Jul 2026· International Journal of Biological Macromolecules· pp.
153591
· 0 citations· 69 references
Medicine
TL;DR
Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.
Abstract
Prolidase is a specialized metalloprotease that overcomes the resistance of proline-containing peptide bonds to proteolysis by exclusively hydrolyzing dipeptides with a C-terminal proline. Despite its potential significance in the life cycle of protozoan parasites, knowledge of prolidase in these organisms remains limited. In this study, the complete coding sequence of Leishmania donovani prolidase (LdProl) was cloned into an expression vector. The recombinant protein was purified using affinity chromatography, and its molecular weight was assessed by size-exclusion chromatography. The purified LdProl exhibited enzymatic activity with a strong substrate preference for Trp-Pro-pNA, and manganese ions were identified as essential cofactors for optimal activity. LdProl also stimulated innate immune mechanisms through elevating pro-inflammatory cytokines and nitric oxide levels, suggesting a role in macrophage-mediated immunomodulation. Fluorescence spectroscopy revealed pH-dependent conformational changes, particularly under strongly acidic conditions, whereas circular dichroism spectroscopy indicated a predominantly α-helical structure that was most stable at neutral pH. Computational modeling demonstrated a conserved three-dimensional structure, metal-binding site, and stable dimeric interface similar to prolidases from other organisms. Docking studies identified Trp-Pro-pNA as the substrate with the highest binding affinity, and molecular dynamics simulations confirmed the formation of stable enzyme-substrate complexes. Virtual screening yielded two lead compounds with higher binding affinities for LdProl than for its human counterpart, along with favorable ADME properties. Molecular dynamics simulations further validated these compounds by demonstrating the stability of enzyme-inhibitor complexes. Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.
Mycobacterium tuberculosis
is extremely dependent upon lipid-hydrolyzing enzymes to utilize the resources of host lipids and survive within the cell. Rv1063c is a conserved hypothetical protein predicted to share sequence homology with the patatin-like family.
In the present study, the
rv1063c
gene was cloned and heterologously expressed in Escherichia coli BL21 (DE3). Functional enzyme was obtained by solubilizing inclusion bodies in 8 mol·L
−1
urea, refolding via gradient dialysis, and purifying through Ni-affinity chromatography.
Bioinformatic homology analysis indicates Rv1063c is homologous to members of the patatin-like family containing a putative Ser52-Asp166 catalytic dyad, though this residue pair has not been experimentally validated in the present study. The recombinant Rv1063c was mainly expressed as inclusion bodies without significant soluble cytoplasmic expression. Biochemical assays demonstrated the recombinant protein exclusively hydrolyzes medium-chain pnitrophenyl esters (C8–12), with maximum hydrolytic activity toward p-NP-C8 (caprylate), followed by C10 and C12, and exhibited the highest activity at 40 °C and pH 7.0. The enzyme exhibited moderate catalytic efficiency but poor thermal stability. All catalytic data in this paper were obtained from artificial p-nitrophenyl ester substrates, which cannot fully reflect natural lipid substrate preference of mycobacteria in vivo.
This work biochemically characterizes Rv1063c as a medium-chain-specific esterase encoded by a protein homologous to the patatin superfamily. Its physiological and pathogenic roles remain undetermined, and further multi-level experiments are required to clarify its biological functions.
Yuming Song, Songsong Dong, Rumeng Zhai et al.· Frontiers in Microbiology· 0 citations
Findings suggest that the studied FPMO may play a role in antibiotic resistance in P. aeruginosa by oxidatively inactivating ampicillin by oxidatively inactivating ampicillin.
Maliheh Mohammadkhani, Shamsozoha Abolmaali, S. D. Astaneh· Iranian Journal of Microbiol...· 0 citations
Mucin-derived peptides constitute attractive antimicrobial candidates, but their clinical application is restricted by limited stability and moderate efficacy. To address these limitations, we modified d-amino-acid-containing peptidomimetics and investigated their Cu(ii) and Zn(ii) complexes with respect to coordination chemistry, structure, proteolytic resistance, and antimicrobial activity. Potentiometric, spectroscopic, and DFT studies revealed that metal binding donor sets are analogous to those of the native peptide, producing only minor local conformational effects without significant global structural rearrangement, as confirmed by circular dichroism analysis. In contrast to the modest structural changes, biological activity was strongly influenced by chirality and metal coordination. The fully d-configured analogue displayed the highest antimicrobial potency, particularly at pH 5.5, and its Zn(ii) and Cu(ii) complexes showed enhanced antibacterial and antifungal effects relative to the native system. Proteolytic assays demonstrated rapid plasma degradation of the native peptide and the partially modified analogue, whereas the fully d-substituted peptidomimetic remained largely intact after 2 h. All compounds exhibited minimal hemolytic and cytotoxic effects. These findings demonstrate that d-amino-acid incorporation combined with metal coordination significantly improves both enzymatic stability and antimicrobial performance of mucin-derived peptides.
A. Ślusarczyk, D. Bellotti, Silvia Leveraro et al.· RSC Advances· 0 citations
Microbial esterases are versatile and stable enzymes with a wide range of biotechnological applications. However, few esterases have been characterized from archaea, an important source of extremophilic enzymes. In this study, we report the biochemical characterization and crystal structure of Ta0887, a novel esterase from the thermoacidophilic archaeon Thermoplasma acidophilum. The protein was successfully cloned, expressed, and purified in Escherichia coli. Light scattering assays revealed that Ta0887 is a monomer in solution. Activity assays using p‐nitrophenyl (p‐NP) esters confirmed its esterase activity, showing a substrate preference for p‐NP hexanoate (C6). Furthermore, the substitution of Ser95 with alanine completely abolished enzymatic activity, thereby confirming its essential role as the nucleophilic residue of the catalytic triad. The enzyme exhibited optimal activity at 65 °C and pH 8.0. Notably, Ta0887 displayed high thermal stability, retaining 66% residual activity after incubation at 80 °C for 2 h, consistent with its thermal denaturation midpoint temperature of 80.6 °C. The crystal structure of Ta0887, resolved at 1.93 Å, revealed an α/β‐hydrolase core domain consisting of an eight‐strand β‐sheet, surrounded by seven α‐helices, and a cap domain comprising four α‐helices. Ta0887 features a large substrate‐binding pocket at the interface between the two domains that contains the conserved residues Ser95, Asp187, and His215 of the catalytic triad. Further analysis indicates that an efficiently packed hydrophobic core is a key feature for the observed thermostability. The findings from this study provide a basis for the future engineering of Ta0887 with the aim of enhancing its potential for industrial and biotechnological applications.
Alejandro Delgado-Rey, M. L. Llamas-García, Gabriela M Montero-Morán et al.· FEBS Open Bio· 0 citations
Structural analysis reveals that ASPRV1-14 possesses distinctly hydrophobic S2/S2' pockets, dictating a strict requirement for hydrophobic residues at the P2/P2' positions of substrates and explaining its resistance to most HIV-1 PR inhibitors, except indinavir.
Xueqian Feng, Zi-Lian Chen, Chao Lan et al.· Acta Biochimica et Biophysic...· 0 citations
Bacteriophage endolysins are peptidoglycan hydrolases currently being clinically developed as antibacterial therapeutics. Despite possessing several advantageous antibacterial properties, many endolysins display only modest thermal stability. This characteristic hinders their therapeutic potential due to limited long-term stability and complex storage requirements. To overcome this limitation, an evolutionary-based protein engineering strategy was employed in a proof-of-concept study to increase the intrinsic stability of an endolysin. Using the multimeric endolysin PlyC as a model, directed evolution was applied to the thermolabile PlyCA catalytic subunit. After screening 18,000 mutants, the lead candidate identified was the point mutant PlyC(PlyCAN211H). The protonated PlyCAH211 side-chain stabilizes the subunit by forming favorable electrostatic field interactions with two acidic residues located in the N-terminal glycosyl hydrolase domain, possibly resulting in an extended linker structure being anchored to the surface of the domain. This mutation improved structural and thermal stability under conditions that maximize the stabilizing effect (pH 6.0) by 3.48°C and 4.10°C, respectively, and increased kinetic stability 18.6-fold over wild-type. Combining PlyCAN211H with a stabilizing mutation (PlyCAT406R) identified in an independent rational-based in silico screen of PlyC additively enhanced thermal stability by 7.46°C at pH 6.0. Accordingly, with a thermal transition temperature of 54.60°C, PlyC(PlyCAN211H,T406R) now represents a highly stable endolysin derived from a mesophilic phage. In addition to in silico screening, this validated directed evolution methodology can now be expanded to other endolysins and bacteriolytic enzymes for the purpose of increasing their thermal stability, thereby improving their practical utility as an antimicrobial biologic.
Ryan D. Heselpoth, Daniel C. Nelson· Journal of Biological Chemis...· 0 citations