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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.

Amino groups derived from naturally abundant amino acids or (di)amines can be used as “shuttles” in nature for oxygen transfer to provide intermediates or products comprising N-O functional groups such as N-hydroxy, oxazine, isoxazolidine, nitro, nitrone, oxime, C-, S-, or N-nitroso, and azoxy units. To this end, molecular oxygen is activated by flavin, heme, or metal cofactor-containing enzymes and transferred to initially obtain N-hydroxy compounds, which can be further functionalized. In this review, we focus on flavin-dependent N-hydroxylating enzymes, which play a major role in the production of secondary metabolites, such as siderophores or antimicrobial agents. Flavoprotein monooxygenases of higher organisms (among others, in humans) can interact with nitrogen-bearing secondary metabolites or are relevant with respect to detoxification metabolism and are thus of importance to understand potential medical applications. Many enzymes that catalyze N-hydroxylation reactions have specific substrate scopes and others are rather relaxed. The subsequent conversion towards various N-O or N-N comprising molecules is also described. Overall, flavin-dependent N-hydroxylating enzymes can accept amines, diamines, amino acids, amino sugars, and amino aromatic compounds and thus provide access to versatile families of compounds containing the N-O motif. Natural roles as well as synthetic applications are highlighted. Key points• N-O and N-N comprising natural and (semi)synthetic products are highlighted.• Flavin-based NMOs with respect to mechanism, structure, and phylogeny are reviewed.• Applications in natural product formation and synthetic approaches are provided.

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 (>95 % ee), demonstrating the simplicity of handling and application of FOYE‐1., Enantioselective hydrogenation: Ene‐reductases (ERs, OYEs) are versatile biocatalysts for selective hydrogenation of activated C=C double bonds. FOYE‐1 was characterized with respect to solvent stability for the conversion of (R)‐carvone into (2R,5R)‐dihydrocarvone at the expense of the synthetic nicotinamide cofactor 1‐benzyl‐1,4‐dihydronicotinamide, the activity of which is compared with that of the natural electron donor NAD(P)H.

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label‐free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC‐MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.

Arylmalonate decarboxylase (AMDase) catalyzes the cofactor‐free asymmetric decarboxylation of prochiral arylmalonic acids and produces the corresponding monoacids with rigorous R selectivity. Alteration of catalytic cysteine residues and of the hydrophobic environment in the active site by protein engineering has previously resulted in the generation of variants with opposite enantioselectivity and improved catalytic performance. The substrate spectrum of AMDase allows it to catalyze the asymmetric decarboxylation of small methylvinylmalonic acid derivatives, implying the possibility to produce short‐chain 2‐methylalkanoic acids with high optical purity after reduction of the nonactivated C=C double bond. Use of diimide as the reductant proved to be a simple strategy to avoid racemization of the stereocenter during reduction. The developed chemoenzymatic sequential cascade with use of R‐ and S‐selective AMDase variants produced optically pure short‐chain 2‐methylalkanoic acids in moderate to full conversion and gave both enantiomers in excellent enantiopurity (up to 83 % isolated yield and 98 % ee).

All protein engineering studies include the stage of identifying and characterizing variants within a mutant library by employing a suitable assay or selection method. A large variety of different assay approaches for different enzymes have been developed in the last few decades, and the throughput performance of these assays vary considerably. Thus, the concept of a protein engineering study must be adapted to the available assay methods. This introductory review chapter describes different assay concepts on selected examples, including selection and screening approaches, detection of pH and cosubstrate changes, coupled enzyme assays, methods using surrogate substrates and selective derivatization. The given examples should guide and inspire the reader when choosing and developing own high-throughput screening approaches.

Oxygenic phototrophs frequently encounter environmental conditions that result in intracellular energy crises. Growth of the unicellular green alga Chlamydomonas reinhardtii in hypoxia in the light depends on acclimatory responses of which the induction of photosynthetic cyclic electron flow is essential. The microalga cannot grow in the absence of molecular oxygen (O2 ) in the dark, although it possesses an elaborate fermentation metabolism. Not much is known about how the microalga senses and signals the lack of O2 or about its survival strategies during energy crises. Recently, nitric oxide (NO) has emerged to be required for the acclimation of C. reinhardtii to hypoxia. In this study, we show that the soluble guanylate cyclase (sGC) CYG12, a homologue of animal NO sensors, is also involved in this response. CYG12 is an active sGC, and post-transcriptional down-regulation of the CYG12 gene impairs hypoxic growth and gene expression in C. reinhardtii. However, it also results in a disturbed photosynthetic apparatus under standard growth conditions and the inability to grow heterotrophically. Transcriptome profiles indicate that the mis-expression of CYG12 results in a perturbation of responses that, in the wild-type, maintain the cellular energy budget. We suggest that CYG12 is required for the proper operation of the photosynthetic apparatus which, in turn, is essential for survival in hypoxia and darkness.

A recombinant enoate reductase was expressed in cyanobacteria and used for the light-catalyzed, enantioselective reduction of C=C bonds. The coupling of oxidoreductases to natural photosynthesis allows asymmetric syntheses fueled by the oxidation of water. Bypassing the addition of sacrificial cosubstrates as electron donors significantly improves the atom efficiency and avoids the formation of undesired side products. Crucial factors for product formation are the availability of NADPH and the amount of active enzyme in the cells. The efficiency of the reaction is comparable to typical whole-cell biotransformations in E. coli. Under optimized conditions, a solution of 100 mg prochiral 2-methylmaleimide was reduced to optically pure 2-methylsuccinimide (99 % ee, 80 % yield of isolated product). High product yields and excellent optical purities demonstrate the synthetic usefulness of light-catalyzed whole-cell biotransformations using recombinant cyanobacteria.

Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer–Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications.

Cisplatin and its analogues are first-line chemotherapeutic agents for the treatment of numerous human cancers. A major inconvenience in their clinical use is their strong tendency to link to sulfur compounds, especially in kidney, ultimately leading to severe nephrotoxicity. To overcome this drawback we prepared a variety of platinum complexes with sulfur ligands and analyzed their biological profiles. Here, a series of six platinum(II) compounds bearing a conserved O,S binding moiety have been synthesized and characterized as experimental anticancer agents. The six compounds differ in the nature of the O,S bidentate β-hydroxydithiocinnamic alkyl ester ligand where both the substituents on the aromatic ring and the length of the alkyl chain may be varied. The two remaining coordination positions at the square-planar platinum(II) center are occupied by a chloride ion and a DMSO molecule. These novel platinum compounds showed an acceptable solubility profile in mixed DMSO-buffer solutions and an appreciable stability at physiological pH as judged from analysis of their time-course UV-visible absorption spectra. Their anti-proliferative and pro-apoptotic activities were tested against the cisplatin-resistant lung cancer cell line A549. Assays revealed significant effects of the sample drugs at low concentrations (in the μmolar range); initial structure-activity-relationships are proposed. The activity of the apoptosis-promoting protein caspase 3/7 was determined; results proved that these novel platinum compounds, under the chosen experimental conditions, preferentially induce apoptosis over necrosis. Reactions with the model proteins cytochrome c, lysozyme and albumin were studied by ESI MS and ICP-OES to gain preliminary mechanistic information. The tested compounds turned out to metalate the mentioned proteins to a large extent. In view of the obtained results these novel platinum complexes qualify themselves as promising cytotoxic agents and merit, in our opinion, a deeper pharmacological evaluation as prospective anticancer agents.

Reactions of cytotoxic platinum drugs with proteins are attracting growing attention for their relevant biological implications. We report here on the reactions of two cis-diphosphane platinum(II) dichlorides (namely cis-bis(trimethylphosphane) platinum(II) dichloride and cis-bis(triethylphosphane) platinum(II) dichloride) with horse heart cytochrome c (cyt c) monitored through advanced ESI MS methods coupled to enzymatic digestion. A remarkable selectivity in terms of adduct stoichiometry is highlighted and the specific metal binding sites are localised on the protein surface.

Optically pure hydroxy amino acids show several bioactivities and are valuable building blocks for the pharmaceutical industry. Fe(II)/α-ketoglutarate dependent dioxygenases catalyze the hydroxylation or sulfoxidation of l-amino acids with high regio- and stereoselectivity. While several β- and γ-specific enzymes have been described, only one δ-specific hydroxylase has been reported so far. Based on its similarity to the known l-leucine 5-hydroxylase from Nostoc punctiforme, an open reading frame from the cyanobacterium Anabaena variabilis was identified as putative l-leucine dioxygenase (AvLDO). Here we report the cloning and characterization of this dioxygenase. The enzyme showed a high preference for acidic conditions and moderate reaction temperatures. AvLDO catalyzed the regio- and stereoselective hydroxylation of several aliphatic amino acids in δ-position. In case of the sulfoxidation of l-methionine, AvLDO produced the opposite diastereomer than isoleucine dioxygenase. AvLDO is thus an interesting addition to the toolbox of Fe(II)/α-ketoglutarate dependent dioxygenases. On the genomic DNA of Anabaena variabilis ATCC 29413, the avldo gene is located on a gene cluster involved (2S,4S)-4-methylproline biosynthesis, which is contained in bioactive peptides often found from cyanobacteria. This fact suggests the metabolic functional role of this amino acid dioxygenase in cyanobacteria.

Twelve Pt(II) complexes with cis-PtP(2)S(2) pharmacophores (where P(2) refers to two monodentate or one bidentate phosphane ligand and S(2) is a dithiolato ligand) were prepared, characterized and evaluated as potential antiproliferative agents. The various compounds were first studied from the structural point of view; afterward, their solubility properties as well as their solution behaviour were analyzed in detail. Antiproliferative properties were specifically evaluated against A2780 human ovarian carcinoma cells, either resistant or sensitive to cisplatin. For comparison purposes similar studies were carried out on four parent cis-dichloro bisphosphane Pt(II)complexes. On the whole, the cis-PtP(2)S(2) compounds displayed significant antiproliferative properties while the cis-PtP(2)Cl(2) (cis-dichloro bisphosphane Pt(II)) compounds revealed quite poor biological performances. To gain further insight into the molecular mechanisms of these bisphosphane Pt(II) compounds, the reactions of selected complexes against the model protein cytochrome c were investigated by ESI-MS and their adduct formation explored. A relevant reactivity with cyt c was obtained only for cis-PtP(2)Cl(2) compounds, whereas cis-PtP(2)S(2) compounds turned out to be nearly unreactive. The obtained results are interpreted and discussed in the frame of the current knowledge of anticancer platinum compounds and their structure-activity-relationships. The observation of appreciable antiproliferative effects for the relatively inert cis-PtP(2)S(2) compounds strongly suggests that these compounds will undergo specific activation within the cellular environment.