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[学术文献 ] Biochemical Investigation and Engineering of a Tardigrade X Family DNA Polymerase for Template-Independent DNA Synthesis 进入全文
ACS Catalysis
The X family of DNA polymerases (PolXs) includes the well-studied mammalian polymerases Polβ, Polλ, Polμ, and terminal deoxynucleotidyl transferase (TdT). The template-independent DNA polymerase activity of TdT has been harnessed for applications in enzymatic de novo DNA synthesis, offering a strategy to overcome the limitations of traditional phosphoramidite-based DNA synthesis methods. Close homologues of the mammalian PolXs are present in other vertebrates, while invertebrate PolXs remain unexplored. In this study, we characterize an invertebrate PolX from the extremotolerant tardigrade Ramazzottius varieornatus (RvPolX), and demonstrate that it possesses modest template-independent DNA polymerase activity, despite limited homology to mammalian PolXs (21% sequence identity with TdT). Through a combination of structural modeling, targeted mutagenesis of active site residues, and high-throughput screening under stringent high salt conditions, we identified a synergistic combination of two mutations (G513A and R522I) that led to a significant increase in activity for the incorporation of all four nucleotides, particularly dATP (∼35-fold), yielding a salt-tolerant polymerase with overall higher activity and substrate promiscuity. Under high salt conditions, the engineered RvPolX displays an activity comparable to TdT and a nucleotide selectivity complementary to TdT. As a template-independent polymerase with a low homology to TdT, RvPolX provides an alternative scaffold for further engineering in various biotechnological applications.
[学术文献 ] Context-aware geometric deep learning for protein sequence design 进入全文
Nature Communications
Protein design and engineering are evolving at an unprecedented pace leveraging the advances in deep learning. Current models nonetheless cannot natively consider non-protein entities within the design process. Here, we introduce a deep learning approach based solely on a geometric transformer of atomic coordinates and element names that predicts protein sequences from backbone scaffolds aware of the restraints imposed by diverse molecular environments. To validate the method, we show that it can produce highly thermostable, catalytically active enzymes with high success rates. This concept is anticipated to improve the versatility of protein design pipelines for crafting desired functions. Advances in deep learning are transforming protein design. Here, authors introduce a method using geometric transformers to predict protein sequences, resulting in highly thermostable and catalytically active enzymes with high success rates.
[学术文献 ] Two different types of hydrolases co-degrade ochratoxin A in a highly efficient degradation strain Lysobacter sp. CW239 进入全文
Journal of Hazardous Materials
Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 μg/L OTA was completely degraded by 1.0 μg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-β-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.
[学术文献 ] TemBERTure: advancing protein thermostability prediction with deep learning and attention mechanisms 进入全文
Bioinformatics Advances
Motivation Understanding protein thermostability is essential for numerous biotechnological applications, but traditional experimental methods are time-consuming, expensive, and error-prone. Recently, deep learning (DL) techniques from natural language processing (NLP) was extended to the field of biology, since the primary sequence of proteins can be viewed as a string of amino acids that follow a physicochemical grammar. Results In this study, we developed TemBERTure, a DL framework that predicts thermostability class and melting temperature from protein sequences. Our findings emphasize the importance of data diversity for training robust models, especially by including sequences from a wider range of organisms. Additionally, we suggest using attention scores from Deep Learning models to gain deeper insights into protein thermostability. Analyzing these scores in conjunction with the 3D protein structure can enhance understanding of the complex interactions among amino acid properties, their positioning, and the surrounding microenvironment. By addressing the limitations of current prediction methods and introducing new exploration avenues, this research paves the way for more accurate and informative protein thermostability predictions, ultimately accelerating advancements in protein engineering.
[学术文献 ] Chemoenzymatic Synthesis of Fluorinated Mycocyclosin Enabled by the Engineered Cytochrome P450-Catalyzed Biaryl Coupling Reaction 进入全文
Journal of the American Chemical Society
Installing fluorine atoms onto natural products holds great promise for the generation of fluorinated molecules with improved or novel pharmacological properties. The enzymatic oxidative carbon–carbon coupling reaction represents a straightforward strategy for synthesizing biaryl architectures, but the exploration of this method for producing fluorine-substituted derivatives of natural products remains elusive. Here, in this study, we report the protein engineering of cytochrome P450 from Mycobacterium tuberculosis (MtCYP121) for the construction of a series of new-to-nature fluorine-substituted Mycocyclosin derivatives. This protocol takes advantage of a “hybrid” chemoenzymatic procedure consisting of tyrosine phenol lyase-catalyzed fluorotyrosine preparation from commercially available fluorophenols, intermolecular chemical condensation to give cyclodityrosines, and an engineered MtCYP121-catalyzed intramolecular biphenol coupling reaction to complete the strained macrocyclic structure. Computational mechanistic studies reveal that MtCYP121 employs Cpd I to abstract a hydrogen atom from the proximal phenolic hydroxyl group of the substrate to trigger the reaction. Then, conformational change makes the two phenolic hydroxyl groups close enough to undergo intramolecular hydrogen atom transfer with the assistance of a pocket water molecule. The final diradical coupling process completes the intramolecular C–C bond formation. The efficiency of the biaryl coupling reaction was found to be influenced by various fluorine substitutions, primarily due to the presence of distinct binding conformations.
[学术文献 ] Biodegradation Characteristics and Mechanism of Aflatoxin B1 by Bacillus amyloliquefaciens from Enzymatic and Multiomics Perspectives 进入全文
Journal of Agricultural and Food Chemistry
Aflatoxin B1 (AFB1), a mycotoxin and natural carcinogen, commonly contaminates cereals and animal feeds, posing serious health risks to human and animal. In this study, Bacillus amyloliquefaciens ZG08 isolated from kimchi could effectively remove 80.93% of AFB1 within 72 h at 37 °C and pH 7.0. Metabolome and transcriptome analysis showed that metabolic processes including glycerophospholipid metabolism and amino acid metabolism were most affected in B. amyloliquefaciens ZG08 exposed to AFB1. The adaptation mechanism likely involved activation of the thioredoxin system to restore intracellular redox equilibrium. The key genes, tpx and gldA, overexpressed in Escherichia coli BL21, achieved degradation rates of 60.15% and 47.16% for 100 μg/kg AFB1 under optimal conditions of 37 °C and pH 8.0 and 45 °C and pH 7.0, respectively. The degradation products, identified as AFD1, were less cytotoxic than AFB1 in HepG2 cells. These findings suggest potential strategies for utilizing probiotics and engineered enzymes in AFB1 detoxification.
