Directed evolution utilizing iterative saturation mutagenesis (ISM) has been applied to the old yellow enzyme homologue YqjM in the quest to broaden its substrate scope, while controlling the enantioselectivity in the bioreduction of a set of substituted cyclopentenone and cyclohexenone derivatives. Guided by the known crystal structure of YqjM, 20 residues were selected as sites for saturation mutagenesis, a pooling strategy based on the method of Phizicky [M. R. Martzen, S. M. McCraith, S. L. Spinelli, F. M. Torres, S. Fields, E. J. Grayhack, E. M. Phizicky, Science 1999, 286, 1153-1155] being used in the GC screening process. The genes of some of the hits were subsequently employed as templates for randomization experiments at the other putative hot spots. Both (R)-and (S)-selective variants were evolved using 3-methylcyclohexenone as the model substrate in the asymmetric bioreduction of the olefinic functionality, only small mutant libraries and thus minimal screening effort being necessary. Some of the best mutants also proved to be excellent catalysts when testing other prochiral substrates without resorting to additional mutagenesis/screening experiments. Thus, the results constitute an important step forward in generalizing the utility of ISM as an efficient method in laboratory evolution of enzymes as catalysts in organic chemistry.
A series of synthetic nicotinamide cofactors were synthesized to replace natural nicotinamide cofactors and promote enoate reductase (ER)-catalyzed reactions without compromising activity or stereoselectivity of the bioreduction process. Conversions and enantioselectivities of up to >99% were obtained for C=C bioreductions and the process was successfully upscaled. Furthermore, high chemoselectivity was observed when employing these nicotinamide cofactor mimics (mNADs) with crude extracts in ER-catalyzed reactions.The asymmetric reduction of conjugated C=C double bonds using enoate reductases (ERs, EC 1.3.1.31) is receiving great interest in preparative organic chemistry.1 § Both authors contributed equally.
Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.
The quest for practical regeneration concepts for nicotinamide-dependent oxidoreductases continues. Recently we proposed the use of visible light to promote the direct reductive regeneration of a flavin-dependent monooxygenase. With this enzyme (PAMO-P3) light-driven enantioselective Baeyer-Villiger oxidations were performed. In spite of the significant reduction in the complexity achieved, catalytic performance of the novel approach did not meet the requirements for an efficient biocatalytic oxygenation system. Driven by this ultimate goal, we further investigated the limiting factors of our particular system. We discovered that oxidative uncoupling of the flavin-regeneration reaction from enzymatic O2-activation accounts for the futile consumption of approximately 95% of the reducing equivalents provided by the sacrificial electron donor, EDTA. Furthermore, it was found that the apparent turnover frequency (TOF) for PAMO-P3 in the present setup is approximately two orders of magnitude lower than in conventional setups that use NADPH as reductant. This finding was traced to sluggish electron transfer kinetics that arose from an impeded interaction between PAMO-P3-bound FAD and the reducing catalyst. The limiting factors and potential approaches for their circumvention are discussed. Furthermore, we broadened the light-driven regeneration approach to the class of flavin-dependent reductases. By using the Old Yellow Enzyme homologue YqjM as a model system, a significantly higher catalytic turnover for the enzyme catalyst was achieved, which we assign to a higher accessibility of the prosthetic group as well as to the absence of oxidative uncoupling.
During the last three decades different types of synthetic metalloenzymes have been prepared, including those based on anchoring appropriate ligands such as diphosphines, phthalocyanines, or dipyridyl moieties covalently or noncovalently onto proteins. [1,2] The respective transition-metal complexes constitute hybrid catalysts that mediate reactions such as asymmetric rhodium-mediated olefin hydrogenation, Diels-Alder cycloadditions, and thioether sulfoxidation.
Von der Natur inspiriert: Ortsspezifische Mutagenese ermöglichte den gezielten Aufbau einer Bindungsstelle mit dem His/His/Asp‐Motiv zur CuII‐Komplexierung in einem robusten Protein (siehe Bild). Das künstliche Metalloenzym katalysiert eine enantioselektive Diels‐Alder‐Reaktion.
Structure‐guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan‐1‐ol to hexanal as the target reaction. A six‐amino‐acid variant was identified with a 20‐fold increased kcat compared to that of the wild‐type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in Tm of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent‐free system. Despite the evolution of choline oxidase towards hexan‐1‐ol, this new variant also showed increased specific activities (by up to 100‐fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.
A simplified procedure for cell-free biocatalytic reductions of conjugated C=C double bonds using old yellow enzymes (OYEs) is reported. Instead of indirectly regenerating YqjM (an OYE homologue from B. subtilis) or NemA (N-ethylmale-A C H T U N G T R E N N U N G imide reductase from E. coli) via regeneration of reduced nicotinamide cofactors, we demonstrate that direct regeneration of catalytically active reduced flavins is an efficient and convenient approach. Reducing equivalents are provided from simple sacrificial electron donors such as ethylenediaminetetra-A C H T U N G T R E N N U N G acetate (EDTA), formate, or phosphite via photocatalytic oxidation. This novel photoenzymatic reaction scheme was characterized. Up to 65% rates of the NADH-driven reaction were obtained while preserving enantioselectivity. The chemoselectivity of the novel approach was exclusive. Even when using crude cell extracts as biocatalyst preparations, only C=C bond reduction was observed while ketone and aldehyde groups remained unaltered. Overall, a simple and practical approach for photobiocatalytic reductions is presented.
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