Insights into E.†coli Cyclopropane Fatty Acid Synthase (CFAS) Towards Enantioselective Carbene Free Biocatalytic Cyclopropanation.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E. coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.

Biocatalysis making waves in organic chemistry.

Biocatalysis has an enormous impact on chemical synthesis. The waves in which biocatalysis has developed, and in doing so changed our perception of what organic chemistry is, were reviewed 20 and 10 years ago. Here we review the consequences of these waves of development. Nowadays, hydrolases are widely used on an industrial scale for the benign synthesis of commodity and bulk chemicals and are fully developed. In addition, further enzyme classes are gaining ever increasing interest. Particularly, enzymes catalysing selective C-C-bond formation reactions and enzymes catalysing selective oxidation and reduction reactions are solving long-standing synthetic challenges in organic chemistry. Combined efforts from molecular biology, systems biology, organic chemistry and chemical engineering will establish a whole new toolbox for chemistry. Recent developments are critically reviewed.

Peroxygenase-Catalysed Selective Oxidation of Silanes to Silanols.

A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h-1 ), catalyst performance (up to 84 s-1 and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity.

Exploring the Temperature Effect on Enantioselectivity of a Baeyer-Villiger Biooxidation by the 2,5-DKCMO Module: The SLM Approach.

Temperature is a crucial parameter for biological and chemical processes. Its effect on enzymatically catalysed reactions has been known for decades, and stereo- and enantiopreference are often temperature-dependent. For the first time, we present the temperature effect on the Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one by the type II Bayer-Villiger monooxygenase, 2,5-DKCMO. In the absence of a reductase and driven by the hydride-donation of a synthetic nicotinamide analogue, the clear trend for a decreasing enantioselectivity at higher temperatures was observed. "Traditional" approaches such as the determination of the enantiomeric ratio (E) appeared unsuitable due to the complexity of the system. To quantify the trend, we chose to use the 'Shape Language Modelling' (SLM), a tool that allows the reaction to be described at all points in a shape prescriptive manner. Thus, without knowing the equation of the reaction, the substrate ee can be estimated that at any conversion.

A Peroxygenase-Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols.

In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.

Stabilisation of the Fatty Acid Decarboxylase from Chlorella variabilis by Caprylic Acid.

The fatty acid photodecarboxylase from Chlorella variabilis NC64 A (CvFAP) catalyses the light-dependent decarboxylation of fatty acids. Photoinactivation of CvFAP still represents one of the major limitations of this interesting enzyme en route to practical application. In this study we demonstrate that the photostability of CvFAP can easily be improved by the administration of medium-chain length carboxylic acids such as caprylic acid indicating that the best way of maintaining CvFAP stability is 'to keep the enzyme busy'.

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer-Villiger oxidations.

Two-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer-Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case.

Biocatalytic Reduction Reactions from a Chemist's Perspective.

Reductions play a key role in organic synthesis, producing chiral products with new functionalities. Enzymes can catalyse such reactions with exquisite stereo-, regio- and chemoselectivity, leading the way to alternative shorter classical synthetic routes towards not only high-added-value compounds but also bulk chemicals. In this review we describe the synthetic state-of-the-art and potential of enzymes that catalyse reductions, ranging from carbonyl, enone and aromatic reductions to reductive aminations.

E. coli Nickel-Iron Hydrogenase 1 Catalyses Non-native Reduction of Flavins: Demonstration for Alkene Hydrogenation by Old Yellow Enzyme Ene-reductases*.

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom-economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25-70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene-reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

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 (>95 % ee), demonstrating the simplicity of handling and application of FOYE-1.

Metals in Biotechnology: Cr-Driven Stereoselective Reduction of Conjugated C=C Double Bonds.

Elemental metals are shown to be suitable sacrificial electron donors to drive the stereoselective reduction of conjugated C=C double bonds using Old Yellow Enzymes as catalysts. Both direct electron transfer from the metal to the enzyme as well as mediated electron transfer is feasible, although the latter excels by higher reaction rates. The general applicability of this new chemoenzymatic reduction method is demonstrated, and current limitations are outlined.

Flavin-dependent N-hydroxylating enzymes: distribution and application.

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. Graphical abstract .

Teaching pediatric otoscopy skills to the medical student in the clinical setting: preceptor perspectives and practice.

BACKGROUND: Acute otitis media (AOM) is the most frequent indication for antibiotic treatment of children in the United States. Its diagnosis relies on visualization of the tympanic membrane, a clinical skill acquired through a deliberate approach. Instruction in pediatric otoscopy begins in medical school. Medical students receive their primary experience with pediatric otoscopy during the required pediatric clerkship, traditionally relying on an immersion, apprentice-type learning model. A better understanding of their preceptors' clinical and teaching practices could lead to improved skill acquisition. This study investigates how pediatric preceptors (PP) and members of the Council on Medical Student Education in Pediatrics (COMSEP) perceive teaching otoscopy. METHODS: A 30-item online survey was administered to a purposeful sample of PP at six institutions in 2017. A comparable 23-item survey was administered to members through the 2018 COMSEP Annual Survey. Only COMSEP members who identified themselves as teaching otoscopy to medical students were asked to complete the otoscopy-related questions on the survey. RESULTS: Survey respondents included 58% of PP (180/310) and 44% (152/348) of COMSEP members. Forty-one percent (62/152) of COMSEP member respondents identified themselves as teaching otoscopy and completed the otoscopy-related questions. The majority agreed that standardized curricula are needed (PP 78%, COMSEP members 97%) and that all graduating medical students should be able to perform pediatric otoscopy (PP 95%, COMSEP members 79%). Most respondents reported usefulness of the American Academy of Pediatrics (AAP) AOM guidelines (PP 95%, COMSEP members 100%). More COMSEP members than PP adhered to the AAP's diagnostic criteria (pediatric preceptors 42%, COMSEP members 93%). The most common barriers to teaching otoscopy were a lack of assistive technology (PP 77%, COMSEP members 56%), presence of cerumen (PP 58%, COMSEP members 60%), time to teach in direct patient care (PP 46%, COMSEP members 48%), and parent anxiety (PP 62%, COMSEP members 54%). CONCLUSIONS: Our study identified systemic and individual practice patterns and barriers to teaching pediatric otoscopy. These results can inform education leaders in supporting and enabling preceptors in their clinical teaching. This approach can be adapted to ensure graduating medical students obtain intended core clinical skills.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

Biocatalytic Oxidation Reactions: A Chemist's Perspective.

Oxidation chemistry using enzymes is approaching maturity and practical applicability in organic synthesis. Oxidoreductases (enzymes catalysing redox reactions) enable chemists to perform highly selective and efficient transformations ranging from simple alcohol oxidations to stereoselective halogenations of non-activated C-H bonds. For many of these reactions, no "classical" chemical counterpart is known. Hence oxidoreductases open up shorter synthesis routes based on a more direct access to the target products. The generally very mild reaction conditions may also reduce the environmental impact of biocatalytic reactions compared to classical counterparts. In this Review, we critically summarise the most important recent developments in the field of biocatalytic oxidation chemistry and identify the most pressing bottlenecks as well as promising solutions.

Biocatalytic C=C Bond Reduction through Carbon Nanodot-Sensitized Regeneration of NADH Analogues.

Light-driven activation of redox enzymes is an emerging route for sustainable chemical synthesis. Among redox enzymes, the family of Old Yellow Enzyme (OYE) dependent on the nicotinamide adenine dinucleotide cofactor (NADH) catalyzes the stereoselective reduction of α,ß-unsaturated hydrocarbons. Here, we report OYE-catalyzed asymmetric hydrogenation through light-driven regeneration of NADH and its analogues (mNADHs) by N-doped carbon nanodots (N-CDs), a zero-dimensional photocatalyst. Our spectroscopic and photoelectrochemical analyses verified the transfer of photo-induced electrons from N-CDs to an organometallic electron mediator (M) for highly regioselective regeneration of cofactors. Light triggered the reduction of NAD+ and mNAD+ s with the cooperation of N-CDs and M, and the reduction behaviors of cofactors were dependent on their own reduction peak potentials. The regenerated cofactors subsequently delivered hydrides to OYE for stereoselective conversions of a broad range of substrates with excellent biocatalytic efficiencies.

A thermophilic-like ene-reductase originating from an acidophilic iron oxidizer.

Ene-reductases originating from extremophiles are gaining importance in the field of biocatalysis due to higher-stability properties. The genome of the acidophilic iron-oxidizing bacterium "Ferrovum" sp. JA12 was found to harbor a thermophilic-like ene-reductase (FOYE-1). The foye-1 gene was ligated into a pET16bp expression vector system, and the enzyme was produced in Escherichia coli BL21 (DE3; pLysS) cells in yields of 10 mg L-1. FOYE-1 showed remarkable activity and rates on N-phenylmaleimide and N-phenyl-2-methylmaleimide (up to 89 U mg-1, >97 % conversion, 95 % (R)-selective) with both nicotinamide cofactors, NADPH and NADH. The catalytic efficiency with NADPH was 27 times higher compared to NADH. At the temperature maximum (50 °C) and pH optimum (6.5), activity was almost doubled to 160 U mg-1. These findings accomplish FOYE-1 for a valuable biocatalyst in the synthesis of succinimides. The appearance of a thermophilic-like ene-reductase in an acidic habitat is discussed with respect to its phylogenetic placement and to the genomic neighborhood of the encoding gene, awarding FOYE-1 a putative involvement in a quorum-sensing process.

Teaching the pediatric ear exam and diagnosis of Acute Otitis Media: a teaching and assessment model in three groups.

BACKGROUND: The serious consequences of inaccurate diagnosis of acute otitis media have led to a call for greater education to develop proficient pediatric otoscopy skills. Despite the clinical and educational needs, peer-reviewed standardized curricula with validated assessment instruments remain limited. This study evaluated a pediatric otoscopy curriculum incorporated into the Pediatric medical student clerkship with use of outcome measures that included assessment of skills with real patients. The objective was to determine whether students who received the intervention would demonstrate significant gains in pediatric otoscopy skills when compared with students with only routine immersion learning exposure. METHODS: During their Pediatric clerkship, an intervention group (IG) of 100 medical students received routine instruction and a curriculum intervention. A non-intervention group (NIG) of 30 students received only routine instruction. Outcome measures included written tests and assessment of skills with real patients. A retention group (RG) consisted of 79 students in the IG who completed a written test at the end of medical school. Paired t-tests were used to compare differences in pre-intervention, post-intervention, and retention scores for the IG, NIG, and RG, while analysis of covariance tests were used to compare differences in scores between the IG and NIG. RESULTS: Pre-intervention scores were similar for the IG and NIG for the written test (mean/SD of 12.9/2.9 for IG and 12.9/1.8 for NIG, p = 0.78) and skills checklist (mean/SD of 11.1/4.4 for IG and 10.9/4.0 for NIG, p = 0.88). The IG had significantly higher post-intervention scores than the NIG for the written test (mean/SD of 22.6/1.7 for IG and 13.9/2.5 for NIG, p < 0.001) and skills checklist (mean/SD of 19.2/3.4 for IG and 11.0/3.8 for NIG, p < 0.001). The IG also had significantly higher gain in scores than the NIG for the written test (mean/SD +9.6/2.8 for IG and +1.0/2.3 for NIG, p < 0.001) and skills checklist (mean/SD of +8.1/4.8 for IG and +0.1/4.5 for NIG, p < 0.001). For the RG, there was a significant decrease (p < 0.001) from the post-intervention scores to retention scores (mean/SD of -7.4/2.7) but a significant increase (p < 0.001) from the pre-intervention score to retention score (mean + 2.6/3.3). CONCLUSIONS: Medical students who received a formal curriculum intervention demonstrated significant gains in pediatric otoscopy skills when compared with students with only routine immersion learning exposure. However, learning gains diminished over time, emphasizing the need for continued practice opportunities to reinforce students' skills. Our study provides a formal curriculum to meet identified educational gaps in the important topic of pediatric otoscopy and offers a model for teaching of other clinical skills using rigorous outcome measures including assessment of skills in real patients.

Cofactor-Free, Direct Photoactivation of Enoate Reductases for the Asymmetric Reduction of C=C Bonds.

Enoate reductases from the family of old yellow enzymes (OYEs) can catalyze stereoselective trans-hydrogenation of activated C=C bonds. Their application is limited by the necessity for a continuous supply of redox equivalents such as nicotinamide cofactors [NAD(P)H]. Visible light-driven activation of OYEs through NAD(P)H-free, direct transfer of photoexcited electrons from xanthene dyes to the prosthetic flavin moiety is reported. Spectroscopic and electrochemical analyses verified spontaneous association of rose bengal and its derivatives with OYEs. Illumination of a white light-emitting-diode triggered photoreduction of OYEs by xanthene dyes, which facilitated the enantioselective reduction of C=C bonds in the absence of NADH. The photoenzymatic conversion of 2-methylcyclohexenone resulted in enantiopure (ee>99 %) (R)-2-methylcyclohexanone with conversion yields as high as 80-90 %. The turnover frequency was significantly affected by the substitution of halogen atoms in xanthene dyes.