Abstract:Herein, we developed an electronic effect-guided rational design strategy to enhance the enantioselectivity of Candida antarctica lipase B (CALB) mutants towards bulky pyridyl(phenyl) methanols. Compared to W104A mutant previously reported with reversed S-stereoselectivity toward sec-alcohols, three mutants (W104C, W104S and W104T) displayed significant improvement of Senantioselectivity in the kinetic resolution (KR) of various phenyl pyridyl methyl acetates due to the increased electronic effects between pyr… Show more
“…The engineering of catalytic‐tetrad residues can increase the enzyme catalytic efficiency (as shown in this study) and reconstruct the active sites for new enzymatic functions in Candida antarctica lipase B (Branneby et al, 2003; Li et al, 2021), halohydrin dehalogenase (Fox et al, 2007), and squalene‐hopene cyclase (Hammer et al, 2015). Together, these suggested that engineering the catalytic tetrad is an alternative strategy to tune enzymatic properties.…”
Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (k cat /K M = 47.3 s −1 •mM −1 ), improved stereoselectivity (from moderate selectivity (de P = 66.7%) to strict (S)-selectivity (de P > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.
“…The engineering of catalytic‐tetrad residues can increase the enzyme catalytic efficiency (as shown in this study) and reconstruct the active sites for new enzymatic functions in Candida antarctica lipase B (Branneby et al, 2003; Li et al, 2021), halohydrin dehalogenase (Fox et al, 2007), and squalene‐hopene cyclase (Hammer et al, 2015). Together, these suggested that engineering the catalytic tetrad is an alternative strategy to tune enzymatic properties.…”
Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (k cat /K M = 47.3 s −1 •mM −1 ), improved stereoselectivity (from moderate selectivity (de P = 66.7%) to strict (S)-selectivity (de P > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.
“…[18] Additionally, the ketone functional group could be efficiently reduced into a methylene or hydroxyl group as demonstrated by examples 5 e and 5 f in 90% and 81% yield, respectively. [19] In conclusion, we have developed a rutheniumcatalyzed direct CÀ H acyloxylation of 2-aroyl pyridines with readily accessible sodium carboxylates under mild conditions. A wide range of aliphatic and aromatic sodium carboxylate coupling partners are well suited substrates in this ruthenium-catalyzed system, offering a variety of substituted acyloxylated 2-aroyl pyridines in 45%-84% yields.…”
Section: Resultsmentioning
confidence: 96%
“…Among a variety of additives that were examined (AgBF 4 , AgNTf, KSbF 6 , KPF 6 , NaPF 6 ), AgSbF 6 proved to be superior. Notably, the desired oxygenation reaction did not occur when [RuCl 2 (p-cymene)] 2 was used as the catalyst in the absence of an additive, which can be rationalized by the in situ generation of a cationic ruthenium(II) catalyst (entries [17][18][19][20][21][22]. The yield increased to 60% upon the addition of 40 mol% AgSbF 6 as an additive (entry 23).…”
A ruthenium‐catalyzed C(sp2)–H acyloxylation of 2‐aroyl pyridine derivatives with simple sodium carboxylate utilizing transformable directing groups is described. This protocol features broad functional group tolerance and chemo‐ and regio‐selectivity, providing the acyloxylation products in 45%‐84%yield. Furthermore, the synthetic utility of this protocol was demonstrated by the late‐stage functionalization of pharmaceutical compounds. Notably, the acyloxylation products could be further transformed into a variety of useful heterocycles under mild conditions.
“…Three polar amino acids including Cys, Ser and Thr with similar size as alanine were introduced at the 104 position to reconstruct the substrate binding pocket with increased polarity, thereby increasing the interactions between polar side chains of residues at 104 and N atom of pyridine-4-yl in the substrate. As a result, the mutants W104C and W104T improved the S -enantioselectivity from 91% to 99% and 98%, respectively, with the similar yield compared to the mutant W104A ( Li et al, 2021 ). Similarly, the substrate binding was rationally modified by simultaneously tuning electronic interactions and steric effects, leading to up to 22-fold enhancement in the enantioselectivity of an esterase BioH towards methyl ( S )-o-chloromandelate ( S -CMM) ( Gu et al, 2015 ).…”
Section: Rational Design Strategy Based On Remodeling Interaction Net...mentioning
confidence: 93%
“…The molecular dynamics simulations showed that, as expected, the mutant V328N formed a stable hydrogen bond with the ester group of the LAC, which was exactly placed at the opposite side in the catalytic pocket compared to Ser264 in the S -selective variant ( Calvó-Tusell et al, 2022 ). Strengthening electronic interactions between substrate and enzyme active site was also used to improve the enantioselectivity of Candida antarctica lipase B (CALB) ( Li et al, 2021 ). It was difficult for CALB to catalyze the hydrolytic kinetic resolution (KR) of bulky racemic phenyl(pyridin-4-yl) methyl acetate due to the steric effect of Trp104.…”
Section: Rational Design Strategy Based On Remodeling Interaction Net...mentioning
The strategy of rational design to engineer enzymes is to predict the potential mutants based on the understanding of the relationships between protein structure and function, and subsequently introduce the mutations using the site-directed mutagenesis. Rational design methods are universal, relatively fast and have the potential to be developed into algorithms that can quantitatively predict the performance of the designed sequences. Compared to the protein stability, it was more challenging to design an enzyme with improved activity or selectivity, due to the complexity of enzyme molecular structure and inadequate understanding of the relationships between enzyme structures and functions. However, with the development of computational force, advanced algorithm and a deeper understanding of enzyme catalytic mechanisms, rational design could significantly simplify the process of engineering enzyme functions and the number of studies applying rational design strategy has been increasing. Here, we reviewed the recent advances of applying the rational design strategy to engineer enzyme functions including activity and enantioselectivity. Five strategies including multiple sequence alignment, strategy based on steric hindrance, strategy based on remodeling interaction network, strategy based on dynamics modification and computational protein design are discussed and the successful cases using these strategies are introduced.
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