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[学术文献 ] Comparing and Combining Alternative Strategies for Enhancing Cytochrome P450 Peroxygenase Activity 进入全文
ACS Catalysis
Cytochrome P450 monooxygenase (CYP) enzymes have advantageous properties over chemical catalysts. However, it is often not feasible to use CYPs in larger-scale synthesis as they require additional cofactors (NAD(P)H) and electron transfer proteins. This could be overcome by transforming CYPs into peroxygenases that use H2O2. Recently, multiple strategies have been reported for converting CYPs into peroxygenases. Mutating the residues of the acid–alcohol pair in the oxygen-binding groove to those found in natural peroxygenases can promote the desired H2O2-driven activity. Another strategy is to enlarge the enzyme’s solvent channels to allow H2O2 easier access into the active site, to enhance peroxygenase activity. Here, we evaluate these different strategies by comparing the peroxygenase activities of the double I-helix mutant D251Q/T252E (the QE mutant) and the F182A mutant of the bacterial enzyme CYP199A4. We also assess whether the peroxygenase activity can be further improved by combining these mutations (to give the F182AQE mutant). The F182A mutant exhibited the highest activity toward a selection of smaller substrates that undergo O-demethylation, S-oxidation, and epoxidation reactions. All the mutants converted 4-vinylbenzoic acid into the (S)-epoxide, with the F182A mutant having the highest stereoselectivity (>99% ee). The F182A mutant was unable to oxidize 4-t-butylbenzoic acid, while the F182AQE mutant could with high activity. The F182A mutation was found to substantially alter the selectivity of the reaction with 4-ethylbenzoic acid, increasing hydroxylation activity over desaturation. The F182A mutant catalyzed significant further oxidation reactions of the primary metabolites before all the substrate had been consumed, demonstrating a relaxed substrate specificity. X-ray crystal structures of the F182A and F182AQE mutants with the substrates revealed changes in substrate binding and solvent access providing insights into these experimental observations.
[学术文献 ] Generative artificial intelligence for enzyme design: Recent advances in models and applications 进入全文
CURRENT OPINION IN GREEN AND SUSTAINABLE CHEMISTRY
Enzyme catalysis is a key enabling technology for green and sustainable production of chemicals. Developing suitable enzymes is at the heart of this technology, which is currently changing by Artificial Intelligence (AI) such as machine learning. AI-based methods were used for enzyme discovery and design. We review the recent advances in generative AI models for enzyme design, with a particular focus on those that have been validated by experiments. Furthermore, we discuss the applications of the enzymes designed by generative AI, including artificial luciferases, non-heme iron (II)-dependent oxygenases, and P450 enzymes. We provide our opinions on several current issues encountered in computational enzyme design. With the fast development of new generative models in enzymes and the implementation of these models by the research community, we believe that the precise design of efficient enzymes with new catalytic functions and/or potential industrial applications will be a mature method in the near future.
[前沿资讯 ] Illinois researchers develop next-generation organic nanozymes and point-of-use system for food and agricultural uses 进入全文
EurekAlert
Nanozymes are synthetic materials that have enzyme-like catalytic properties, and they are broadly used for biomedical purposes, such as disease diagnostics. However, inorganic nanozymes are generally toxic, expensive, and complicated to produce, making them unsuitable for the agricultural and food industries. A University of Illinois Urbana-Champaign research team has developed organic-material-based nanozymes that are non-toxic, environmentally friendly, and cost effective. In two new studies, they introduce next-generation organic nanozymes and explore a point-of-use platform for molecule detection in agricultural products.“The first generation of organic-compound-based (OC) nanozymes had some minor drawbacks, so our research group worked to make improvements. The previous OC nanozymes required the use of particle stabilizing polymers having repeatable functional groups, which assured stability of the nanozyme’s nanoscale framework, but didn’t achieve a sufficiently small particle size,” said lead author Dong Hoon Lee, who completed his Ph.D. from the Department of Agricultural and Biological Engineering (ABE), part of the College of Agricultural, Consumer and Environmental Sciences and The Grainger College of Engineering at the U. of I. In the new iteration, they used a core amino acid (L- alanine) and polyethylene glycol as constituent materials and a novel particle synthesis technique that allowed them to bring the particle size down to less than 100 nanometers. This nanozyme resembles the physical framework and mimics the catalytic activity of target enzymes.
[学术文献 ] Production of a δ-Lactam from Glucose through Integrating Biological and Chemical Catalysis 进入全文
ACS SUSTAINABLE CHEMISTRY & ENGINEERING
We present a new strategy for the production of a δ-lactam from glucose that integrates biological production of triacetic acid lactone (TAL, 4-hydroxy-6-methyl-2H-2-one) with catalytic transformation of TAL into 6-methylpiperidin-2-one (MPO) through metabolic engineering, isomerization, amination, and catalytic hydrogenation/hydrogenolysis. We developed a sustainable and antibiotic-free fed-batch fermentation using genetically modified Rhodotorula toruloides IFO0880. This process achieved a yield of 2-hydroxy-6-methyl-4H-pyran-4-one (2H4P) at 0.05 g/g of glucose, corresponding to a 9.9 g/L titer. By adjusting the pH of the fermentation broth to 2, 2H4P was quantitatively converted into TAL. The TAL in the fermentation broth was directly converted by aminolysis into 4-hydroxy-6-methylpyridin-2(1H)-one (HMPO), which achieved an 18.5% yield with 94.3% purity. The HMPO yield was lower in the fermentation broth than in a clean feedstock (32.2%), suggesting that the biological impurities are inhibitors in this reaction. Further investigation revealed that lower pH levels and reduced TAL concentrations in the fermentation broth significantly decreased HMPO yields. Subsequently, the precipitated HMPO was filtered and dried and then subjected to the final catalytic conversion in H2O solvent, achieving a MPO yield of 91.8%. This integrated approach demonstrated the direct use of TAL in the filtered aqueous fermentation broth without the need to isolate TAL.
[学术文献 ] Computational design of serine hydrolases 进入全文
Science
The design of enzymes with complex active sites that mediate multistep reactions remains an outstanding challenge. With serine hydrolases as a model system, we combined the generative capabilities of RFdiffusion with an ensemble generation method for assessing active site preorganization to design enzymes starting from minimal active site descriptions. Experimental characterization revealed catalytic efficiencies (kcat/Km) up to 2.2x105 M−1 s−1 and crystal structures that closely match the design models (Cα RMSDs < 1 Å). Selection for structural compatibility across the reaction coordinate enabled identification of new catalysts in low-throughput screens with five different folds distinct from those of natural serine hydrolases. Our de novo approach provides insight into the geometric basis of catalysis and a roadmap for designing enzymes that catalyze multistep transformations.
[学术文献 ] A metagenomic ‘dark matter’ enzyme catalyses oxidative cellulose conversion 进入全文
Nature
The breakdown of cellulose is one of the most important reactions in nature1,2 and is central to biomass conversion to fuels and chemicals3. However, the microfibrillar organization of cellulose and its complex interactions with other components of the plant cell wall poses a major challenge for enzymatic conversion4. Here, by mining the metagenomic ‘dark matter’ (unclassified DNA with unknown function) of a microbial community specialized in lignocellulose degradation, we discovered a metalloenzyme that oxidatively cleaves cellulose. This metalloenzyme acts on cellulose through an exo-type mechanism with C1 regioselectivity, resulting exclusively in cellobionic acid as a product. The crystal structure reveals a catalytic copper buried in a compact jelly-roll scaffold that features a flattened cellulose binding site. This metalloenzyme exhibits a homodimeric configuration that enables in situ hydrogen peroxide generation by one subunit while the other is productively interacting with cellulose. The secretome of an engineered strain of the fungus Trichoderma reesei expressing this metalloenzyme boosted the glucose release from pretreated lignocellulosic biomass under industrially relevant conditions, demonstrating its biotechnological potential. This discovery modifies the current understanding of bacterial redox enzymatic systems devoted to overcoming biomass recalcitrance5,6,7. Furthermore, it enables the conversion of agro-industrial residues into value-added bioproducts, thereby contributing to the transition to a sustainable and bio-based economy.
