Xiang Sheng

Direct activation of α,β-unsaturated ketones for enantioselective transformations has remained rare to enzyme catalysis due to their lower electrophilicity and challenging steric environment. In this work, we establish an enantioselective Friedel–Crafts alkylation of α,β-unsaturated cyclic ketones with indoles using a fully genetically encoded artificial enzyme featuring a catalytic primary amine residue. This abiological catalytic center enables iminium ion formation with cyclic enones, thereby unlocking a new reaction manifold for biocatalysis. This study offers a versatile platform for enantioselective transformations of α,β-unsaturated ketones, advancing the integration of new-to-nature catalysis into biocatalytic synthesis.

A Per-ARNT-Sim (PAS) domain exhibits oxidative N-demethylation activity toward dimethylamine (DMA); however, the detailed reaction mechanism remains elusive, thus hindering the rational engineering of the enzyme. In this work, we conducted molecular dynamics (MD) simulations and quantum mechanical/molecular mechanics (QM/MM) calculations to investigate the PAS domain-catalyzed DMA N-demethylation reaction. Our results indicate that the protonated substrate DMA is the most plausible proton donor during the formation of compound I (Cpd I, iron(IV)-oxo porphyrin cation radical). The calculated energy barrier for Cpd I generation is 14.4 kcal/mol, which is the rate-determining step of the reaction. Following its generation, Cpd I can rapidly oxidize the substrate via an electron-transfer-initiated hydrogen atom abstraction. The chemoselectivity favoring methyl hydroxylation over amine oxidation is mainly determined by the protein environment. Our computations provide mechanistic insights into the heme-dependent oxidative N-demethylase (HODM)-catalyzed reaction and offer information for the rational design of new PAS enzymes.

Cellobiose dehydrogenase (CDH) is a typical biomass catalyst with significant potential for applications such as lignocellulose degradation, bioremediation, and bioelectrocatalysis. Its two-domain flavoheme structure enables the construction of direct electron transfer (DET)-based bioelectrochemical devices. However, the natural enzyme suffers from low electron transfer efficiency and oxygen reactivity that leads to H2O2 formation, hindering the performance of CDH. Here, we established a region-specific engineering strategy to enhance electron transfer efficiency and minimize oxygen interference in CDH. Through structure-based site-directed mutagenesis, molecular docking, and molecular dynamics (MD) simulations, the G1 variant (A165C-W316H-S560V) achieved a 10-fold increase in electron transfer rate and a 4.5-fold increase in catalytic activity, with H2O2 formation below the detection limit under the tested conditions. Rapid kinetics and electrochemical characterization further revealed the apparent kinetic behaviors of the mutants. The G1 variant displayed a 20-fold increase in catalytic current density compared to WT. The region-specific rational engineering strategies presented here may provide valuable insights into interdomain electron transfer, CYT domain surface electron transfer, and oxygen-dependent side reactivity simultaneously within CDH and offer a possible approach for improving the DET efficiency of other multi-domain oxidoreductases.

Molecular dynamics (MD)-based methods to predict the enantioselectivity or product specificity of enzymes typically use a near attack conformation (NAC) approach. The computational efficiency of this ground-state approach, taking minutes to hours, allows to in silico screen thousands of enzyme variants. During the computational design and screening of new variants of limonene epoxide hydrolase for improved stereoselectivity, it was noticed that, while this approach gave satisfactory prediction accuracy for most substrates, the enantioselectivity predictions were far less accurate for variants designed for cis-stilbene oxide. Here, to investigate whether inaccurate predictions with the NAC-based approach were typical for this substrate, first, a larger data set was generated by experimentally testing and computationally predicting the enantioselectivity of an additional 18 enzyme variants for cis-stilbene oxide. Subsequent MD simulations confirmed that a NAC analysis resulted in inaccurate enantioselectivity predictions throughout the data set. Density functional theory (DFT) calculations using the B3LYP-D3(BJ) functional, with an active site cluster model of >300 atoms, provided a rationale for why the NAC approach gave poor predictions with cis-stilbene oxide. The aromatic rings of the substrate influence the nucleophilic attack on the epoxide group, by providing a blend of stereoelectronic activation and steric hindrance to the transition state, which is not considered in the NAC approach. With this substrate, the DFT accurately predicted the enantioselectivity. The results offer valuable insights into the applicability of the NAC approach for predicting enzyme selectivity, suggesting that more expensive methodologies, which do explicitly consider the transition state structure, can also become required when bulky or stereoelectronically activating groups are bound to the reacting atoms.

Indole diketopiperazine (DKP) alkaloids constitute an important class of fungal natural products with diverse biological activities. Although dimeric indole DKPs have been isolated from natural sources and often exhibit enhanced bioactivities compared with their monomeric counterparts, enzymatic dimerization of indole DKPs remains rare and mechanistically poorly understood. Here, we investigate the enzymatic dimerization of 6-hydroxy-deoxybrevianamide E (6-OH-DoE), an indole DKP intermediate selected based on reports of naturally occurring dimeric derivatives. In vitro assays revealed that dye-decolorizing peroxidases (DyPs) catalyze the regioselective dimerization of 6-OH-DoE, affording di-6-OH-DoE through exclusive C7–C7′ coupling on the indole moiety. Combined experimental and computational analyses indicate that the reaction proceeds through a hydroxyl-to-carbon radical relay pathway, initiated by oxidation of the indole hydroxyl group, followed by intramolecular radical transfer to the C7 position, rather than direct C–H hydrogen-atom abstraction. This mechanistic proposal was further validated by substrate analogue studies and radical-probing experiments. Furthermore, a homologous DyP enzyme from the native producer was shown to catalyze the same transformation. Collectively, this study elucidates the enzymatic mechanism of indole DKP dimerization and expands the catalytic repertoire of DyP peroxidases, highlighting their potential as biocatalysts for regioselective radical-mediated C–C bond formation in indole DKP scaffolds.

The multienzyme cascade conversion of CO ₂ to methanol is a promising route for sustainable chemical synthesis, yet the reduction of formate to formaldehyde remains its major bottleneck. Here, we use quantum chemical calculations to elucidate the catalytic mechanism of formaldehyde dehydrogenase from Burkholderia multivorans ( Bm FaldDH). The lowest‐energy pathway initiates with formate activation via a proton‐relay network involving His52, Ser49, and the NADH ribose, followed by a concerted yet asynchronous proton‐hydride transfer to yield methanediol with a barrier of 16.0 kcal mol ⁻¹ . Interestingly, calculations show that subsequent dehydration of methanediol to formaldehyde is more favorable in solution than within the active site, with barriers of 16.2 and 24.0 kcal mol ⁻¹ , respectively. This lower barrier in solution is mediated by a water‐cluster model in which three water molecules facilitate proton relay, and two stabilize the methanediol. Furthermore, binding energy calculations indicate that methanediol has a relatively weak binding affinity for Bm FaldDH, suggesting that it readily dissociates into solution. Overall, this study demonstrates that the limited efficiency of Bm FaldDH in catalyzing formate reduction stems from its incompetence to catalyze methanediol dehydration within its active pocket. These findings provide a mechanistic insight for engineering efficient enzymes for the formate‐to‐formaldehyde conversion.

Enzymatic direct hydroxylation of steroid skeletons is vital for generation of physiologically and pharmacologically valuable compounds due to their structural and functional diversity. In this study, we discovered that ketosteroid isomerase (KSI) exhibited peroxidation activity using ferrocene and its analogues as electron mediators. KSI effectively converted steroidal and bicyclic enone substrates into hydroxylation products with 20–65% isolated yields in the presence of PPh3, providing an alternative approach to hydroxylation at the allyl position of steroidal and bicyclic enones. The mechanism studies suggested that the ferrocenium cation and radical intermediate were generated via a single-electron transfer process, and a reaction mechanism was proposed. This study not only demonstrates the catalytic diversity of KSIs via ferrocene-mediated electron transfer but also showcases valuable catalytic promiscuity of ketosteroid isomerases for design of enzymatic reactions and development of synthetic methods.

Steroidal medicines are a vital class of pharmaceuticals, with their active ingredients primarily derived from natural steroids. A key step in their biosynthesis involves the isomerization of ∆⁵-3-ketosteroids to ∆⁴-3-ketosteroids, catalyzed by ketosteroid isomerase (KSI). In this study, we discover that KSI exhibits an unexpected catalytic promiscuity, efficiently converting cyclic enones into homo- and hetero-addition products. Crystal structures of KSI variants and mutagenesis reveal the homo-dimeric interface as another active center. Molecular dynamics simulations and quantum mechanics/molecular mechanics calculations suggest a cascade catalytic mechanism by a single enzyme, in which isomerization occurs in the monomeric pocket, while the addition reaction preferentially occurs at the homo-dimeric interface due to reduced steric hindrance. These results not only provide valuable insights into the catalytic diversity of KSIs, but also inspire further exploration of new-to-nature and diverse catalytic properties of enzymes, thus expanding their applications in biocatalysis and drug development.