Substrate Promiscuity Research Articles

Uncovering Hydroxynitrile Lyase Variants with Promiscuous Diastereoselective Nitroaldolase Activity toward the Highly Stereocontrolled Synthesis of Anti β-Nitroalcohols

Uncovering Hydroxynitrile Lyase Variants with Promiscuous Diastereoselective Nitroaldolase Activity toward the Highly Stereocontrolled Synthesis of <i>Anti</i> β-Nitroalcohols

The total biosynthesis route of rosmarinic acid in Sarcandra glabra based on transcriptome sequencing

Sarcandra glabra is a widely distributed and valuable plant in food and daily chemical industries, and is also a common-used medicinal plant for treating inflammatory diseases and tumors. Rosmarinic acid (RA) with significant pharmacological activity is an abundant and important constituent in S. glabra, however, little information about key enzymes involving the biosynthesis of RA in S. glabra is available and the underlying biosynthesis mechanisms of RA in S. glabra remain undeciphered. Therefore, in this study, by full-length transcriptome sequencing analyses of S. glabra, we screened the RA biosynthesis candidate genes based on sequence similarity and conducted enzymatic function characterization in vitro and in vivo. As a result, a complete set of 7 kinds of enzymes (SgPALs, SgC4H, Sg4CL, SgTATs, SgHPPRs, SgRAS and SgC3H) involving the biosynthesis route of RA from phenylalanine and tyrosine, were identified and fully characterized. This research systematically revealed the complete biosynthesis route of RA in S. glabra, which helps us better understand the process of RA synthesis and accumulation, especially the substrate promiscuities of SgRAS and SgC3H provide the molecular biological basis for the efficient biosynthesis of specific and abundant RA in S. glabra. The 7 kinds of key enzymes revealed in this study can be utilized as tool enzymes for production of RA by synthetic biology methods.

Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme

Incorporation of irreversible steps in pathway design enhances the overall thermodynamic favorability and often leads to better bioconversion yield given functional enzymes. Using this concept, here we constructed the first non-natural itaconate biosynthesis pathway driven by thioester hydrolysis. Itaconate is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Production of itaconate has long relied on the decarboxylation of TCA cycle intermediate cis-aconitate as the only biosynthetic route. Inspired by nature's design of itaconate detoxification, here we engineered a novel itaconate producing pathway orthogonal to native metabolism with no requirement of auxotrophic knock-out. The reversed degradation pathway initiates with pyruvate and acetyl-CoA condensation forming (S)-citramalyl-CoA, followed by its dehydration and isomerization into itaconyl-CoA then hydrolysis into itaconate. Phenylacetyl-CoA thioesterase (PaaI) from Escherichiacoli was identified via screening to deliver the highest itaconate formation efficiency when coupled to the reversible activity of citramalate lyase and itaconyl-CoA hydratase. The preference of PaaI towards itaconyl-CoA hydrolysis over acetyl-CoA and (S)-citramalyl-CoA also minimized the inevitable precursor loss due to enzyme promiscuity. With acetate recycling, acetyl-CoA conservation, and condition optimization, we achieved a final itaconate titer of 1 g/L using the thioesterase driven pathway, which is a significant improvement compared to the original degradation pathway based on CoA transferase. This study illustrates the significance of thermodynamic favorability as a design principle in pathway engineering.

Evolution and origins of rubisco

Rubisco (D-ribulose 1,5-bisphosphate carboxylase/oxygenase) is the most abundant enzyme in the world, constituting up to half of the soluble protein content in plant leaves. Such is its ubiquity that its chemical fingerprint can be detected in the geological record spanning billions of years. Rubisco catalyses the conversion of inorganic CO2 into organic sugars, which underpin almost all of the biosphere, including our entire food chain. Due to its central role in the global carbon cycle, rubisco has been the subject of intense research for over 50 years. Rubisco is often considered inefficient due to its slow rate of carboxylation compared with other central metabolism enzymes, and its promiscuous oxygenase activity, which competes with the productive carboxylation reaction. It is hoped that engineering improved CO2 fixation will have significant advantages in agriculture and climate change mitigation. However, rubisco has proven difficult to engineer, with decades of efforts yielding limited results. Recent research has focused on reconstructing the evolutionary trajectory of rubisco to help elucidate its cryptic origins. Such evolutionary studies have led to a better understanding of both the origins of more complex rubisco forms and the broader relationship between rubisco's structure and function.

Substrate promiscuity of key resistance P450s confers clothianidin resistance while increasing chlorfenapyr potency in malaria vectors

Novel insecticides were recently introduced to counter pyrethroid resistance threats in African malaria vectors. To prolong their effectiveness, potential cross-resistance from promiscuous pyrethroid metabolic resistance mechanisms must be elucidated. Here, we demonstrate that the duplicated P450s CYP6P9a/-b, proficient pyrethroid metabolizers, reduce neonicotinoid efficacy in Anopheles funestus while enhancing the potency of chlorfenapyr. Transgenic expression of CYP6P9a/-b in Drosophila confirmed that flies expressing both genes were significantly more resistant to neonicotinoids than controls, whereas the contrasting pattern was observed for chlorfenapyr. This result was also confirmed by RNAi knockdown experiments. Invitro expression of recombinant CYP6P9a and metabolism assays established that it significantly depletes both clothianidin and chlorfenapyr, with metabolism of chlorfenapyr producing the insecticidally active intermediate metabolite tralopyril. This study highlights the risk of cross-resistance between pyrethroid and neonicotinoid and reveals that chlorfenapyr-based control interventions such as Interceptor G2 could remain efficient against some P450-based resistant mosquitoes.

Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor

The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.

Old Yellow Enzymes as Oxime Reductases: New Variants by Substrate‐Based Enzyme Engineering

AbstractThe reduction of oximes was recently identified as a promiscuous activity of Old Yellow Enzymes (OYEs). This reaction involves a two‐step reduction of α‐oxime‐ß‐ketoesters to the corresponding amines, which spontaneously dimerise to yield pyrazine derivatives. This biotransformation is currently limited to substrates with small substituents like methyl/ethyl on the keto moiety. We used a structure‐based approach to engineer 12‐oxophytodienoate reductase 3 (OPR3) from Solanum lycopersicum as a prototypical OYE to accept oximes with bulkier substituents. To this end, three single and two double variants were prepared and tested on six oxime substrates. The engineered variants indeed showed activity on some of the bulkier substrates, which had not been converted at all by the wild‐type enzyme, including the diester compound diethyl‐2‐(hydroximino) malonate. While we were unable to identify variants capable of converting substrates with branched and aromatic substituents, the results demonstrate the validity of our engineering approach, suggesting potential pathways for expanding the substrate scope of OYEs.

Unraveling the role of cytochrome P450 enzymes in oleanane triterpenoid biosynthesis in arjuna tree.

Triterpenoids (C30-isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated β-amyrin-derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C-2α and C-23 hydroxylases/oxidases than C-28 oxidases. CYP716A233 and CYP716A432 catalyzed β-amyrin/erythrodiol C-28 oxidation to produce oleanolic acid. C-2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C-23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C-23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C-23 hydroxylation. Overall, P450 substrate selectivity suggested that C-28 oxidation is the first P450-catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C-2α/C-23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds.

PARENT COMMUNICATION PATTERNS AND RISK SEXUAL BEHAVIOR IN LATE ADOLESCENT

Background: Risky sexual behaviour in adolescents can have a detrimental impact on adolescent development and health, such as exposure to sexually transmitted diseases including HIV/AIDS, unwanted pregnancy, and abortion. Many factors can cause risky sexual behaviour, family factors, especially parental communication patterns are one of the most important influences, because parents are the environment that has the strongest preventive efforts in keeping adolescents involved in promiscuous activities. This study aims to find the relationship between parental communication patterns and risky sexual behaviour in late adolescents. Method: Observational analytic with cross-sectional research design. The sample size was 300 respondents with consecutive sampling technique. The independent variable is communication pattern. The dependent variable was risky sexual behaviour in late adolescents. The research data used instruments in the form of questionnaires and analysed using the Spearman's rank test with a significance level of p &lt; 0.05. Result: Parental communication patterns in late adolescents were dominated by authoritative communication patterns at 56% and 63% of respondents have low-risk sexual behaviour. Spearman's rank test results p value &lt; α, p = 0.000 and r = -0.239. Conclusion : Parental communication patterns have a relationship with risky sexual behaviour in late adolescents.

An efficient flavonoid glycosyltransferase NjUGT73B1 from Nardostachys jatamansi of alpine Himalayas discovered by structure-based protein clustering

Tilianin and linarin, two rare glycosylated flavonoids in the aromatic endangered medicinal plant Nardostachys jatamansi (D.on)DC., play an important role in the fields of medicine, cosmetics, food and dye industries. However, there remains a lack of comprehensive understanding regarding their biosynthetic pathway. In this study, the phytochemical investigation of N. jatamansi resulted in the isolation of linarin. With help of AlphaFold2 to cluster the entire glycosyltransferase family based on predicted structure similarities, we successfully identified a flavonoid glycosyltransferase NjUGT73B1, which could efficiently catalyze the glucosylation of acacetin at 7-OH to produce tilianin, also the key precursor in the biosynthesis of linarin. Additionally, NjUGT73B1 displayed a high degree of substrate promiscuity, enabling glucosylation at 7-OH of many flavonoids. Molecular modeling and site-directed mutagenesis revealed that H19, H21, H370, F126, and F127 play the crucial roles in the glycosylation ability of NjUGT73B1. Notably, comparation with the wild NjUGT73B1, mutant H19K led to a 50% increase in the activity of producing tilianin from acacetin.

Expanding the substrate selectivity of the fimsbactin biosynthetic adenylation domain, FbsH.

Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria Acinetobacter baumannii produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report on structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early-stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.

MPEK: a multitask deep learning framework based on pretrained language models for enzymatic reaction kinetic parameters prediction.

Enzymatic reaction kinetics are central in analyzing enzymatic reaction mechanisms and target-enzyme optimization, and thus in biomanufacturing and other industries. The enzyme turnover number (kcat) and Michaelis constant (Km), key kinetic parameters for measuring enzyme catalytic efficiency, are crucial for analyzing enzymatic reaction mechanisms and the directed evolution of target enzymes. Experimental determination of kcat and Km is costly in terms of time, labor, and cost. To consider the intrinsic connection between kcat and Km and further improve the prediction performance, we propose a universal pretrained multitask deep learning model, MPEK, to predict these parameters simultaneously while considering pH, temperature, and organismal information. Through testing on the same kcat and Km test datasets, MPEK demonstrated superior prediction performance over the previous models. Specifically, MPEK achieved the Pearson coefficient of 0.808 for predicting kcat, improving ca. 14.6% and 7.6% compared to the DLKcat and UniKP models, and it achieved the Pearson coefficient of 0.777 for predicting Km, improving ca. 34.9% and 53.3% compared to the Kroll_model and UniKP models. More importantly, MPEK was able to reveal enzyme promiscuity and was sensitive to slight changes in the mutant enzyme sequence. In addition, in three case studies, it was shown that MPEK has the potential for assisted enzyme mining and directed evolution. To facilitate in silico evaluation of enzyme catalytic efficiency, we have established a web server implementing this model, which can be accessed at http://mathtc.nscc-tj.cn/mpek.

Rational modification of Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase for lacto-N-neotetraose synthesis with reduced long-chain derivatives

Lacto-N-neotetraose (LNnT), as a neutral core structure within human milk oligosaccharides (HMOs), has garnered widespread attention due to its exceptional physiological functions. In the process of LNnT synthesis using cellular factory approaches, substrate promiscuity of glycosyltransferases leads to the production of longer oligosaccharide derivatives. Here, rational modification of β1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis (LgtA) effectively decreased the concentration of long-chain LNnT derivatives. Specifically, the optimal β1,4-galactosyltransferase (β1,4-GalT) was selected from seven known candidates, enabling the efficient synthesis of LNnT in Escherichia coli BL21(DE3). Furthermore, the influence of lactose concentration on the distribution patterns of LNnT and its longer derivatives was investigated. The modification of LgtA was conducted with computational assistance, involving alanine scanning based on molecular docking to identify the substrate binding pocket and implementing large steric hindrance on crucial amino acids to obstruct LNnT entry. The implementation of saturation mutagenesis at positions 223 and 228 of LgtA yielded advantageous mutant variants that did not affect LNnT synthesis while significantly reducing the production of longer oligosaccharide derivatives. The most effective mutant, N223I, reduced the molar ratio of long derivatives by nearly 70%, showcasing promising prospects for LNnT production with diminished byproducts.

The mechanism of mammalian proton-coupled peptide transporters.

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

The mechanism of mammalian proton-coupled peptide transporters

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

Identification and Characterization of Thiamine Diphosphate-Dependent Lyases with an Unusual CDG Motif.

The thiamine diphosphate (ThDP)-binding motif, characterized by the canonical GDG(X)24-27N sequence, is highly conserved among ThDP-dependent enzymes. We investigated a ThDP-dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α-keto acids as donors. Sterically hindered aromatic aldehydes or non-activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Å reveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long-sought genuine tetrahedral intermediates formed with pyruvate and 2-oxobutyrate in the pre-decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP-binding motif and unlock a family of lyases that can perform valuable carboligation reactions.

Identification and Characterization of Thiamine Diphosphate‐Dependent Lyases with an Unusual CDG Motif

AbstractThe thiamine diphosphate (ThDP)‐binding motif, characterized by the canonical GDG(X)24‐27N sequence, is highly conserved among ThDP‐dependent enzymes. We investigated a ThDP‐dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α‐keto acids as donors. Sterically hindered aromatic aldehydes or non‐activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Å reveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long‐sought genuine tetrahedral intermediates formed with pyruvate and 2‐oxobutyrate in the pre‐decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP‐binding motif and unlock a family of lyases that can perform valuable carboligation reactions.

Drug metabolism of ciprofloxacin, ivacaftor, and raloxifene by Pseudomonas aeruginosa cytochrome P450 CYP107S1

Drug metabolism is one of the main processes governing the pharmacokinetics and toxicity of drugs via their chemical biotransformation and elimination. In humans, the liver, enriched with cytochrome P450 (CYP) enzymes, plays a major metabolic and detoxification role. The gut microbiome and its complex community of microorganisms can also contribute to some extent to drug metabolism. However, during an infection when pathogenic microorganisms invade the host, our knowledge of the impact on drug metabolism by this pathobiome remains limited. The intrinsic resistance mechanisms and rapid metabolic adaptation to new environments often allow the human bacterial pathogens to persist, despite the many antibiotic therapies available. Here, we demonstrate that a bacterial CYP enzyme, CYP107S1, from Pseudomonas aeruginosa, a predominant bacterial pathogen in cystic fibrosis (CF) patients, can metabolize multiple drugs from different classes. CYP107S1 demonstrated high substrate promiscuity and allosteric properties much like human hepatic CYP3A4. Our findings demonstrated binding and metabolism by the recombinant CYP107S1 of fluoroquinolone antibiotics (ciprofloxacin and fleroxacin), a CF transmembrane conductance regulator potentiator (ivacaftor), and a SERM antimicrobial adjuvant (raloxifene). Our in vitro metabolism data were further corroborated by molecular docking of each drug to the heme active site using a CYP107S1 homology model. Our findings raise the potential for microbial pathogens modulating drug concentrations locally at the site of infection, if not systemically, via CYP-mediated biotransformation reactions. To our knowledge, this is the first report of a CYP enzyme from a known bacterial pathogen that is capable of metabolizing clinically utilized drugs.

Substrate Characterization for Hydrolysis of Multiple Types of Aromatic Esters by Promiscuous Aminopeptidases.

Synthetic aromatic esters, widely employed in agriculture, food, and chemical industries, have become emerging environmental pollutants due to their strong hydrophobicity and poor bioavailability. This study attempted to address this issue by extracellularly expressing the promiscuous aminopeptidase (Aps) from Pseudomonas aeruginosa GF31 in B. subtilis, achieving an impressive enzyme activity of 13.7 U/mg. Notably, we have demonstrated, for the first time, the Aps-mediated degradation of diverse aromatic esters, including but not limited to pyrethroids, phthalates, and parabens. A biochemical characterization of Aps reveals its esterase properties and a broader spectrum of substrate profiles. The degradation rates of p-nitrobenzene esters (p-NB) with different side chain structures vary under the action of Aps, showing a preference for substrates with relatively longer alkyl side chains. The structure-dependent degradability aligns well with the binding energies between Aps and p-NB. Molecular docking and enzyme-substrate interaction elucidate that hydrogen bonding, hydrophobic interactions, and π-π stacking collectively stabilize the enzyme-substrate conformation, promoting substrate hydrolysis. These findings provide new insights into the enzymatic degradation of aromatic ester pollutants, laying a foundation for the further development and modification of promiscuous enzymes.

Atroposelective Synthesis of Axial Biaryls by Dynamic Kinetic Resolution Using Engineered Imine Reductases.

Axially chiral biaryl compounds are ubiquitous scaffolds in natural products, bioactive molecules, chiral ligands and catalysts, but biocatalytic methods for their asymmetric synthesis are limited. Herein, we report a highly efficient biocatalytic route for the atroposelective synthesis of biaryls by dynamic kinetic resolution (DKR). This DKR approach features a transient six-membered aza-acetal-bridge-promoted racemization followed by an imine reductase (IRED)-catalyzed stereoselective reduction to construct the axial chirality under ambient conditions. Directed evolution of an IRED from Streptomyces sp. GF3546 provided a variant (S-IRED-Ss-M11) capable of catalyzing the DKR process to access a variety of biaryl aminoalcohols in high yields and excellent enantioselectivities (up to 98 % yield and >99 : 1 enantiomeric ratio). Molecular dynamics simulation studies on the S-IRED-Ss-M11 variant revealed the origin of its improved activity and atroposelectivity. By exploiting the substrate promiscuity of IREDs and the power of directed evolution, our work further extends the biocatalysts' toolbox to construct challenging axially chiral molecules.