 U $mg^{−1}$). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with $\it Dc$VAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts

Enantioselective epoxidation by flavoprotein monooxygenases supported by organic solvents

Styrene and indole monooxygenases (SMO and IMO) are two-component flavoprotein monooxygenases composed of a nicotinamide adenine dinucleotide (NADH)-dependent flavin adenine dinucleotide (FAD)-reductase (StyB or IndB) and a monooxygenase (StyA or IndA). The latter uses reduced FAD to activate oxygen and to oxygenate the substrate while releasing water. We circumvented the need for the reductase by direct FAD reduction in solution using the NAD(P)H-mimic 1-benzyl-1,4-dihydronicotinamide (BNAH) to fuel monooxygenases without NADH requirement. Herein, we report on the hitherto unknown solvent tolerance for the indole monooxygenase from $\textit {Gemmobacter nectariphilus}$ DSM15620 ($\it Gn$IndA) and the styrene monooxygenase from $\textit {Gordonia rubripertincta}$ CWB2 ($\it Gr$StyA). These enzymes were shown to convert bulky and rather hydrophobic styrene derivatives in the presence of organic cosolvents. Subsequently, BNAH-driven biotransformation was furthermore optimized with regard to the applied cosolvent and its concentration as well as FAD and BNAH concentration. We herein demonstrate that $\it Gn$IndA and $\it Gr$StyA enable selective epoxidations of allylic double bonds (up to 217 mU $mg^{−1}$) in the presence of organic solvents such as tetrahydrofuran, acetonitrile, or several alcohols. Notably, $\it Gn$IndA was found to resist methanol concentrations up to 25 vol.%. Furthermore, a diverse substrate preference was determined for both enzymes, making their distinct use very interesting. In general, our results seem representative for many IMOs as was corroborated by in silico mutagenetic studies

Asymmetric reduction of (R\it R)‐carvone through a thermostable and organic‐solvent‐tolerant ene‐reductase

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 $\it F$OYE‐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 $\it F$OYE‐1 to produce valuable compounds. A maximum activity of (319.7$\pm$3.2) U mg$^{−1}$ with NADPH or of (206.7$\pm$3.4) U mg$^{−1}$ with 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) for the reduction of $\it N$‐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: $\it F$OYE‐1 showed excellent performance in most systems with up to 20 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 200 mL biotransformation of 750 mg ($\it R$)‐carvone afforded 495 mg of (2$\it R$,5$\it R$)‐dihydrocarvone (>95 % $\it ee$), demonstrating the simplicity of handling and application of $\it F$OYE‐1