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Evolving towards a greener and better tomorrow

A team led by ITQB NOVA scientists identified the mutations that improve the efficiency of an important catalyst in the green industry

Evolution is a natural process about the survival of the fittest. What if we could guide it? Could we create organisms with improved functions that will help us move towards a greener and more sustainable future? That is exactly what a team of scientists from ITQB NOVA and Zymvol Biomodeling (a biotech company), together with Universitat Autònoma de Barcelona, were able to achieve in a paper published in ACS Catalysis. They describe the molecular strategies behind the evolution of an enzyme from using inorganic metal ions to an organic substrate. This type of enzymes are widely used in the production of biofuels and in the treatment of water and soils.

Multicopper oxidases (MCOs) are enzymes present in all domains of life. The catalytic sites of these enzymes have copper and display a wide range of substrates and catalytic rates. They are divided into two classes – laccases and metallo-oxidases – that are still difficult to distinguish. Laccases are enzymes that oxidize several compounds and have massive potential for biotechnological applications in green chemistry, bioremediation, and biorefinery fields. Conversely, metallo-oxidases exhibit high activity for transient metals such as copper, iron, and manganese, and are important in cellular metal homeostasis systems (control of the levels of metal).

A previous paper published by the team reported the optimization of a specific MCO for a typical laccase substrate, ABTS (2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid), through several rounds of evolution (insertion of mutations at different stages). In this study, they used a combination of mutagenesis with structural, kinetic, and in silico analysis to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase into a laccase, which might have great potential in more sustainable industrial processes.

The mutations introduced during evolutionary engineering lead to conformational changes in the catalytic site of the enzyme, towards states more suitable for recognizing and stabilizing ABTS. They showed that most mutations are far away from the active site. These mutations act as small allosteric molecules, given that they induce changes in the motion and pathways between the residues, optimizing the distances to the active site, resulting in a more focused and dynamic relationship with the substrate. Additionally, in alignment with previous findings, they demonstrated that early mutations in evolution played a critical role by generating or enhancing the positive role of subsequent mutations.

From left to right: Tiago Cordeiro, Lígia O. Martins, Vânia Brissos, Patrícia Borges and Carlos Frazão

“This undertaking contributed to advancing our knowledge on the features and mechanisms behind the different substrate specificity in MCOs, understanding evolutionary dynamics, and fostering the design of new proteins”, states Lígia O. Martins, head of the Microbial and Enzyme Technology lab, and leader of the team.

According to Vânia Brissos, from the same lab, “biocatalysis is a vital tool for establishing future circular bio-economies, and its application has constantly been increasing in a range of industries”. “Understanding how to enhance or make flexible sites more rigid, opens new opportunities for protein engineering, which are fundamental to biocatalysis techniques”, adds her colleague Patrícia Borges.

“We showed that these mutations scattered across the enzyme collectively improve the catalysis to this particular substrate”, says Carlos Frazão, head of the Structural Biology Lab.

Tiago Cordeiro, PI of the Dynamic Structural Biology lab, concludes “this spots the importance of experimental and computational analysis in providing insights into the changes involved in the evolution of enzyme specificity.”

 

Original Paper

ACS Catalysis: https://doi.org/10.1021/acscatal.2c00336

Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase

Vânia Brissos, Patrícia T. Borges, Reyes Núñez-Franco, Maria Fátima Lucas, Carlos Frazão, Emanuele Monza, Laura Masgrau, Tiago N. Cordeiro, and Lígia O. Martins

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