Dynamic loops towards an efficient biocatalysis
Oeiras, June 2020
The application of enzymes offers an enormous potential to provide cleaner and more sustainable industrial processes and the development of novel biomedical applications. They are particularly attractive to various biotechnological applications, from food and beverage industries to biomedical diagnosis, biosensors and biofuel cells. For this, multicopper oxidases, also known as laccases, are very suitable and versatile biocatalysts, and understanding their structural and biochemical properties is key. Now, ITQB NOVA researchers have unravelled unique structural aspects related to catalysis in these enzymes.
Lacquer is a natural coating material that is polymerized by laccases and that has been used as a protective and decorative coating material in traditional crafts for at least six thousand years in Japan and China. They were first described based on the observation of the rapid hardening of the latex (containing phenols and laccase) released from Japanese lacquer trees in the presence of air. Under the action of laccase, the substrates oxidize and polymerize coupled to the reduction of oxygen to water. However, the mechanism of electron transfer from substrates and the details of substrate binding sites in these enzymes remain to be fully elucidated. To fill in this gap, a team of researchers at ITQB NOVA have solved the structure of a bacterial laccase from Aquifex aeolicus as well as the molecular determinants that underlies their catalytic activity.
The study was published in ACS Catalysis and resulted from the collaboration between three ITQB NOVA labs: the Microbial and Enzyme Technology Lab, led by Lígia Martins, the Dynamic Structural Biology Lab, led by Tiago N. Cordeiro, and the Structural Biology Lab, led by Carlos Frazão. This in-house synergy allowed unprecedented access to laccases’ structural aspects related to catalysis.
The mutagenesis studies were performed by Vânia Brissos, in Lígia Martins lab, the X-ray diffraction was performed by Patrícia Borges and Carlos Frazão, and Small-angle X-ray scattering (SAXS)/Rosetta simulations were carried out by Guillem Hernandez and Tiago Cordeiro. Molecular dynamic simulations, done by Zymvol Biomodeling, were also part of this research.
The combination of SAXS, Rosetta, and molecular dynamics simulations probed the conformational landscape of a long Met-loop – a hallmark of these enzymes –, invisible in the crystal structure close to the catalytic centre. “Accurate prediction of loop structures remains challenging, especially for long segments with sizable conformational space. To the best of our knowledge, this is the first study on the conformational landscape of a low complexity Met-rich region, with a role in catalysis”, says Tiago N. Cordeiro.
Loop opening and closure is known to be essential to hang and stabilize substrates, setting up the active site for efficient catalysis. These results indicate that the Met-loop and its dynamics display key regulatory function, capable of controlling the access of substrates and determining the chemical environment of the enzyme’s activity. “This has fundamental implications in identifying elements with a role in discerning substrate specificity and catalytic rates among multicopper oxidases. These motifs are also important targets of engineering for the development of industrial and biomedical applications", highlights Lígia O. Martins.
Furthermore, the structural characterization underlies McoA thermostability since the crystal structure has the most tightly compact and hydrophobic core among its counterparts of other origins. “The studies of hyperthermophilic laccases are worthwhile exploring to improve the enzyme selectivity and design new robust hyper-thermostable enzymes for suitable biotechnological applications”, adds Carlos Frazão.
The ITQB NOVA researchers will continue to pursue this path using high-resolution structural studies with nuclear magnetic resonance, combined with molecular dynamics simulations, to provide new insights into Met-rich elements dynamic conformational landscape and substrate binding.
The work was performed within the frame of FCT funded project FitZymes (PTDC/BII-BBF/29564/2017) and the B-LigZymes project from the European Union’s Horizon 2020 Research and Innovation Programme, both coordinated by Lígia Martins and developed in partnership with the teams led by Carlos Frazão and Tiago Cordeiro, from ITQB NOVA, and Emanuele Monza and Maria Fátima Lucas, at Zymvol Biomodeling in Barcelona.
ITQB NOVA researchers - Lígia Martins, Vânia Brissos, Carlos Frazão, Patrícia Borges, Tiago Cordeiro and Guillem Hernandez.
ACS Catalysis | doi.org/10.1021/acscatal.0c01623
Methionine-Rich Loop of Multicopper Oxidase McoA Follows Open-to-Close Transitions with a Role in Enzyme Catalysis
Patrícia T. Borges, Vânia Brissos, Guillem Hernandez, Laura Masgrau, Maria Fátima Lucas, Emanuele Monza, Carlos Frazão*, Tiago N. Cordeiro*, and Lígia O. Martins*
