Directed Mutagenesis of Catalytic Residues in L-Asparaginase II from Salmonella paratyphi: Structural, Functional and Stability Analysis Using In-Vitro, Docking and Simulation Studies.
Jul 2026· Applied Biochemistry and Biotechnology· 0 citations· 14 references
Medicine
TL;DR
Findings demonstrate that rational site-directed mutagenesis can effectively enhance both the catalytic efficiency and thermal stability of recSalA and indicate that the introduced mutations preserve the overall protein architecture.
The rational engineering of an α-L-rhamnosidase (DthRha) to address limitations and enhance its performance for flavonoid production highlights the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.
Haoyu Jia, Luran Wang, Tong Yan et al.· International Journal of Bio...· 0 citations
Aspartase from Escherichia coli (AspA) catalyzes the direct conversion of acrylic acid to β-alanine; however, its substrate specificity and low catalytic efficiency limit its broader application. We engineered an AspA mutant capable of efficiently catalyzing the amination of acrylic acid for β-alanine synthesis, using Rosetta Enzyme Design to computationally redesign the Cβ-binding region of the acrylic acid binding site in AspA. Based on energy scores, structural configurations, and hydrogen bonding networks, 51 candidate variants with penalty scores below 30 were selected for mutant construction and performance testing; >70% of these variants exhibited enhanced catalytic activity in acrylic acid’s hydrogen amination. Four mutants achieved over 3-fold improved activity. The optimal mutant, M1 (T190I-M324I-K327L-N329C), demonstrated a 7.3-fold increased specific enzyme activity and a 13.0-fold improved kcat/Km compared with the wild type. Conformational changes in the S-loop and enhanced hydrophobic interactions near the active site contributed significantly to M1’s enhanced activity. Upon reaction optimization, the conversion of β-alanine synthesis using M1 in whole-cell catalysis increased from 5% with the wild type to 90% with M1. This study provides a reference for the biocatalytic synthesis of β-alanine, significantly enhancing the conversion of acrylic acid and demonstrating the enzyme’s potential for industrial applications.
Long-Xian Li, Baodi Ma, Yi Xu· Catalysts· 0 citations
Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the β7-β8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the β7-β8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.
Erich G. Walkenhauer, Noah Cox-Tigre, Manish Chaubey et al.· bioRxiv· 0 citations
1-Naphthol-2-hydroxylase (1NH, group-A flavoprotein monooxygenase) from Carbaryl-degrading Pseudomonas sp. C5pp catalyzes ortho-hydroxylation of 1-naphthol to 1,2-dihydroxynaphthalene. Homology model-guided site-directed-mutagenesis identified residues involved in FAD-binding, substrate-entry, catalysis and dimerization. Alanine substitution of R55, Q128, W298 and D318 caused activity loss with decreased flavin content, indicating roles in FAD-binding. Bulky substitutions in tunnel lumen (G207F, M260F, M380F and M383F) abolished activity, supporting its role in substrate-entry. Mutations at the active-site showed that H57 forms principal substrate-binding residue which is oriented by N125, while L229 and P325 contribute to substrate positioning. The apparent coupling efficiency (hydroxylation efficiency) of mutants remained comparable to that of wildtype enzyme, indicating that these substitutions affected activity without altering coupling between NADH oxidation and product formation. The C-terminal domain was crucial for folding and dimerization, while H541A/Y542A led to monomeric 1NH with ∼52% activity. These findings provide insights into role of domains and the catalytic mechanism of 1NH.
Tushar Dhamale, S. S. Dhakar, P. Phale· Archives of Biochemistry and...· 0 citations
Hydxymycotrienin A is identified as a promising, bioavailable ‘privileged scaffold’ that offers a non-covalent therapeutic strategy for NSCLC, characterizing Hydroxymycotrienin A as a promising, bioavailable ‘privileged scaffold’ that offers a non-covalent therapeutic strategy.
Adeline Celina Rufus, Sidharth Kumar N, M. Ramasamy et al.· Frontiers in Bioinformatics· 0 citations
Arginine-specific gingipain B (RgpB), a key cysteine protease from Porphyromonas gingivalis, is associated with several systemic diseases. It is synthesized as a zymogen bound to a propeptide inhibitor that blocks its catalytic activity until activation in the extracellular environment. To identify inhibitory peptide variants with enhanced affinity, mutants of the propeptide inhibitory loop were designed. A total of 52 mutants were generated and, for each model, four independent molecular dynamics replicas were performed, followed by binding free energy calculations using MM/GBSA. Most variants exhibited more favorable binding affinities than the wild-type (WT) loop (−109.8 ± 4.9 kcal mol−1). Among them, V126K (−128.1 ± 5.6 kcal mol−1), E131D (−120.6 ± 8.2 kcal mol−1), and N132R (−135.8 ± 4.6 kcal mol−1) emerged as promising, with the first two showing statistically significant improvements using ANOVA followed by Tukey's post-hoc test (p = 0.001). Thermodynamic integration calculations were consistent with increased binding affinity for the selected variants. Thermodynamic integration calculations further confirmed a significant increase in the relative binding affinity for the selected variants compared to the wild type. These results provide a solid basis for future in vitro validation and establish an in silico framework to accelerate the rational design of propeptide-based inhibitors and the development of novel therapeutic approaches targeting RgpB.
S. Tapia, O. Yáñez, Olimpo García-Beltrán et al.· RSC Advances· 0 citations
