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In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the β-hydroxyacid dehydrogenases superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes. [ABSTRACT FROM AUTHOR]

This study explored the enantiocomplementary bioreduction of substituted 1-(arylsulfanyl)propan-2-ones in batch mode using four wild-type yeast strains and two different recombinant alcohol dehydrogenases from Lactobacillus kefir and Rhodococcus aetherivorans. The selected yeast strains and recombinant alcohol dehydrogenases as whole-cell biocatalysts resulted in the corresponding 1-(arylsulfanyl)propan-2-ols with moderate to excellent conversions (60–99%) and high selectivities (ee > 95%). The best bioreductions—in terms of conversion (>90%) and enantiomeric excess (>99% ee)—at preparative scale resulted in the expected chiral alcohols with similar conversion and selectivity to the screening reactions. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase, and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, demonstrate complex formation between TclI, TclJ, and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. [ABSTRACT FROM AUTHOR]

Gluconobacter strains perform incomplete oxidation of various sugars and alcohols, employing regio- and stereoselective membrane-bound dehydrogenases oriented toward the periplasmic space. This oxidative fermentation process is utilized industrially. The ketogluconate production pathway, characteristic of these strains, begins with the conversion of d -glucose to d -gluconate, which then diverges and splits into 2 pathways producing 5-keto- d -gluconate and 2-keto- d -gluconate and subsequently 2,5-diketo- d -gluconate. These transformations are facilitated by membrane-bound d -glucose dehydrogenase, glycerol dehydrogenase, d -gluconate dehydrogenase, and 2-keto- d -gluconate dehydrogenase. The variance in end products across Gluconobacter strains stems from the diversity of enzymes and their activities. This review synthesizes biochemical and genetic knowledge with biotechnological applications, highlighting recent advances in metabolic engineering and the development of an efficient production process focusing on enzymes relevant to the ketogluconate production pathway in Gluconobacter strains. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is a natural producer of 2,3-butanediol (2,3-BDO) and has acquired "Generally Regarded as Safe" status. It is reported to produce 2,3-BDO from synthetic sugars as well as complex and economic sugar sources such as molasses. However, the rate-limiting step in the formation of 2,3-BDO is its conversion from acetoin to 2,3-BDO by the enzyme butanediol dehydrogenase (2,3-BDH). Such 2,3-BDHs were screened based on higher affinity (lower Km) towards acetoin as substrate. The in silico docking studies were conducted for further validation, and they showed a high interaction profile for the PpBDH protein towards acetoin. Heterologous expression of these genes was studied in engineered Bacillus subtilis (BS1A1). In this study, it was seen that 2,3-BDH from Paenibacillus polymyxa ZJ-9 was reported to have higher enzyme activity levels, and in the fermentation studies, it was seen that the ratio of 2,3-BDO to acetoin was increased by 80.25%. The insights encourage further bioprocess optimization for increasing the fermentative production of 2,3-BDO. Our results provide a potential strategy to avoid the back conversion of 2,3-BDO to acetoin in an engineered Bacillus system. [ABSTRACT FROM AUTHOR]

Background: Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily. Results: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold. Conclusions: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions. [ABSTRACT FROM AUTHOR]

Anthropogenic climate change has been caused by over‐exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell‐free enzyme cascade generating pyruvate as the central intermediate, the NAD+‐dependent, selective oxidation of D‐glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24‐fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi‐rational engineering improved the substrate selectivity of the enzyme up to 21‐fold, thus minimizing side reactions in the one‐pot enzyme cascade. The final biotransformation of D‐glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications. [ABSTRACT FROM AUTHOR]

The use of immobilized alcohol dehydrogenases (ADHs) offers numerous advantages, especially in the reaction conditions required by industrial applications. Looking for more efficient and cost-effective methods of ADH immobilization, in this study we explored silica-based supports as an alternative to the use of functionalized polymeric resins. Three commercially available ADHs were immobilized by adsorption and covalent bond formation. The obtained supported biocatalysts were applied for the bioreduction of acetophenone and some derivatives with good yields and excellent enantioselectivity. The important intermediate (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol was obtained with a high enantiomeric excess (>99%) by using the highest performing immobilized ADH sample. The reusability of this biocatalyst was investigated in a flow system for five consecutive runs; the experiments showed that the biocatalyst could be recycled without a loss of activity and enantioselectivity. Finally, cross-linking with the glutaraldehyde of the supported biocatalyst was also carried out to prevent the leaching of the enzyme during the catalytic reactions. [ABSTRACT FROM AUTHOR]

Short-chain dehydrogenases/reductases (SDRs) are one of the most prevalent enzyme families distributed among the sequenced microorganisms. Despite the presence of a conserved catalytic tetrad and high structural similarity, these enzymes exhibit different substrate specificities. The insufficient knowledge regarding the amino acids underlying substrate specificity hinders the understanding of the SDRs' roles in diverse and significant biological processes. Here, we performed bioinformatic analysis, molecular modeling, and mutagenesis studies to identify the key residues that regulate the substrate specificities of two homologous microbial SDRs (i.e., DesE and KduD). Further, we investigated the impact of altering the physicochemical properties of these amino acids on enzyme activity. Interestingly, molecular dynamics simulations also suggest a critical role of enzyme conformational flexibility in substrate recognition and catalysis. Overall, our findings improve the understanding of microbial SDR substrate specificity and shed light on future rational design of more efficient and effective biocatalysts. [ABSTRACT FROM AUTHOR]

Oxidoreductases are enzymes with distinctive characteristics that favor their use in different areas, such as agriculture, environmental management, medicine, and analytical chemistry. Among these enzymes, oxidases, dehydrogenases, peroxidases, and oxygenases are very interesting. Because their substrate diversity, they can be used in different biocatalytic processes by homogeneous and heterogeneous catalysis. Immobilization of these enzymes has favored their use in the solution of different biotechnological problems, with a notable increase in the study and optimization of this technology in the last years. In this review, the main structural and catalytical features of oxidoreductases, their substrate specificity, immobilization, and usage in biocatalytic processes, such as bioconversion, bioremediation, and biosensors obtainment, are presented. [ABSTRACT FROM AUTHOR]

The enzymatic reduction of carbon dioxide presents limited applicability due to denaturation and the impossibility of biocatalyst recovery; disadvantages that can be minimized by its immobilization. Here, a recyclable bio‐composed system was constructed by in‐situ encapsulation under mild conditions using formate dehydrogenase in a ZIF‐8 metalorganic framework (MOF) in the presence of magnetite. The partial dissolution of ZIF‐8 in the enzyme's operation medium can be relatively inhibited if the concentration of magnetic support used exceeds 10 mg mL−1. The bio‐friendly environment for immobilization does not harm the integrity of the biocatalyst, and the production of formic acid is improved 3.4‐fold compared to the free enzyme because the MOFs act as concentrators of the enzymatic cofactor. Furthermore, the bio‐composed system retains 86 % of its activity after a long time of five cycles, thus indicating an excellent magnetic recovery and a good reusability. [ABSTRACT FROM AUTHOR]

The review discloses the historical and technological evolution of enzyme‐based field‐effect transistors (EnFETs) underlying the importance of gate electrode modification toward the implementation of novel FETs configurations such as extended‐gate FET (EG‐FETs) or EG organic FETs (EG‐OFETs). The working principle of the EnFETs as postulated by Bergveld in 1970, who defined the EnFET as an ion‐selective FET (ISFET) modified with enzyme‐membrane, is also discussed considering the analytical equations related to the EnFET output response. For each category, namely EnFETs, EG‐FETs, and EG‐OFETs, we reviewed the key devices' configurations that addressed the research in this field in the last 40 years with particular attention to the analytical figures of merit. [ABSTRACT FROM AUTHOR]

Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO2 to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies. [ABSTRACT FROM AUTHOR]

The study objective was to determine the relationship of selected enzyme activities with carbon sequestration and N, P, K, Mg, Zn and Cu contents in Phaeozem soils. Soil samples were taken from a 10 ha area. A selection of their physical and chemical properties and the contents of the available forms of selected macro- and microelements were determined. The activities of dehydrogenases (DEH), catalase (CAT), peroxidases (PER), alkaline (AlP) and acid (AcP) phosphatase, β-glucosidase (BG) and proteases (PR) were also determined. The relationship between enzymatic soil fertility indices (AlP/AcP, BIF, GMea, TEI, BA12 and BA13) and selected soil parameters was also determined. The research used principal component analysis (PCA) to distinguish significantly correlated parameters of a Phaeozem used for agricultural purposes. The study area showed low TOC and K contents and average P and Mg contents. Significant positive correlations were found between the TOC content and activity of the tested enzymes, evidencing that soil enzymes are an important parameter in carbon sequestration and soil nutrient dynamics. [ABSTRACT FROM AUTHOR]

A minimal light‐driven approach was established for studying enzymatic CO2 conversion spectroscopically. The system consists of a photosensitizer Eosin Y, EDTA as a sacrificial electron donor and substrate source, and formate dehydrogenase from Rhodobacter capsulatus (RcFDH) as a biocatalyst. This simplified three‐component system provides a photo‐triggered control for in situ characterization of the entire catalytic reaction. Direct reduction of RcFDH by the photosensitizer without additional electron carriers was confirmed via UV‐Vis spectroscopy, while GC‐MS and IR spectroscopy were used to follow photoinduced CO2 generation from EDTA and its subsequent enzymatic reduction, yielding the product formate. Photo‐driven and in vitro, dye‐based CO2 reduction was inhibited by azide under a mixed (competitive‐non‐competitive) inhibition mode. IR spectroscopy reveals displacement of the competitively‐bound azide by CO2, reflecting an interaction of both with the active site cofactor. This work comprises a proof‐of‐concept for a new approach to employ light for regulating the reaction of formate dehydrogenases and other CO2 reductases. [ABSTRACT FROM AUTHOR]

Application of thermoplastic fiber-reinforced lightweight composite materials provides a wide range of advantages that are of particular importance for the mobility sector. UD tapes composed of unidirectionally (UD) oriented inorganic fibers embedded in a thermoplastic matrix represent light-weight materials with high tensile strength. This publication addresses recycling aspects of novel UD tape made of a combination of basalt fibers and different PLA (polylactic acid) formulations. The kinetics of enzyme-based separation of polymer from the fiber were investigated. Different types of UD tapes with a thickness of 270-290 μm reinforced with basalt fiber weight ratios ranging between 51 and 63% were incubated at 37 °C in buffer solution (pH 7.4) containing proteinase K. The influence of enzyme concentration, tape weight per incubation tube, proteinase K activators, and tape types on the rate of enzymatic decomposition was investigated. Enzyme activity was measured by analyzing lactate concentration with lactate dehydrogenase and by measuring weight loss of the composite material. The rate of lactate release increased in the first 30 min of incubation and remained stable for at least 90 min. Weight loss of 4% within 4 h was achieved for a tape with 56% (w/w) fiber content. For a sample with a surface area of 3 cm² in a buffer volume of 10 mL, the rate of lactate release as a function of enzyme concentration reached saturation at 300 μg enzyme/mL. With this enzyme concentration, the rate of lactate release increased in a linear manner for tape surface areas between 1 and 5 cm². Four tapes with different PLA types were treated with the enzyme for 17 h. Weight loss ranged between 7 and 24%. Urea at a concentration of 0.5% (w/v) increased lactate release by a factor of 9. Pretreatment of tapes in alkaline medium before enzymatic degradation increased weight loss to 14% compared to 5% without pretreatment. It is concluded that enzymatic PLA hydrolysis from UD tapes is a promising technology for the release of basalt fibers after alkaline pretreatment or for the final cleaning of basalt fibers. [ABSTRACT FROM AUTHOR]

Medium‐chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non‐canonical 1,4‐reduction of an α,β‐unsaturated iminium moiety. Comparison with structures of plant‐derived ADHs suggest the 1,4‐iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2‐aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2‐iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism. [ABSTRACT FROM AUTHOR]

Upgrading of the bio-based platform chemical 5-hydroxymethylfurfural (HMF) into high-value derivatives is an important research topic, particularly in green and sustainable chemistry. Herein, we applied a new, highly HMF-tolerant strain, Enterobacter ludwigii YYP3, as a whole-cell biocatalyst for efficient reduction of HMF to 2,5-bis(hydroxymethyl)furan (BHMF). Upon process optimization, within only 3 h, BHMF was produced with a yield >99% and 98.5% selectivity using 100 mM HMF, resulting in the highest space time yield (4.2 g L−1 h−1) among the currently reported HMF bioreduction processes. In a fed-batch conversion, E. ludwigii YYP3 achieved large-scale production of 290 mM BHMF within 9 h and retained its high catalytic activity for three runs (27 h), suggesting an excellent cycling stability. Based on genome and transcriptome analysis, the molecular mechanism underlying the high HMF tolerance of E. ludwigii YYP3 was explored, primarily through the downregulation of genes related to amino acid biosynthetic and metabolic processes and upregulation of genes associated with DNA replication, recombination, and repair; biofilm formation; and redox homeostasis. Meanwhile, two novel short-chain dehydrogenase/reductase family oxidoreductases ElSDR-ykvO and ElSDR-SSP1627 were identified as target enzymes responsible for conversion of HMF to less toxic BHMF in E. ludwigii YYP3. Combined with structure and mutation analysis, the catalytic mechanisms of target enzymes were determined to be based on the active sites Ser, Tyr, and Lys. Our work not only confirms that E. ludwigii YYP3 has promising application prospects in large-scale production of BHMF, but also provides novel insights into understanding the molecular mechanism of HMF reduction. [ABSTRACT FROM AUTHOR]

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon‐felt‐based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD+), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD+ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD+ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches. [ABSTRACT FROM AUTHOR]

The evolutionary benefit accounting for widespread conservation of oligomeric structures in proteins lacking evidence of intersubunit cooperativity remains unclear. Here, crystal and cryo‐EM structures, and enzymological data, demonstrate that a conserved tetramer interface maintains the active‐site structure in one such class of proteins, the short‐chain dehydrogenase/reductase (SDR) superfamily. Phylogenetic comparisons support a significantly longer polypeptide being required to maintain an equivalent active‐site structure in the context of a single subunit. Oligomerization therefore enhances evolutionary fitness by reducing the metabolic cost of enzyme biosynthesis. The large surface area of the structure‐stabilizing oligomeric interface yields a synergistic gain in fitness by increasing tolerance to activity‐enhancing yet destabilizing mutations. We demonstrate that two paralogous SDR superfamily enzymes with different specificities can form mixed heterotetramers that combine their individual enzymological properties. This suggests that oligomerization can also diversify the functions generated by a given metabolic investment, enhancing the fitness advantage provided by this architectural strategy. Synopsis: Many proteins form evolutionarily conserved oligomers in the absence of obvious functional interactions between subunits. Structural and phylogenetic analyses demonstrate that these interactions maintain active‐site structure in the short‐chain dehydrogenase/reductase superfamily. A cryo‐EM structure of a short‐chain dehydrogenase/reductase (SDR) superfamily enzyme shows dynamic opening of its tetramerization interface coupled to disordering of its active site.The same homotetramer structure is conserved in the vast majority of SDR superfamily enzymes with structures deposited in the PDB.The maintenance of active‐site structure using homooligomeric interactions enables a shorter protein subunit to form an active enzyme.Increased evolutionary fitness can therefore be conferred by homooligomeric interactions due to reduced metabolic investment. [ABSTRACT FROM AUTHOR]

Background: The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags. Results: In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g− 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg− 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade. Conclusion: CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9. [ABSTRACT FROM AUTHOR]

The cascade catalysis of old yellow enzyme, alcohol dehydrogenase and glucose dehydrogenase has become a promising approach for one pot, two-step reduction of (E/Z)-citral to (S)-citronellol, serving as a chiral alcohol with rose fragrance. During the multi-enzymatic cascade catalysis, old yellow enzyme is responsible for the reduction of the conjugated C=C and the introduction of the chiral center, requiring high activity and (S)-enantioselectiviy. Herein, to improve the activity of the old yellow enzyme from Providencia stuartii (NemR-PS) with strict (S)-enantioselectivity, the semi-rational design on its substrate binding pocket was performed through a combination of homology modeling, molecular docking analysis, alanine scanning and iterative saturation mutagenesis. The NemR-PS variant D275G/F351A with improved activity was obtained and then purified for characterization, obeying the substrate inhibition kinetics. Compared with the wild type, the parameters Ki and Kcat/Km were increased from 39.79 mM and 2.09 s−1mM−1 to 128.50 mM and 5.01 s−1mM−1, respectively. Moreover, the variant D275G/F351A maintained strict (S)-enantioselectivity, avoiding the trade-off effect between activity and enantioselectivity. Either the enzyme NemR-PS or the variant D275G/F351A was co-expressed with alcohol dehydrogenase from Yokenella sp. WZY002 (YsADH) and glucose dehydrogenase from Bacillus megaterium (BmGDHM6). In contrast to the whole-cell biocatalyst co-expressing NemR-PS, that co-expressing the variant D275G/F351A shortened the reaction time from 36 h to 12 h in the reduction of 400 mM (E/Z)-citral. In the manner of substrate constant feeding, the accumulated product concentration reached up to 500 mM and completely eliminate the residual intermediate and by-product, suggesting the effectiveness of protein engineering and substrate engineering to improve catalytic efficiency. [ABSTRACT FROM AUTHOR]

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha, 17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs. [ABSTRACT FROM AUTHOR]

A series of optically active β‐hydroxy sulfones has been obtained through an oxosulfonylation‐stereoselective reduction sequence in aqueous medium. Firstly, β‐keto sulfones were synthesized from arylacetylenes and sodium sulfinates to subsequently develop the carbonyl reduction in a highly selective fashion using alcohol dehydrogenases as biocatalysts. Optimization of the chemical oxosulfonylation reaction was investigated, finding inexpensive iron(III) chloride hexahydrate (FeCl3 ⋅ 6H2O) as the catalyst of choice. The selection of isopropanol in the alcohol‐water media resulted in high compatibility with the enzymatic process for enzyme cofactor recycling purposes, providing a straightforward access to both (R)‐ and (S)‐β‐hydroxy sulfones. The practical usefulness of this transformation was illustrated by describing the synthesis of a chiral intermediate of Apremilast. Interestingly, the development of a chemoenzymatic cascade approach avoided the isolation of β‐keto sulfone intermediates, which allowed the preparation of chiral β‐hydroxy sulfones in high conversion values (83–94 %) and excellent optical purities (94 to >99 % ee). [ABSTRACT FROM AUTHOR]

[Display omitted] • An efficient biocatalyst for preparation of N -heterocyclic primary amines was developed. • Mutant M4-2 exhibited broad substrate scope, high activity, and excellent stereoselectivity. • First report of the biocatalytic synthesis of this type of N -heterocyclic primary amines. • Provided engineering guidance for the acceptance evolution of AmDH family members. Chiral N -heterocyclic primary amines are highly attractive synthons in the pharmaceutical industry. Amine dehydrogenases (AmDHs)-catalyzed direct asymmetric reductive amination of the readily available N -heterocyclic ketones represents a promising approach for synthesizing N -heterocyclic primary amines. However, the limited substrate acceptance of AmDHs restricts their application in biocatalytic synthesis. Here, we engineered the substrate acceptance of L- Es AmDH from Exiguobacterium sibiricum to access a panel of structurally diverse N -heterocyclic ketones. Through reverse substrate design and combining with substrate walking strategy, two active mutants with extended substrate specificity toward N -Boc-4-acetylpiperidine were identified, and two additional rounds of iterative site mutagenesis further increased the activity by 116.3-fold. The optimal mutant M4-2 (L49G/V303A/L307A/T143A) exhibited significantly expanded N -Boc, N -Bn, and N -Cbz-substituted heterocyclic ketones scope, and its practical biocatalytic synthesis performance was verified in the gram-scale synthesis of (R)-4-aminoethyl-1-Boc-piperidine, achieving 95 % conversion, >99 % ee , and an overall isolated yield of 86 % (9.8 g). Our study lays the foundation for the collective biocatalytic synthesis of structurally diverse N -heterocyclic primary amines and gives referable guidance for engineering AmDH family members into versatile biocatalysts. [ABSTRACT FROM AUTHOR]

Ni,Fe‐containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe‐cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS‐VCh. Its structure reveals the typical CODH scaffold, but contains an iron‐sulfur‐oxo hybrid‐cluster. Although closely related to true CODHs, CooS‐VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS‐VCh undergoes a redox‐dependent restructuring between a reduced [4Fe‐3S]‐cluster and an oxidized [4Fe‐2S‐S*‐2O‐2(H2O)]‐cluster. Hydroxylamine, a slow‐turnover substrate of CooS‐VCh, oxidizes the hybrid‐cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O‐cluster in place of the Ni,Fe‐heterocubane‐cluster of CODHs. [ABSTRACT FROM AUTHOR]

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High‐resolution structural analysis of these enzymes is crucial in order to understand their mechanisms and modulate their properties by targeted engineering. Although cryo‐electron microscopy (cryoEM) has revolutionized structural biology, its applicability to high‐resolution structural analysis of comparatively small enzymes has so far been largely unexplored. Here, it is shown that cryoEM can reveal the structures of plant borneol dehydrogenases of ∼120 kDa at or below 2 Å resolution, paving the way for the rapid development of new biocatalysts that can provide access to bioactive terpenes and terpenoids. [ABSTRACT FROM AUTHOR]

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs. [ABSTRACT FROM AUTHOR]

Polymeric materials containing ionic liquid fragments, like those found in bulk ILs, are excellent solid media for the immobilisation of biocatalysts. Herein, the entrapment of the enzymatic system formed by alcohol dehydrogenase from Rhodococcus ruber (ADH-A) overexpressed in E. coli and its coenzyme has been studied. The activity, stability and reusability of these preparations have been investigated in the bioreduction of prochiral ketones finding excellent levels of conversion and selectivity. Interestingly, the immobilised enzyme remained active and exhibited excellent stability in aqueous solutions after several recycling uses. More importantly, these biopolymer materials retained most of their activity after consecutive reaction cycles, prolonged storage and under flow conditions. [ABSTRACT FROM AUTHOR]

Biocatalytic approaches have been investigated in order to isolate the enantiomers of Wieland–Miescher ketone (1) and of its alcoholic derivatives (cis‐2 and trans‐3). Specifically, two enzymes from our in‐house metagenomic collection of oxidoreductases, IS2‐SDR and Dm7α‐HSDH, catalyzed the kinetic resolution of the starting racemic ketone 1 or its complete conversion into two diastereomeric products, respectively. Moreover, the kinetic resolution of the racemic cis‐alcohol (2) was very efficiently obtained (E≅2.000) by lipase PS catalyzed acetylation in dry acetone. All the products were isolated with ee≥95 %. Simple chemical elaborations of some of them allowed to isolate the missing enantiomers. [ABSTRACT FROM AUTHOR]

NADPH-dependent oxidoreductases are crucial biocatalysts for the industrial production of chiral compounds. For in situ recycling of required expensive cofactors in this biosynthetic process, NAD(P)+-dependent formate dehydrogenases (FDHs) are wanted to be employed as redox biocatalysts due to their greener and process friendly nature. However, their utilization is limited by their undesired cofactor preference that strongly prone to NAD+ more than NADP+ and catalytic efficiency. To mine NADP+-dependent FDHs in nature by the guidance of bioinformatic tools or re-engineering of their NAD+-dependent equivalents to get an applicable recycler are attractive topics in bioengineering. It can be said that, up to now, the attempts to switch the cofactor preference of the FDHs generally have resulted in NADP+-dependent enzymes that have not to catch the desired catalytic efficiencies or stability. In this review, all studies about the native NADP+-dependent FDHs and also engineered equivalents that reconstructed with different protein engineering approaches for altering the coenzyme specificity are outlined. To switch the coenzyme preference of FDHs or to find the native NADP+-dependent FDHs will be the hot topics in bioengineering until finding a feasible regenerator. Therefore, this study will be a useful guide to get a pathway for designing or discovering novel NADP+-dependent FDHs. [ABSTRACT FROM AUTHOR]