Synthesis of C-N bonds by nicotinamide-dependent oxidoreductase: an overview.

Compounds containing chiral C-N bonds play a vital role in the composition of biologically active natural products and small pharmaceutical molecules. Therefore, the development of efficient and convenient methods for synthesizing compounds containing chiral C-N bonds is a crucial area of research. Nicotinamide-dependent oxidoreductases (NDOs) emerge as promising biocatalysts for asymmetric synthesis of chiral C-N bonds due to their mild reaction conditions, exceptional stereoselectivity, high atom economy, and environmentally friendly nature. This review aims to present the structural characteristics and catalytic mechanisms of various NDOs, including imine reductases/ketimine reductases, reductive aminases, EneIRED, and amino acid dehydrogenases. Additionally, the review highlights protein engineering strategies employed to modify the stereoselectivity, substrate specificity, and cofactor preference of NDOs. Furthermore, the applications of NDOs in synthesizing essential medicinal chemicals, such as noncanonical amino acids and chiral amine compounds, are extensively examined. Finally, the review outlines future perspectives by addressing challenges and discussing the potential of utilizing NDOs to establish efficient biosynthesis platforms for C-N bond synthesis. In conclusion, NDOs provide an economical, efficient, and environmentally friendly toolbox for asymmetric synthesis of C-N bonds, thus contributing significantly to the field of pharmaceutical chemical development.

Facile incorporation of non-canonical heme ligands in myoglobin through chemical protein synthesis.

The incorporation of non-canonical amino acids (ncAAs) into the metal coordination environments of proteins has endowed metalloproteins with enhanced properties and novel activities, particularly in hemoproteins. In this work, we disclose a scalable synthetic strategy that enables the production of myoglobin (Mb) variants with non-canonical heme ligands, i.e., HoCys and f4Tyr. The ncAA-containing Mb* variants (with H64V/V68A mutations) were obtained through two consecutive native chemical ligations and a subsequent desulfurization step, with overall isolated yield up to 28.6 % in over 10-milligram scales. After refolding and heme b cofactor reconstitution, the synthetic Mb* variants showed typical electronic absorption bands. When subjected to the catalysis of the cyclopropanation of styrene, both synthetic variants, however, were not as competent as the His-ligated Mb*. We envisioned that the synthetic method reported herein would be useful for incorporating a variety of ncAAs with diverse structures and properties into Mb for varied purposes.

Channel Engineering of a Glutamate Exporter.

Mechanosensitive channel MscCG2 is involved in glutamate excretion in most C. glutamicum strains. Improving the excretion efficiency of MscCG2 is beneficial to the production of glutamate. In this study, structure-based rational design was carried out to obtain an improved efflux ability of exporter MscCG2 and its mechanistic advance via two strategies: widening the channel entrance for smoother entry of glutamate and reducing the electronegativity at the entrance of the channels to minimize the rejection of negatively charged glutamate entry. The designed variants were found to enhance glutamate excretion by 2 to 3.3-fold in the early phase and 1.1-fold to 1.5-fold in the late phase of fermentation. The enhanced glutamate excretion was further confirmed by using glutamate toxic analog 4-fluoroglutamate (4-FG) and Glu-Glu peptide uptake and glutamate export assay. Molecular dynamic (MD) simulations revealed that the amino acid substitutions indeed enlarged the channel entrance and reduced the repulsion of glutamate when entering the channel. The finding of this study is important for understanding the underlying structure-function relationship and the mechanism of glutamate secretion to improve glutamate efflux efficiency of glutamate exporter.

A Novel Tetravalent CD95/Fas Fusion Protein With Superior CD95L/FasL Antagonism.

Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo.

Red-Shifting B12-Dependent Photoreceptor Protein via Optical Coupling for Inducible Living Materials.

Cobalamin (B12)-dependent photoreceptors are gaining traction in materials synthetic biology, especially for optically controlling cell-to-cell adhesion in living materials. However, these proteins are mostly responsive to green light, limiting their deep-tissue applications. Here, we present a general strategy for shifting photoresponse of B12-dependent photoreceptor CarHC from green to red/far-red light via optical coupling. Using thiol-maleimide click chemistry, we labeled cysteine-containing CarHC mutants with SulfoCyanine5 (Cy5), a red light-capturing fluorophore. The resulting photoreceptors not only retained the ability to tetramerize in the presence of adenosylcobalamin (AdoB12), but also gained sensitivity to red light; labeled tetramers disassembled on red light exposure. Using genetically encoded click chemistry, we assembled the red-shifted proteins into hydrogels that degraded rapidly in response to red light. Furthermore, Saccharomyces cerevisiae cells were genetically engineered to display CarHC variants, which, alongside in situ Cy5 labeling, led to living materials that could assemble and disassemble in response to AdoB12 and red light, respectively. These results illustrate the CarHC spectrally tuned by optical coupling as a versatile motif for dynamically controlling cell-to-cell interactions within engineered living materials. Given their prevalence and ecological diversity in nature, this spectral tuning method will expand the use of B12-dependent photoreceptors in optogenetics and living materials.

Collagen-targeted Protein Nanomicelles for the Imaging of Non-Alcoholic Steatohepatitis.

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding - thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. STATEMENT OF SIGNIFICANCE: : Direct molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. Compared with that in control mice, the protein in these mice clears from the serum faster and accumulates significantly more in fibrotic livers. This work advances the development of targeted protein probes for in vivo fibrosis imaging.

Tuning the Functionality of Designer Translating Organelles with Orthogonal tRNA Synthetase/tRNA Pairs.

Site-specific incorporation of noncanonical amino acids (ncAAs) can be realized by genetic code expansion (GCE) technology. Different orthogonal tRNA synthetase/tRNA (RS/tRNA) pairs have been developed to introduce a ncAA at the desired site, delivering a wide variety of functionalities that can be installed into selected proteins. Cytoplasmic expression of RS/tRNA pairs can cause a problem with background ncAA incorporation into host proteins. The application of orthogonally translating organelles (OTOs), inspired by the concept of phase separation, provides a solution for this issue in mammalian cells, allowing site-specific and protein-selective ncAA incorporation. So far, only Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase (PylRS) has been used within OTOs, limiting the method's potential. Here, we explored the implementation of four other widely used orthogonal RS/tRNA pairs with OTOs, which, to our surprise, were unsuccessful in generating mRNA-selective GCE. Next, we tested several experimental solutions and developed a new chimeric phenylalanyl-RS/tRNA pair that enables ncAA incorporation in OTOs in a site-specific and protein-selective manner. Our work reveals unaccounted design constraints in the spatial engineering of enzyme functions using designer organelles and presents a strategy to overcome those in vivo. We then discuss current limitations and future directions of in-cell engineering in general and protein engineering using GCE specifically.

[A dual-enzyme cascade for production of indigo from L-tryptophan].

Indigo, as a water-soluble non-azo colorant, is widely used in textile, food, pharmaceutical and other industrial fields. Currently, indigo is primarily synthesized by chemical methods, which causes environmental pollution, potential safety hazards, and other issues. Therefore, there is an urgent need to find a safer and greener synthetic method. In this study, a dual-enzyme cascade pathway was constructed with the tryptophan synthase (tryptophanase, EcTnaA) from Escherichia coli and flavin-dependent monooxygenase (flavin-dependent monooxygenase, MaFMO) from Methylophaga aminisulfidivorans to synthesize indigo with L-tryptophan as substrate. A recombinant strain EM-IND01 was obtained. The beneficial mutant MaFMOD197E was obtained by protein engineering of the rate-limiting enzyme MaFMO. MaFMOD197E showed the specific activity and kcat/Km value 2.36 times and 1.34 times higher than that of the wild type, respectively. Furthermore, MaFMOD197E was introduced into the strain EM-IND01 to construct the strain EM-IND02. After the fermentation conditions were optimized, the strain achieved the indigo titer of (1 288.59±7.50) mg/L, the yield of 0.86 mg/mg L-tryptophan, and the productivity of 26.85 mg/(L·h) in a 5 L fermenter. Protein engineering was used to obtain mutants with increased MaFMO activity in this study, which laid a foundation for industrial production of indigo.

Rational design of disulfide bonds to increase thermostability of Rhodococcus opacus catechol 1,2 dioxygenase.

Catechol 1,2 dioxygenase is a versatile enzyme with several potential applications. However, due to its low thermostability, its industrial potential is not being met. In this study, the thermostability of a mesophilic catechol 1,2 dioxygenase from the species Rhodococcus opacus was enhanced via the introduction of disulphide bonds into its structure. Engineered designs (56) were obtained using computational prediction applications, with a set of hypothesized selection criteria narrowing the list to 9. Following recombinant production and purification, several of the designs demonstrated substantially improved protein thermostability. Notably, variant K96C-D278C yielded improvements including a 4.6°C increase in T50, a 725% increase in half-life, a 5.5°C increase in Tm, and a >10-fold increase in total turnover number compared to wild type. Stacking of best designs was not productive. Overall, current state-of-the-art prediction algorithms were effective for design of disulfide-thermostabilized catechol 1,2 dioxygenase.

An anti-sortilin affibody-peptide fusion inhibits sortilin-mediated progranulin degradation.

Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.

Bivalent target-binding bioPROTACs induce potent degradation of oncogenic SHP2.

Targeted protein degradation is an emergent and rapidly evolving therapeutic strategy. In particular, biologics-based targeted degradation modalities (bioPROTACs) are relatively under explored compared to small molecules. Here, we investigate how target affinity, cellular localization, and valency of bioPROTACs impact efficacy of targeted degradation of the oncogenic phosphatase src-homology 2 containing protein tyrosine phosphatase-2 (SHP2). We identify bivalent recruitment of SHP2 by bioPROTACs as a broadly applicable strategy to improve potency. Moreover, we demonstrate that SHP2-targeted bioPROTACs can effectively counteract gain-of-function SHP2 mutants present in cancer, which are otherwise challenging to selectively target with small molecule constructs. Overall, this study demonstrates the utility of bioPROTACs for challenging targets, and further explicates design principles for therapeutic bioPROTACs.

Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.

Exosome Loaded Protein Hydrogel for Enhanced Gelation Kinetics and Wound Healing.

Exosomes are being increasingly explored in biomedical research for wound healing applications. Exosomes can improve blood circulation and endocrine signaling, resulting in enhanced cell regeneration. However, exosome treatments suffer from low retention and bioavailability of exosomes at the wound site. Hydrogels are a popular tool for drug delivery due to their ability to encapsulate drugs in their network and allow for targeted release. Recently, hydrogels have proven to be an effective method to provide increased rates of wound healing when combined with exosomes that can be applied noninvasively. We have designed a series of single-domain protein-based hydrogels capable of physical cross-linking and upper critical solution temperature (UCST) behavior. Hydrogel variant Q5, previously designed with improved UCST behavior and a significantly enhanced gelation rate, is selected as a candidate for encapsulation release of exosomes dubbed Q5Exo. Q5Exo exhibits low critical gelation times and significant decreases in wound healing times in a diabetic mouse wound model showing promise as an exosome-based drug delivery tool and for future hybrid, noninvasive protein-exosome design.

Structural basis for substrate flexibility of the O-methyltransferase MpaG' involved in mycophenolic acid biosynthesis.

MpaG' is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase involved in the compartmentalized biosynthesis of mycophenolic acid (MPA), a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. MpaG' catalyzes the 5-O-methylation of three precursors in MPA biosynthesis including demethylmycophenolic acid (DMMPA), 4-farnesyl-3,5-dihydroxy-6-methylphthalide (FDHMP), and an intermediate containing three fewer carbon atoms compared to FDHMP (FDHMP-3C) with different catalytic efficiencies. Here, we report the crystal structures of S-adenosyl-L-homocysteine (SAH)/DMMPA-bound MpaG', SAH/FDHMP-3C-bound MpaG', and SAH/FDHMP-bound MpaG' to understand the catalytic mechanism of MpaG' and structural basis for its substrate flexibility. Structural and biochemical analyses reveal that MpaG' utilizes the catalytic dyad H306-E362 to deprotonate the C5 hydroxyl group of the substrates for the following methylation. The three substrates with differently modified farnesyl moieties are well accommodated in a large semi-open substrate binding pocket with the orientation of their phthalide moiety almost identical. Based on the structure-directed mutagenesis, a single mutant MpaG'Q267A is engineered with significantly improved catalytic efficiency for all three substrates. This study expands the mechanistic understanding and the pocket engineering strategy for O-methyltransferases involved in fungal natural product biosynthesis. Our research also highlights the potential of O-methyltransferases to modify diverse substrates by protein design and engineering.

Crystal structure and enzyme engineering of the broad substrate spectrum l-amino acid oxidase 4 from the fungus Hebeloma cylindrosporum.

l-Amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to α-keto acids. Recombinant production of LAAOs with broad substrate spectrum remains a formidable challenge. We previously achieved this for the highly active and thermostable LAAO4 of Hebeloma cylindrosporum (HcLAAO4). Here, we crystallized a proteolytically truncated surface entropy reduction variant of HcLAAO4 and solved its structure in substrate-free form and in complex with diverse substrates. The ability to support the aliphatic portion of a substrate's side chain by an overall hydrophobic active site is responsible for the broad substrate spectrum of HcLAAO4, including l-amino acids with big aromatic, acidic and basic side chains. Based on the structural findings, we generated an E288H variant with increased activity toward pharmaceutical building blocks of high interest.

A Dual-Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase.

Industrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade-off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate-degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase-m5b, which exhibited 13.3 °C higher Tm and 4.2 times higher catalytic activity than wild-type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αß/ßα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications.

An Engineered Imine Reductase for Highly Diastereo- and Enantioselective Synthesis of ß-Branched Amines with Contiguous Stereocenters.

ß-Branched chiral amines with contiguous stereocenters are valuable building blocks for preparing various biologically active molecules. However, their asymmetric synthesis remains challenging. Herein, we report a highly diastereo- and enantioselective biocatalytic approach for preparing a broad range of ß-branched chiral amines starting from their corresponding racemic ketones. This involves a dynamic kinetic resolution-asymmetric reductive amination process catalyzed using only an imine reductase. Four rounds of protein engineering endowed wild-type PocIRED with higher reactivity, better stereoselectivity, and a broader substrate scope. Using the engineered enzyme, various chiral amine products were synthesized with up to >99.9% ee, >99:1 dr, and >99% conversion. The practicability of the developed biocatalytic method was confirmed by producing a key intermediate of tofacitinib in 74% yield, >99.9% ee, and 98:2 dr at a challenging substrate loading of 110 g L-1. Our study provides a highly capable imine reductase and a protocol for developing an efficient biocatalytic dynamic kinetic resolution-asymmetric reductive amination reaction system.

Truncation of a novel C-terminal domain of a ß-glucanase improves its thermal stability and specific activity.

Enzymes that degrade ß-glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo-ß-1,3(4)-glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum, was cloned and expressed in Pichia pastoris. Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C-terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR-mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A-ΔC, exhibited significantly enhanced thermal stability at 50°C, with D-values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A-ΔC demonstrated a 20%-25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full-length enzymes. Notably, specific activity against cereal ß-glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress-tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo-ß-1,3(4)-glucanase. Additionally, it sheds light on a group of endo-ß-1,3(4)-glucanases from Antarctic rock-inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function.

A comprehensive review on fructosyl peptide oxidase as an important enzyme for present hemoglobin A1c assays.

Glycated proteins are generated by binding of glucose to the proteins in blood stream through a nonenzymatic reaction. Hemoglobin A1c (HbA1c) is a glycated protein with glucose at the N-terminal of ß-chain. HbA1c is extensively used as an indicator for assessing the blood glucose concentration in diabetes patients. There are different conventional clinical methods for the detection of HbA1c. However, enzymatic detection method has newly obtained great attention for its high precision and cost-effectiveness. Today, fructosyl peptide oxidase (FPOX) plays a key role in the enzymatic measurement of HbA1c, and different companies have marketed HbA1c assay systems based on FPOX. Recent investigations show that FPOX could be used in assaying HbA1 without requiring HbA1c primary digestion. It could also be applied as a biosensor for HbA1c detection. In this review, we have discussed the recent improvements of FPOX properties, different methods of FPOX purification, solubility, and immobilization, and also the use of FPOX in HbA1c biosensors.

Crystal structure of urethanase from Candida parapsilosis and insights into the substrate-binding through in silico mutagenesis and improves the catalytic activity and stability.

Ethyl carbamate (EC) is classified as a Class 2A carcinogen, and is present in various fermented foods, posing a threat to human health. Urethanase (EC 3.5.1.75) can catalyze EC to produce ethanol, CO2 and NH3. The urethanase (cpUH) from Candida parapsilosis can hydrolyze EC, but its low affinity and poor stability hinder its application. Here, the structure of cpUH from Candida parapsilosis was determined with a resolution of 2.66 Å. Through sequence alignment and site-directed mutagenesis, it was confirmed that cpUH contained the catalytic triad Ser-cisSer-Lys of the amidase family. Then, the structure-oriented engineering mutant N194V of urethanase was obtained. Its urethanase activity increased by 6.12 %, the catalytic efficiency (kcat/Km) increased by 21.04 %, and the enzyme stability was also enhanced. Modeling and molecular docking analysis showed that the variant N194V changed the number of hydrogen bonds between the substrate and the catalytic residue, resulting in enhanced catalytic ability. MD simulation also demonstrated that the introduction of hydrophobic amino acid Val reduced the RMSD value and increased protein stability. The findings of this study suggest that the N194V variant exhibits significant potential for industrial applications due to its enhanced affinity for substrate binding, improved catalytic efficiency, and increased enzyme stability.