Biology subjects

Souza, C. S.

Publications and source records attributed to Souza, C. S..

3 recordsLinked to original sources

bioRxiv2025

Computer-guided enzyme engineering of PET hydrolase mutants towards improved PET affinity

Polyethylene Terephthalate (PET) is an extensively used plastic whose durability and resistance to degradation contribute to growing environmental pollution and concerns. Enzymatic PET degradation, particularly via PETase from Ideonella sakaiensis, has emerged as a sustainable approach due to its ability to depolymerize PET under mild conditions. While research has largely focused on enhancing the enzymes thermal stability through distal mutations, less attention has been given to active-site engineering aimed at directly improving catalytic efficiency. Here, we used an automated in silico protein engineering platform called Gene Discovery and Enzyme Engineering (GDEE), designed to systematically explore mutations at the active site. By leveraging FastPETase (FP) as scaffold, we perform a high throughput generation of thousands of variants, evaluated them via docking studies with a PET substrate analogue, and ranked candidates based on binding affinity and catalytic geometry. We identified S238Y as a key mutation that enhanced PET film degrading performance at 40 {degrees}C when inserted in two of the most active PETase variants reported to date: 2.2-fold increase in the FP scaffold and 3.4-increase in the ThermoStable-PETase (TSP) background. Compared to wild type PETase, FP S238Y showed a 14.8-fold increase in bulk activity, translating into 9.4-fold more TPA and 20-fold more MHET by UPLC, while TSP S238Y reached a 25.8-fold increase (14.4-fold more TPA and 42.6-fold more MHET). This mutation also enhanced catalytic efficiency and resistance to enzyme concentration inhibition, especially in the TSP scaffold. Molecular dynamics confirm position 238 as a relevant modulator of ligand stabilisation. These findings underscore the potential of targeted active-site engineering, combined with structure-guided prediction, to accelerate the development of efficient mesophilic biocatalysts for plastic waste remediation.

bioinformatics↗

bioRxiv2025

GDEE: A Structure-Based Platform for Gene Discovery and Enzyme Engineering

Enzymes are extensively utilized for catalyzing the production of value-added compounds in diverse sectors with significant economic impact. However, challenges such as poor protein expression, low catalytic activity, substrate/co-factor limitations, and toxicity of final products limit the efficiency of these biocatalysts. Protein engineering offers a solution by re-designing enzyme catalytic properties to enhance biosynthetic pathways. In this study, we present an automated platform for gene discovery and enzyme engineering, aimed at overcoming these bottlenecks. The GDEE platform finds and optimizes enzymes responsible for rate-limiting steps in biosynthetic pathways, with objectives ranging from improving catalytic efficiency to enabling novel transformations. The platform operates through four key steps: an initial sequence step that either sources natural enzyme sequences or generates mutant variants, followed by an atomistic protein structure prediction step, a docking step, and a binding energy evaluator, obtaining a set of variant enzyme sequences that are prioritized for experimental validation. Additionally, machine learning-based re-scoring of binding poses to improve binding affinities significantly enhances the accuracy of predicting the effect of mutations on binding, highlighting its potential in metabolic engineering applications.

bioinformatics↗

bioRxiv2021

New targets for drug design: Importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has triggered a global pandemic with devastating consequences for health-care and social-economic systems. Thus, the understanding of fundamental aspects of SARS-CoV-2 is of extreme importance. In this work, we have focused our attention on the viral ribonuclease (RNase) nsp14, since this protein was considered one of the most interferon antagonists from SARS-CoV-2, and affects viral replication. This RNase is a multifunctional protein that harbors two distinct activities, an N-terminal 3-to-5 exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both with critical roles in coronaviruses life cycle. Namely, SARS-CoV-2 nsp14 ExoN knockout mutants are non-viable, indicating nsp14 as a prominent target for the development of antiviral drugs. Nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein, which has a pleiotropic function during viral replication. In this study, we have performed the first biochemical characterization of the complex nsp14-nsp10 from SARS-CoV-2. Here we confirm the 3-5 exoribonuclease and MTase activities of nsp14 in this new Coronavirus, and the critical role of nsp10 in upregulating the nsp14 ExoN activity in vitro. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity. The nsp14 MTase activity also seems to be independent of the presence of nsp10 cofactor, contrarily to nsp14 ExoN. Until now, there is no available structure for the SARS-CoV-2 nsp14-nsp10 complex. As such, we have modelled the SARS-CoV-2 nsp14-nsp10 complex based on the 3D structure of the complex from SARS-CoV (PDB ID 5C8S). We also have managed to map key nsp10 residues involved in its interaction with nsp14, all of which are also shown to be essential for stimulation of the nsp14 ExoN activity. This reinforces the idea that a stable interaction between nsp10 and nsp14 is strictly required for the nsp14-mediated ExoN activity of SARS-CoV-2, as observed for SARS-CoV. We have studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function contrasting to the functionality of these conserved catalytic residues in SARS-CoV, and in the Middle East respiratory syndrome coronavirus (MERS). The differences here revealed can have important implications regarding the specific pathogenesis of SARS-CoV-2. The nsp10-nsp14 interface is a recognized attractive target for antivirals against SARS-CoV-2 and other coronaviruses. This work has unravelled a basis for discovering inhibitors targeting the specific amino acids here reported, in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

microbiology↗