Oxalate Oxidase for In Situ H O -Generation in Unspecific Peroxygenase-Catalysed Drug Oxyfunctionalisations.

H O -driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H O for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO as a by-product and uses oxalate as a cheap sacrificial electron donor.

Whole-cell screening of oxidative enzymes using genetically encoded sensors.

Biocatalysis is increasingly used for synthetic purposes in the chemical and especially the pharmaceutical industry. Enzyme discovery and optimization which is frequently needed to improve biocatalytic performance rely on high-throughput methods for activity determination. These methods should ideally be generic and applicable to entire enzyme families.

A Perspective on Synthetic Biology in Drug Discovery and Development-Current Impact and Future Opportunities.

The global impact of synthetic biology has been accelerating, because of the plummeting cost of DNA synthesis, advances in genetic engineering, growing understanding of genome organization, and explosion in data science. However, much of the discipline's application in the pharmaceutical industry remains enigmatic. In this review, we highlight recent examples of the impact of synthetic biology on target validation, assay development, hit finding, lead optimization, and chemical synthesis, through to the development of cellular therapeutics.

Enzymatic Late-Stage Modifications: Better Late Than Never.

Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late-stage modification often superior to conventional de novo synthesis.

Mechanistic and Crystallographic Studies of Azoreductase AzoA from A01.

The azoreductase AzoA from the alkali-tolerant A01 has been studied to reveal its structural and mechanistic details. For this, a recombinant expression system was developed which yields impressive amounts of fully active enzyme. The purified holo enzyme is remarkably solvent-tolerant and thermostable with an apparent melting temperature of 71 °C.

Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme.

Detoxifying enzymes such as flavin-containing monooxygenases deal with a huge array of highly diverse xenobiotics and toxic compounds. In addition to being of high physiological relevance, these drug-metabolizing enzymes are useful catalysts for synthetic chemistry. Despite the wealth of studies, the molecular basis of their relaxed substrate selectivity remains an open question.

Characterization of a thermostable flavin-containing monooxygenase from Nitrincola lacisaponensis (NiFMO).

The flavin-containing monooxygenases (FMOs) play an important role in drug metabolism but they also have a high potential in industrial biotransformations. Among the hitherto characterized FMOs, there was no thermostable representative, while such biocatalyst would be valuable for FMO-based applications. Through a targeted genome mining approach, we have identified a gene encoding for a putative FMO from Nitrincola lacisaponensis, an alkaliphilic extremophile bacterium.

Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol.

Glycerol is a major byproduct of biodiesel production, and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here, we describe the comprehensive structural and biochemical characterization of this enzyme.

Dispersal limitation and thermodynamic constraints govern spatial structure of permafrost microbial communities.

Understanding drivers of permafrost microbial community composition is critical for understanding permafrost microbiology and predicting ecosystem responses to thaw. We hypothesize that permafrost communities are shaped by physical constraints imposed by prolonged freezing, and exhibit spatial distributions that reflect dispersal limitation and selective pressures associated with these physical constraints. To test this, we characterized patterns of environmental variation and microbial community composition in permafrost across an Alaskan boreal forest landscape.

Kinetic Resolution of sec-Thiols by Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase.

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec-thiols by using structure-guided engineering of 5-(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec-thiols, thus yielding the corresponding thioketones and nonreacted R-configured thiols with excellent enantioselectivities (E≥200).

Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes.

Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states.

Molecular Basis for Converting (2S)-Methylsuccinyl-CoA Dehydrogenase into an Oxidase.

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen.

Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A.

The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[H]-choline as a substrate. The limiting rate constants k and k at saturating substrate were well separated (k/k>9), and were >15-fold slower than for wild-type choline oxidase.

Baeyer-Villiger Monooxygenase FMO5 as Entry Point in Drug Metabolism.

Flavin-containing monooxygenases (FMOs) are emerging as effective players in oxidative drug metabolism. Until recently, the functions of the five human FMO isoforms were mostly linked to their capability of oxygenating molecules containing soft N- and S-nucleophiles. However, the human FMO isoform 5 was recently shown to feature an atypical activity as Baeyer-Villiger monooxygenase.

Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase.

A number of oxidoreductases from the VAO/-cresol methylhydroxylase flavoprotein family catalyze the oxidation of -substituted phenols. One of the best-studied is vanillyl-alcohol oxidase (VAO) from the fungus For oxidation of phenols by VAO to occur, they must first be bound in the active site of the enzyme in their phenolate anion form. The crystal structure of VAO reveals that two tyrosine residues, Tyr-108 and Tyr-503, are positioned to facilitate this deprotonation.

Crystal structures and atomic model of NADPH oxidase.

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of NOX5.

Engineering Cyclohexanone Monooxygenase for the Production of Methyl Propanoate.

A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate.

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase.

Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity. However, the low stability of many Baeyer-Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported.

In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus.

In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron.

Alcohol oxidation by flavoenzymes.

This review article describes the occurrence, general properties, and substrate specificity of the flavoenzymes belonging to the glucose-methanol-choline oxidoreductase superfamily and the l-α-hydroxyacid dehydrogenase family. Most of these enzymes catalyze the oxidations of hydroxyl groups, yielding carbonyl moieties. Over the years, carbanion, hydride transfer, and radical mechanisms have been discussed for these enzymes, and the main experimental evidences supporting these mechanisms are presented here.

Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH.

Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase.

Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N(5)-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus.

Monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C).

New oxidase from Bjerkandera arthroconidial anamorph that oxidizes both phenolic and nonphenolic benzyl alcohols.

A new flavooxidase is described from a Bjerkandera arthroconidial anamorph. Its physicochemical characteristics, a monomeric enzyme containing non-covalently bound flavin adenine dinucleotide (FAD), and several catalytic properties, such as oxidation of aromatic and polyunsaturated aliphatic primary alcohols, are similar to those of Pleurotus eryngii aryl-alcohol oxidase (AAO). However, it also efficiently oxidizes phenolic benzyl and cinnamyl alcohols that are typical substrates of vanillyl-alcohol oxidase (VAO), a flavooxidase from a different family, characterized by its multimeric nature and presence of covalently-bound FAD.