Explore the vast range of titles available.

The family of macrophage migration inhibitory factor (MIF) proteins in humans consist of MIF, its functional homolog D-dopachrome tautomerase (D-DT, also known as MIF-2) and the relatively unknown protein named DDT-like (DDTL). MIF is a pleiotropic cytokine with multiple properties in tissue homeostasis and pathology. MIF was initially found to associate with inflammatory responses and therefore established a reputation as a pro-inflammatory cytokine. However, increasing evidence demonstrates that MIF influences many different intra- and extracellular molecular processes important for the maintenance of cellular homeostasis, such as promotion of cellular survival, antioxidant signaling, and wound repair. In contrast, studies on D-DT are scarce and on DDTL almost nonexistent and their functions remain to be further investigated as it is yet unclear how similar they are compared to MIF. Importantly, the many and sometimes opposing functions of MIF suggest that targeting MIF therapeutically should be considered carefully, taking into account timing and severity of tissue injury. In this review, we focus on the latest discoveries regarding the role of MIF family members in tissue injury, inflammation and repair, and highlight the possibilities of interventions with therapeutics targeting or mimicking MIF family proteins.

The selective enzymatic reduction of nitroaliphatic and nitroaromatic compounds to aliphatic amines and amino-, azoxy- and azo-aromatics, respectively, remains a persisting challenge for biocatalysis. Here we demonstrate the light-powered, selective photoenzymatic synthesis of aliphatic amines and amino-, azoxy- and azo-aromatics from the corresponding nitro compounds. The nitroreductase from Bacillus amyloliquefaciens , in synergy with a photocatalytic system based on chlorophyll, promotes selective conversions of electronically-diverse nitroarenes into a series of aromatic amino, azoxy and azo products with excellent yield (up to 97%). The exploitation of an alternative nitroreductase from Enterobacter cloacae enables the tailoring of a photoenzymatic system for the challenging synthesis of aliphatic amines from nitroalkenes and nitroalkanes (up to 90% yield). This photoenzymatic reduction overcomes the competing bio-Nef reaction, typically hindering the complete enzymatic reduction of nitroaliphatics. The results highlight the usefulness of nitroreductases to create selective photoenzymatic systems for the synthesis of precious chemicals, and the effectiveness of chlorophyll as an innocuous photocatalyst, enabling the use of sunlight to drive the photobiocatalytic reactions. The selective enzymatic reduction of nitroaliphatic and nitroaromatic compounds is challenging. Here, the authors report selective (sun)light-driven photoenzymatic reduction of a wide variety of nitro compounds to aliphatic amines and amino-, azoxy- and azo-aromatics using flavin-dependent nitroreductases and chlorophyll as photocatalyst

Biocatalysis contributes significantly to the development of more sustainable synthetic pathways by using mild reaction conditions and water as a solvent. However, many relevant classes of compounds, including privileged groups in drug design, are not yet accessible via enzymatic pathways. In this context, the development of an enzymatic route to indazoles remains an unmet challenge. Here, we present a nitroreductase-triggered indazole formation, in which 2-nitrobenzylamine derivatives are converted to reactive nitrosobenzylamine intermediates that spontaneously cyclize and aromatize to indazoles. Two nitroreductases accept a series of 2-nitrobenzylamine derivatives with excellent conversions (up to >99 %). In the case of N -substituted nitrosobenzylamines, 2 H -indazoles are formed, whereas other derivatives led to 1 H -indazoles. The synthetic value of the nitroreductase-triggered indazole formation is further demonstrated by successful coupling with an imine reductase in a sequential cascade reaction on a 50 mg scale. With this cascade, 2 H -indazoles are accessible from cheap 2-nitrobenzaldehyde and primary amines, resulting in up to 85 % conversion and 68 % isolated yield. An enzymatic route to indazoles, scaffolds present in many drugs, has not been developed yet. Here, the authors report nitroreductase-triggered indazole formation, in which 2-nitrobenzylamine derivatives are converted to reactive nitrosobenzylamine intermediates that spontaneously cyclize and aromatize to indazoles.

The Michael-type addition reaction is widely used in organic synthesis for carbon–carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon–carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon–carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence–function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary ‘Michaelases’. These ‘Michaelases’ catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives. The Michael-type addition reaction is used for carbon-carbon bond formation; however biocatalytic methods for this reaction are rare. Here, the authors generate and exploit mutability landscapes of 4-oxalocrotonate tautomerase to direct the redesign of this promiscuous enzyme into enantio-complementary Michaelases.

Photoenzymes are light-powered biocatalysts that typically rely on the excitation of cofactors or unnatural amino acids for their catalytic activities 1 , 2 . A notable natural example is the fatty acid photodecarboxylase, which uses light energy to convert aliphatic carboxylic acids to achiral hydrocarbons 3 . Here we report a method for the design of a non-natural photodecarboxylase based on the excitation of enzyme-bound catalytic intermediates, rather than reliance on cofactor excitation 4 . Iminium ions 5 , transiently generated from enals within the active site of an engineered class I aldolase 6 , can absorb violet light and function as single-electron oxidants. Activation of chiral carboxylic acids, followed by decarboxylation, generates two radicals that undergo stereospecific cross-coupling, yielding products with two stereocentres. Using the appropriate enantiopure chiral substrate, the desired diastereoisomeric product is selectively obtained with complete enantiocontrol. This finding underscores the ability of the active site to transfer stereochemical information from the chiral radical precursor into the product, effectively addressing the long-standing problem of rapid racemization of chiral radicals. The resulting ‘memory of chirality’ scenario 7 is a rarity in enantioselective radical chemistry. We report a method for the design of a non-natural photodecarboxylase based on the excitation of enzyme-bound catalytic intermediates, rather than reliance on cofactor excitation.

Macrophage migration inhibitory factor (MIF) is a cytokine found to be associated with chronic obstructive pulmonary disease (COPD). However, there is no consensus on how MIF levels differ in COPD compared to control conditions and there are no reports on MIF expression in lung tissue. Here we studied gene expression of members of the MIF family MIF , D-Dopachrome Tautomerase ( DDT ) and DDT-like ( DDTL ) in a lung tissue dataset with 1087 subjects and identified single nucleotide polymorphisms (SNPs) regulating their gene expression. We found higher MIF and DDT expression in COPD patients compared to non-COPD subjects and found 71 SNPs significantly influencing gene expression of MIF and DDTL . Furthermore, the platform used to measure MIF (microarray or RNAseq) was found to influence the splice variants detected and subsequently the direction of the SNP effects on MIF expression. Among the SNPs found to regulate MIF expression, the major LD block identified was linked to rs5844572, a SNP previously found to be associated with lower diffusion capacity in COPD. This suggests that MIF may be contributing to the pathogenesis of COPD, as SNPs that influence MIF expression are also associated with symptoms of COPD. Our study shows that MIF levels are affected not only by disease but also by genetic diversity (i.e. SNPs). Since none of our significant eSNPs for MIF or DDTL have been described in GWAS for COPD or lung function, MIF expression in COPD patients is more likely a consequence of disease-related factors rather than a cause of the disease.

Peroxygenases are attractive biocatalysts for the selective introduction of oxygen into organic molecules under mild conditions with hydrogen peroxide as the oxygen source. In addition to the identification of primary peroxygenases, different classes of enzymes were shown to display promiscuous peroxygenase activity. Even though enzymes with peroxygenase activity are promising industrial biocatalysts, further optimization of their properties is required for their effective use in industrial applications. Here we give a comprehensive overview of enzymes with peroxygenase activity and review diverse strategies, including directed evolution, rational approaches and the assistance of small functional molecules to improve the expression, catalytic activity, substrate scope or selectivity of these promising enzymes. Furthermore, we discuss the exploration of modified or unnatural cofactors to design artificial peroxygenases for desired reactions. The rapidly expanding field of hydrogen peroxide-utilizing enzymes bears a great potential to provide biocatalysts for selective oxyfunctionalization chemistry, contributing to the development of environmentally friendly and sustainable oxidation processes. Peroxygenases are promising enzymes to contribute to more environmentally friendly and sustainable oxidation reactions in industry. This Review Article gives a comprehensive overview about enzymes with peroxygenase activity and strategies to engineer them for desired reactions and enhanced performances.

The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo -3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain). Substituted aspartic acids are highly valuable as tools for biological research and as chiral building blocks for pharmaceuticals. Here, engineering of the enzyme methylaspartate ammonia lyase to accept a large variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives is described.

Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (49 kDa each), and requires Mg^sup 2+^ and K^sup +^ ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k ^sub cat^=78 s^sup -1^ and K ^sub m^=16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale.[PUBLICATION ABSTRACT]

The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each recognizes and expels a broad spectrum of chemically unrelated drugs from the cell. The Escherichia coli Mdr transporter MdfA is able to transport differentially charged substrates in exchange for protons. This includes neutral compounds, namely chloramphenicol and thiamphenicol, and lipophilic cations such as tetraphenylphosphonium and ethidium. Here we show that the chloramphenicol and thiamphenicol transport reactions are electrogenic, whereas the transport of several monovalent cationic substrates is electroneutral. Therefore, unlike with positively charged substrates, the transmembrane electrical potential (negative inside) constitutes a major part of the driving force for the transport of electroneutral substrates by MdfA. These results demonstrate an unprecedented ability of a single secondary transporter to catalyze discrete transport reactions that differ in their electrogenicity and are governed by different components of the proton motive force.