The regulation of chromatin configuration at AGAMOUS locus by LFR-SYD-containing complex is critical for reproductive organ development in Arabidopsis.

Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are evolutionarily conserved, multi-subunit machinery that play vital roles in the regulation of gene expression by controlling nucleosome positioning and occupancy. However, little is known about the subunit composition of SPLAYED (SYD)-containing SWI/SNF complexes in plants. Here, we show that the Arabidopsis thaliana Leaf and Flower Related (LFR) is a subunit of SYD-containing SWI/SNF complexes. LFR interacts directly with multiple SWI/SNF subunits, including the catalytic ATPase subunit SYD, in vitro and in vivo. Phenotypic analyses of lfr-2 mutant flowers revealed that LFR is important for proper filament and pistil development, resembling the function of SYD. Transcriptome profiling revealed that LFR and SYD shared a subset of co-regulated genes. We further demonstrate that the LFR and SYD interdependently activate the transcription of AGAMOUS (AG), a C-class floral organ identity gene, by regulating the occupation of nucleosome, chromatin loop, histone modification, and Pol II enrichment on the AG locus. Furthermore, the chromosome conformation capture (3C) assay revealed that the gene loop at AG locus is negatively correlated with the AG expression level, and LFR-SYD was functional to demolish the AG chromatin loop to promote its transcription. Collectively, these results provide insight into the molecular mechanism of the Arabidopsis SYD-SWI/SNF complex in the control of higher chromatin conformation of the floral identity gene essential to plant reproductive organ development.

Bioinformatics Identification and Experimental Validation of a Prognostic Model for the Survival of Lung Squamous Cell Carcinoma Patients.

Lung squamous cell carcinoma (LUSC) kills more than four million people yearly. Creating more trustworthy tumor molecular markers for LUSC early detection, diagnosis, prognosis, and customized treatment is essential. Cuproptosis, a novel form of cell death, opened up a new field of study for searching for trustworthy tumor indicators. Our goal was to build a risk model to assess drug sensitivity, monitor immune function, and predict prognosis in LUSC patients. The 19 cuproptosis-related genes were found in the literature, and patient genomic and clinical information was collected using the Cancer Genomic Atlas (TCGA) database. The LUSC patients were grouped using unsupervised clustering techniques, and 7626 differentially expressed genes were identified. Using univariate COX analysis, LASSO regression analysis, and multivariate COX analysis, a prognostic model for LUSC patients was developed. The tumor immune escape was evaluated using the Tumor Immune Dysfunction and Exclusion (TIDE) method. The R packages 'pRRophetic,' 'ggpubr,' and 'ggplot2' were utilized to examine drug sensitivity. For modeling, a 6-cuproptosis-based gene signature was found. Patients with high-risk LUSC had significantly worse survival rates than those with low-risk conditions. The possibility of tumor immunological escape was increased in patients with higher risk scores due to more immune cell inactivation. For patients with high-risk LUSC, we discovered seven potent potential drugs (AZD6482, CHIR.99021, CMK, Embelin, FTI.277, Imatinib, and Pazopanib). In conclusion, the cuproptosis-based genes predictive risk model can be utilized to predict outcomes, track immune function, and evaluate medication sensitivity in LUSC patients.

Integration of Single-Cell and Bulk RNA-seq Data to Identify the Cancer-Associated Fibroblast Subtypes and Risk Model in Glioma.

Cancer-associated fibroblasts (CAFs) are an important component of the stroma. Studies showed that CAFs were pivotally in glioma progression which have long been considered a promising therapeutic target. Therefore, the identification of prognostic CAF markers might facilitate the development of novel diagnostic and therapeutic approaches. A total of 1333 glioma samples were obtained from the TCGA and CGGA datasets. The EPIC, MCP-counter, and xCell algorithms were used to evaluate the relative proportion of CAFs in glioma. CAF markers were identified by the single-cell RNA-seq datasets (GSE141383) from the Tumor Immune Single-Cell Hub database. Unsupervised consensus clustering was used to divide the glioma patients into different distinct subgroups. The least absolute shrinkage and selection operator regression model was utilized to establish a CAF-related signature (CRS). Finally, the prognostic CAF markers were further validated in clinical specimens by RT‒qPCR. Combined single-cell RNA-seq analysis and differential expression analysis of samples with high and low proportions of CAFs revealed 23 prognostic CAF markers. By using unsupervised consensus clustering, glioma patients were divided into two distinct subtypes. Subsequently, based on 18 differentially expressed prognostic CAF markers between the two CAF subtypes, we developed and validated a new CRS model (including PCOLCE, TIMP1, and CLIC1). The nomogram and calibration curves indicated that the CRS was an accurate prognostic marker for glioma. In addition, patients in the high-CRS score group had higher immune infiltration and tumor mutation burden levels. Moreover, the CRS score had the potential to predict the response to immune checkpoint blockade (ICB) therapy and chemotherapy. Finally, the expression profiles of three CAF markers were verified by RT‒qPCR. In general, our study classified glioma patients into distinct subgroups based on CAF markers, which will facilitate the development of individualized therapy. We also provided insights into the role of the CRS in predicting the response to ICB and chemotherapy in glioma patients.

Spatial and temporal dynamics of base flow in semi-arid montane watersheds and the effects of landscape patterns and topography.

Base flow (BF) is harder to predict than other hydrological signatures. The lack of hydrologically relevant information or adequately broad spectrum of typically selected catchment attributes (particularly landscape and topography) hinders the explanatory power. Our goals were to identify the most influential controls on base flow spatially and temporally and to elucidate the response relationships. Base flow in 19 semi-arid sub-watersheds was separated by digital filtering. One hundred and fourteen sub-watershed attributes were related to base flow using random forest regression. The main results were as follows: (1) Annual BF significantly declined since 1999 due to decreased precipitation, increased air temperature, afforestation, urban expansion, and increasing water consumption. Annual base flow index (BFI), varying between 0.319 and 0.695, showed less noticeable temporal trends. (2) Precipitation (P) and underlying carbonate rocks primarily controlled the spatial variation of annual BF and total flow (TF), with the impacts being positive. Landscape was less influential. After the abrupt runoff decline, landscape composition rather than configuration exerted greater impacts on spatial BF and TF, and the importance of forest increased, whereas landscape configuration was decisive for BFI during the whole observation period. The absence of significant links between landscape configuration and water quantity may result from a scale issue. Concave profile curvatures were found to be topographic variables more important than slopes. The impact of soil was the least. This study would benefit the selection of catchment attributes and spatial extents to quantify these attributes in building BF predicting models in future studies.

TCP7 interacts with Nuclear Factor-Ys to promote flowering by directly regulating SOC1 in Arabidopsis.

The success of plant reproduction depends on the timely transition from the vegetative phase to reproductive growth, a process often referred to as flowering. Although several plant-specific transcription factors belonging to the Teosinte Branched 1/Cycloidea/Proliferating Cell Factor (TCP) family are reportedly involved in the regulation of flowering in Arabidopsis, the molecular mechanisms, especially for Class I TCP members, are poorly understood. Here, we genetically identified Class I TCP7 as a positive regulator of flowering time. Protein interaction analysis indicated that TCP7 interacted with several Nuclear Factor-Ys (NF-Ys), known as the 'pioneer' transcription factors; CONSTANS (CO), a main photoperiod regulator of flowering. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was differentially expressed in the dominant-negative mutant of TCP7 (lcu) and the loss-of-function mutant of Class I TCP members (septuple). Additionally, we obtained genetic and molecular evidence showing that TCP7 directly activates the flowering integrator gene, SOC1. Moreover, TCP7 synergistically activated SOC1 expression upon interacting with CO and NF-Ys in vivo. Collectively, our results provide compelling evidence that TCP7 synergistically interacts with NF-Ys to activate the transcriptional expression of the flowering integrator gene SOC1.

Impact of PROTAC Linker Plasticity on the Solution Conformations and Dissociation of the Ternary Complex.

The conformational behavior of a small molecule free in solution is important to understand the free energy of binding to its target. This could be of special interest for proteolysis-targeting chimeras (PROTACs) due to their often flexible and lengthy linkers and the need to induce a ternary complex. Here, we report on the molecular dynamics (MD) simulations of two PROTACs, MZ1 and dBET6, revealing different linker conformational behaviors. The simulation of MZ1 in dimethyl sulfoxide (DMSO) agrees well with the nuclear magnetic resonance study, providing strong support for the relevance of our simulations. To further understand the role of linker plasticity in the formation of a ternary complex, the dissociation of the complex von Hippel-Lindau-MZ1-BRD4 is investigated in detail by steered simulations and is shown to follow a two-step pathway. Interestingly, both MZ1 and dBET6 display in water, a tendency toward an intramolecular lipophilic interaction between the two warheads. The hydrophobic contact of the two warheads would prevent them from binding to their respective proteins and might have an effect on the efficacy of induced cellular protein degradation. However, conformations featuring this hydrophobic contact of the two warheads are calculated to be marginally more favorable.

Wearable Sunlight-Triggered Bimorph Textile Actuators.

Photothermal bimorph actuators have attracted considerable attention in intelligent devices because of their cordless control and lightweight and easy preparation. However, current photothermal bimorph actuators are mostly based on films or papers driven by near-infrared sources, which are deficient in flexibility and adaptability, restricting their potential in wearable applications. Herein, a bimorph textile actuator that can be scalably fabricated with a traditional textile route and autonomously triggered by sunlight is reported. The active layer and passive layer of the bimorph are constructed by polypropylene tape and a MXene-modified polyamide filament. Because of the opposite thermal expansion and MXene-enhanced photothermal efficiency (>260%) of the bimorph, the textile actuator presents effective deformation (1.38 cm-1) under low sunlight power (100 mW/cm2). This work provides a new pathway for wearable sunlight-triggered actuators and finds attractive applications for smart textiles.

Identifying spatial influence of urban elements on road-deposited sediment and the associated phosphorus by coupling Geodetector and Bayesian Networks.

Elevated particles and phosphorus washed from road-deposited sediment (RDS) are noteworthy causes of eutrophication in urban water bodies. Identifying how urban elements (e.g., dwellings, roads) spatially influence RDS and the associated phosphorus can help pinpoint the primary management areas for RDS pollution and therefore effectively mitigate this problem. This study investigated spatial influence of urban elements on RDS build-up load and phosphorus load in Hanyang district of Wuhan city in central China. Bayesian Networks (BNs), combined with geographical detector (Geodetector) and correlation analysis, were applied to quantify spatial association between kernel density of urban elements, RDS build-up load and phosphorus load in RDS. Results showed that (1) areas with higher density of factories related elements usually had elevated level of RDS build-up load, aluminum-bound phosphorus (Al-P), occluded phosphorus (Oc-P), organophosphorus (Or-P). Higher load of RDS associated iron-bound phosphorus (Fe-P) and apatite phosphorus (Ca-P) usually occurred where dwellings, catering, and entertainment related elements were concentrated. (2) Urban elements mainly showed positive correlation with RDS build-up load, Fe-P, Ca-P, De-P (detrital apatite phosphorus), while they chiefly showed negative correlation with Ex-P (exchangeable phosphorus), Al-P, Oc-P, and Or-P. Bus stations, dwellings, and factories related elements had relatively strong determinant power over spatial stratified heterogeneity of RDS and RDS-associated phosphorus. (3) Geodetector and correlation analysis could boost factors filtering and construction of network structures in the process of developing BNs models. The developed BNs resulted in sound prediction of <150 µm RDS build-up load and phosphorus load, given that the prediction accuracy of models ranged from 0.532 to 0.657. These findings demonstrate that urban elements are useful spatial predictors of RDS pollution, and coupling Geodetector and BNs is promising in RDS pollution prediction and supporting urban nonpoint source pollution management.

OsLFR is essential for early endosperm and embryo development by interacting with SWI/SNF complex members in Oryza sativa.

Rice (Oryza sativa L.) endosperm provides the developing embryo with nutrients and provides human beings with a staple food. The embryo eventually develops into a new sporophyte generation. Despite their important roles, the molecular mechanisms underlying early-stage endosperm and embryo development remain elusive. Here, we established the fundamental functions of rice OsLFR, an ortholog of the Arabidopsis SWI/SNF chromatin-remodeling complex (CRC) component LFR. OsLFR was expressed primarily in the rice spikelets and seeds, and the OsLFR protein was localized to the nucleus. We conducted genetic, cellular and molecular analyses of loss-of-function mutants and transgenic rescue lines. OsLFR depletion resulted in homozygous lethality in the early seed stage through endosperm and embryo defects, which could be successfully recovered by the OsLFR genomic sequence. Cytological observations revealed that the oslfr endosperm had relatively fewer free nuclei, had abnormal and arrested cellularization, and demonstrated premature programed cell death: the embryo was reduced in size and failed to differentiate. Transcriptome profiling showed that many genes, involved in DNA replication, cell cycle, cell wall assembly and cell death, were differentially expressed in a knockout mutant of OsLFR (oslfr-1), which was consistent with the observed seed defects. Protein-protein interaction analysis showed that OsLFR physically interacts with several putative rice SWI/SNF CRC components. Our findings demonstrate that OsLFR, possibly as one component of the SWI/SNF CRC, is an essential regulator of rice seed development, and provide further insights into the regulatory mechanism of early-stage rice endosperm and embryo development.

Mat_peptide: comprehensive annotation of mature peptides from polyproteins in five virus families.

MOTIVATION: Sequence repositories have few well-annotated virus mature peptide sequences. Therefore post-translational proteolytic processing of polyproteins into mature peptides (MPs) has been performed in silico, with a new computational method, for over 200 species in 5 pathogenic virus families (Caliciviridae, Coronaviridae, Flaviviridae, Picornaviridae and Togaviridae). RESULTS: Using pairwise alignment with reference sequences, MPs have been annotated and their sequences made available for search, analysis and download. At publication the method had produced 156 216 sequences, a large portion of the protein sequences now available in https://www.viprbrc.org. It represents a new and comprehensive mature peptide collection. AVAILABILITY AND IMPLEMENTATION: The data are available at the Virus Pathogen Resource https://www.viprbrc.org, and the software at https://github.com/VirusBRC/vipr_mat_peptide.

Leadership, management, quality, and innovation in organ donation: 2019 Kunming recommendations for One Belt & One Road countries.

The findings and recommendations of the 2019 consensus conference in organ donation, held in Kunming, China, are here reported. The main objective of the conference was to gather relevant information from experts involved in the field. The data and opinions provided allowed to propose a series of recommendations for "One Belt & One Road Countries" on how to achieve self-sufficiency in organ donation. Leadership in organ donation should be results-oriented and goal-driven based on the principles of excellence, empowerment, and engagement, providing the means, resources, and strategies necessary to reach the goal in earnest. Management includes good governance and transparency of a national registry of patients in the waiting list, donors, transplants, transplant teams, quality, and safety programs with continuous educational training of health care professionals. Mandatory monitoring, auditing and evaluation of quality must be incorporated into donation practices as relevant points in innovation, as well as the adoption of already established and novel processes and technologies. Achievement of self-sufficiency in organ donation is a crucial step to fight against transplant tourism and to prevent organ trafficking. Based on recommendations arising from the conference, each country could review and develop individualized action plans adjusted to its own circumstances and reality.

Discovery of USP7 small-molecule allosteric inhibitors.

Ubiquitin specific protease-7 (USP7) is considered an attractive target for cancer therapy by promoting degradation of the tumor suppressor p53 and negatively affecting the immune response to tumors. However, the development of selective non-covalent USP7 inhibitors has proven challenging. In this work we report the NMR characterization of a weak binder from SPR screening of an in-house fragment library which reveals that it binds to the allosteric palm site of the catalytic domain. Molecular modeling combined with 1HNMR saturation transfer difference and NOESY experiments enabled structure-based design of additional compounds showing IC50 values in the low-micromolar range with good selectivity over the closest homolog USP47. The most potent analogue represents a promising starting point for the development of novel, selective USP7 inhibitors.

LFR is functionally associated with AS2 to mediate leaf development in Arabidopsis.

Leaves are essential organs for plants. We previously identified a functional gene possibly encoding a component of the SWI/SNF complex named Leaf and Flower Related (LFR) in Arabidopsis thaliana. Loss-of-function mutants of LFR displayed obvious defects in leaf morphogenesis, indicating its vital role in leaf development. Here an allelic null mutant of ASYMMETRIC LEAVES2 (AS2), as2-6, was isolated as an enhancer of lfr-1 in petiole length, vasculature pattern and leaf margin development. The lfr as2 double-mutants showed enhanced ectopic expression of BREVIPEDICELLUS (BP) compared with each of the single-mutants, which is consistent with their synergistic genetic enhancement in multiple BP-dependent development processes. Moreover, LFR and several putative subunits of the SWI/SNF complex interacted physically with AS2. LFR associated with BP chromatin in an AS1-AS2-dependent manner to promote the nucleosome occupancy for appropriate BP repression in leaves. Taken together, our findings reveal that LFR and the SWI/SNF complex play roles in leaf development at least partly by repressing BP transcription as interacting factors of AS2, which expounds our understanding of BP repression at the chromatin structure level in leaf development.

Enhancing the atypical esterase promiscuity of the γ-lactamase Sspg from Sulfolobus solfataricus by substrate screening.

Promiscuous enzymes can be modified by protein engineering, which enables the catalysis of non-native substrates. γ-lactamase Sspg from Sulfolobus solfataricus is an enzyme with high activity, high stability, and pronounced tolerance of high concentrations of the γ-lactam substrate. These characteristics suggest Sspg as a robust enzymatic catalyst for the preparation of optically pure γ-lactam. This study investigated the modification of this enzyme to expand its application toward resolving chiral esters. γ-Lactamase-esterase conversion was performed by employing a three-step method: initial sequence alignment, followed by substrate screening, and protein engineering based on the obtained substrate-enzyme docking results. This process of fine-tuning of chemical groups on substrates has been termed "substrate screening." Steric hindrance and chemical reactivity of the substrate are major concerns during this step, since both are determining factors for the enzyme-substrate interaction. By employing this three-step method, γ-lactamase Sspg was successfully converted into an esterase with high enantioselectivity towards phenylglycidate substrates (E value > 300). However, since both wild-type Sspg and Sspg mutants did not hydrolyze para-nitrophenyl substrates (pNPs), this esterase activity was termed "atypical esterase activity." The γ-lactamase activity and stability of the Sspg mutants were not severely compromised. The proposed method can be applied to find novel multi-functional enzyme catalysts within existing enzyme pools.

Influenza Research Database: An integrated bioinformatics resource for influenza virus research.

The Influenza Research Database (IRD) is a U.S. National Institute of Allergy and Infectious Diseases (NIAID)-sponsored Bioinformatics Resource Center dedicated to providing bioinformatics support for influenza virus research. IRD facilitates the research and development of vaccines, diagnostics and therapeutics against influenza virus by providing a comprehensive collection of influenza-related data integrated from various sources, a growing suite of analysis and visualization tools for data mining and hypothesis generation, personal workbench spaces for data storage and sharing, and active user community support. Here, we describe the recent improvements in IRD including the use of cloud and high performance computing resources, analysis and visualization of user-provided sequence data with associated metadata, predictions of novel variant proteins, annotations of phenotype-associated sequence markers and their predicted phenotypic effects, hemagglutinin (HA) clade classifications, an automated tool for HA subtype numbering conversion, linkouts to disease event data and the addition of host factor and antiviral drug components. All data and tools are freely available without restriction from the IRD website at https://www.fludb.org.

TCP7 functions redundantly with several Class I TCPs and regulates endoreplication in Arabidopsis.

TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR) proteins, a family of plant-specific transcription factors, play important roles in many developmental processes. However, genetic and functional redundancy among class I TCP limits the analysis of their biological roles. Here, we identified a dominant-negative mutant of Arabidopsis thaliana TCP7 named leaf curling-upward (lcu), which exhibits smaller leaf cells and shorter hypocotyls than the wild type, due to defective endoreplication. A septuple loss-of-function mutant of TCP7, TCP8, TCP14, TCP15, TCP21, TCP22, and TCP23 displayed similar developmental defects to those of lcu. Genome-wide RNA-sequencing showed that lcu and the septuple mutant share many misexpressed genes. Intriguingly, TCP7 directly targets the CYCLIN D1;1 (CYCD1;1) locus and activates its transcription. We determined that the C-terminus of TCP7 accounts for its transcriptional activation activity. Furthermore, the mutant protein LCU exhibited reduced transcriptional activation activity due to the introduction of an EAR-like repressive domain at its C-terminus. Together, these observations indicate that TCP7 plays important roles during leaf and hypocotyl development, redundantly, with at least six class I TCPs, and regulates the expression of CYCD1;1 to affect endoreplication in Arabidopsis.

Calcium-sensing receptor-mediated mitogen-activated protein kinase pathway improves the status of transplanted mouse embryonic stem cells in rats with acute myocardial infarction.

Several studies have identified the critical role of calcium-sensing receptors (CaSRs) in cardiac ischaemia/reperfusion injury and cardiac hypertrophy and have demonstrated that CaSRs induce myocardial apoptosis by activating MAPKs. Using acute myocardial infarction rat models, we found that a combination therapy of CaSR inhibition and embryonic stem cell (ESC) transplantation after acute myocardial infarction (AMI) leads to a dramatic reduction in the infarct size; a significant increase in the maximum rising and falling rate (+dp/dtmax and -dp/dtmax, respectively) of left ventricular pressure; a significant decrease in left ventricular end-diastolic pressure; a significant decrease in the mRNA expression level of CaSR, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, p-ERK, p-JNK and p-P38 protein together with apoptosis indexes in the C and E groups; and a significant decrease in cTnT levels as well as LDH and CK activity. These findings indicate that cardiac function could be enhanced significantly by combination therapy with CaSR inhibition and ESC transplantation; the effect was better than ESC transplantation alone, and the mechanism might be associated with a reduction in cell apoptosis via the inhibition of the MAPK pathway. Apoptosis could be reduced through CaSR, which regulates the MAPK pathway and apoptosis-related protein. Our study indicated that CaSR inhibitors have a pivotal role in the treatment of AMI.

Enriching screening libraries with bioactive fragment space.

By deconvoluting 238,073 bioactive molecules in the ChEMBL library into extended Murcko ring systems, we identified a set of 2245 ring systems present in at least 10 molecules. These ring systems belong to 2221 clusters by ECFP4 fingerprints with a minimum intracluster similarity of 0.8. Their overlap with ring systems in commercial libraries was further quantified. Our findings suggest that success of a small fragment library is driven by the convergence of effective coverage of bioactive ring systems (e.g., 10% coverage by 1000 fragments vs. 40% by 2million HTS compounds), high enrichment of bioactive ring systems, and low molecular complexity enhancing the probability of a match with the protein targets. Reconciling with the previous studies, bioactive ring systems are underrepresented in screening libraries. As such, we propose a library of virtual fragments with key functionalities via fragmentation of bioactive molecules. Its utility is exemplified by a prospective application on protein kinase CK2, resulting in the discovery of a series of novel inhibitors with the most potent compound having an IC50 of 0.5µM and a ligand efficiency of 0.41kcal/mol per heavy atom.

Current kinase inhibitors cover a tiny fraction of fragment space.

We analyze the chemical space coverage of kinase inhibitors in the public domain from a fragment point of view. A set of 26,668 kinase inhibitors from the ChEMBL database of bioactive molecules were decomposed automatically by fragmentation at rotatable bonds. Remarkably, about half of the resulting 10,302 fragments originate from inaccessible libraries, as they are not present in commercially available compounds. By mapping to the established kinase pharmacophore models, privileged fragments in sub-pockets are identified, for example, the 5681 ring-containing fragments capable of forming bi-dentate hydrogen bonds with the hinge region in the ATP binding site. Surprisingly, hinge-binding fragments in current kinase inhibitors cover only 1% of the potential hinge-binders obtained by decomposing a library of nearly 7.5 million commercially available compounds, which indicates that a large fraction of chemical space is unexplored.

Identification and regulation of the catalytic promiscuity of (-)-γ-lactamase from Microbacterium hydrocarbonoxydans.

Mhg, a previously reported (-)-γ-lactamase from Microbacterium hydrocarbonoxydans, was identified to have perhydrolase activity by combining structure similarity search with activity assays. Kinetic studies illustrated that perhydrolysis was the native activity owing to lower K m and higher k cat/K m values. Experimental evidence showed that both hydrolysis and perhydrolysis reactions took place at the same active center. Engineering of the putative substrate-binding pocket revealed that Leu233 site played a vital role in the aspects of selective catalysis, soluble protein expression level and optimum temperature shift, etc. The mutants L233A, L233P, and L233T retained (-)-γ-lactamase activity but lost perhydrolase activity, while L233M only kept perhydrolase activity. Substitutions of Leu233 could dramatically influence the state of expressed protein. Computational analysis explicitly explained the relationships between mutations and γ-lactamase activity changes. Our investigations demonstrated that it was an efficient method to identify the enzyme catalytic promiscuity by combining 3D structure alignment with activity validations, and engineering of substrate-binding pocket could serve as a promising way to regulate activities of promiscuous enzymes.