xtal

Duarte, M, Carvalho AL, Ferreira MC, Caires B, Romão MJ, Prates JAM, Najmudin S, Bayer EA, Fontes CMGA, Bule P.  2025.  Tripartite binding mode of cohesin-dockerin complexes from Ruminococcus flavefaciens involving naturally truncated dockerins, 2025. 301(7):110325. AbstractWebsite

Polysaccharides in plant cell walls serve as a rich carbon and energy source, yet their structural complexity presents a barrier to efficient degradation. To address this, anaerobic microorganisms like R. flavefaciens have developed sophisticated multi-enzyme complexes known as cellulosomes, which enable the efficient breakdown of these recalcitrant polysaccharides. These complexes are assembled through high-affinity interactions between cohesin (Coh) modules in scaffoldin proteins and dockerin (Doc) modules in cellulosomal enzymes. R. flavefaciens FD-1 harbors one of the most intricate cellulosomes described to date, comprising over 200 Doc-containing proteins encoded in its genome. Despite substantial research on this cellulosome, the role of a group of truncated but functional dockerins, known as group-2 Docs, remains unclear. In this study, we present a detailed structural and binding analysis of a Coh-Doc complex involving the cohesin from the cell-anchoring scaffoldin ScaE and a group-2 Doc that bears only one of the two Ca+2-coordinating loops that characterise the canonical Docs. Our findings reveal a novel tripartite binding mechanism, in which the cohesin can simultaneously bind two distinct dockerin units in three alternative conformations. This discovery provides new insights into the modular versatility of the R. flavefaciens cellulosome and sheds light on the mechanisms that enhance its efficiency in polysaccharide degradation.

XTAL at the 11th ENURS meeting

Past June 6th, XTAL members Maria João Romão, Ana Luísa Carvalho, Cristiano Mota, Guilherme Alves, Fernando Ribeiro, Carolina Videira, Guilherme Oliveira and Marco Graça attended the 11th ENURS meeting (https://www.itqb.unl.pt/meetings-and-courses/enurs), at ITQB-NOVA, Oeiras, Portugal.
MSc student Carolina Videira was selected for Oral Comunication, entitled " Structural insights into the catalytic reduction site of human aldehyde oxidase".

XTAL at the 11th ENURS meeting

Past June 6th, XTAL members Maria João Romão, Ana Luísa Carvalho, Cristiano Mota, Guilherme Alves, Fernando Ribeiro, Carolina Videira, Guilherme Oliveira and Marco Graça attended the 11th ENURS meeting (https://www.itqb.unl.pt/meetings-and-courses/enurs), at ITQB-NOVA, Oeiras, Portugal.
MSc student Carolina Videira was selected for Oral Comunication, entitled " Structural insights into the catalytic reduction site of human aldehyde oxidase".

Caseiro, C, McGregor NGS, Alves VD, Carvalho AL, Romão MJ, Davies GJ, Fontes CMGA, Bule P.  2024.  Family GH157 enzyme exhibits broad linkage tolerance and a dual endo/exo- β -glucanase activity on β-glucans, 2024. :137402. AbstractWebsite

The structural and chemical diversity of β-glucans is reflected on the variety of essential biological roles tackled by these polysaccharides. This natural heterogeneity requires an elaborate assortment of enzymatic mechanisms to assemble, degrade or modify, as well as to extract their full biotechnological potential. Recent metagenomic efforts have provided an unprecedented growth in potential new biocatalysts, most of which remain unconfirmed or uncharacterized. Here we report the first biochemical and structural characterization of two bacterial β-glucanases from the recently created glycoside hydrolase family 157 (LaGH157 and BcGH157) and investigate their molecular basis for substrate hydrolysis. Structural analysis by X-ray crystallography revealed that GH157 enzymes belong to clan GH-A, possessing a (β/α)8-barrel fold catalytic domain, two β-sandwich accessory domains and two conserved catalytic glutamates residues, with relative positions compatible with a retaining mechanism of hydrolysis. Specificity screening and enzyme kinetics suggest that the enzymes prefer mixed-linkage glucans over β-1,3-glucans. Activity screening showed that both enzymes exhibit pH optimum at 6.5 and temperature optimum for LaGH157 and BcGH157 at 25 °C and 48 °C, respectively. Product analysis with HPAEC-PAD and LC-MS revealed that both enzymes are endo-1,3(4)-β-glucanases, capable of cleaving β-1,3 and β-1,4-linked glucoses, when preceded by a β-1,3 linkage. Moreover, BcGH157 needs a minimum of 4 subsites occupied for hydrolysis to occur, while LaGH157 only requires 3 subsites. Additionally, LaGH157 possesses exohydrolytic activity on β-1,3 and branching β-1,6 linkages. This unusual bifunctional endo-1,3(4)/exo-1,3–1,6 activity constitutes an expansion on our understanding of β-glucan deconstruction, with the potential to inspire future applications.

Guilherme Vilela-Alves presented his PhD Thesis Plan

Today, our PhD student Guilherme Vilela-Alves, supervised by Cristiano Mota and Maria João Romão, from XTAL, and Harmut Luecke, from the CryoEM group at NOVA FCT, has publicly presented his PhD Thesis Plan. The whole group is proud of you, Guilherme! Great work and stimulating discussion!

The XTAL is back to the European Researchers' Night!

This year's activity was titled: Studying proteins in 3D can help reduce atmospheric CO2

Ana Luísa Carvalho and Cristiano Mota represented the XTAL at ECM34 in Padova

The 34th European Crystallographic Meeting (ECM34,
Oliveira, AR, Mota C, Vilela-Alves G, Manuel RR, Pedrosa N, Fourmond V, Klymanska K, Léger C, Guigliarelli B, Romão MJ, Cardoso Pereira IA.  2024.  An allosteric redox switch involved in oxygen protection in a CO2 reductase, 2024. Nat Chem Biol. 20(1):111-119. AbstractWebsite

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

Vilela-Alves, G, Manuel RR, Viegas A, Carpentier P, Biaso F, Guigliarelli B, Pereira IC, Romão MJ, Mota C.  2024.  Substrate-dependent oxidative inactivation of a W-dependent formate dehydrogenase involving selenocysteine displacement, 2024. Chemical Science. :-.: The Royal Society of Chemistry AbstractWebsite

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards innovative strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be better understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the oxygen sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a different species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyse the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate.

Ribeiro, DO, Bonnardel F, Palma AS, Carvalho ALM, Perez S.  2024.  CBMcarb-DB: interface of the three-dimensional landscape of carbohydrate-binding modules, 2024/06/26. Carbohydrate Chemistry: Chemical and Biological Approaches Volume 46. 46(Pilar Rauter, Amélia, Queneau, Yves, Palma, Angelina Sá, Eds.).: Royal Society of Chemistry Abstract

Carbohydrate-binding-modules (CBMs) are discrete auxiliary protein modules with a non-catalytic carbohydrate-binding function and that exhibit a great diversity of binding specificities. CBMcarb-DB is a curated database that classifies the three-dimensional structures of CBM–carbohydrate complexes determined by single-crystal X-ray diffraction methods and solution NMR spectroscopy. We designed the database architecture and the navigation tools to query the database with the Protein Data Bank (PDB), UniProtKB, and GlyTouCan (universal glycan repository) identifiers. Special attention was devoted to describing the bound glycans using simple graphical representation and numerical format for cross-referencing to other glycosciences and functional data databases. CBMcarb-DB provides detailed information on CBMs and their bound oligosaccharides and features their interactions using several open-access applications. We also describe how the curated information provided by CBMcarb-DB can be integrated with AI algorithms of 3D structure prediction, facilitating structure–function studies. Also in this chapter, we discuss the exciting convergence of CBMcarb-DB with the glycan array repository, which serves as a valuable resource for investigating the specific binding interactions between glycans and various biomolecular targets. The interaction of the two fields represents a significant milestone in glycosciences. CBMcarb-DB is freely available at https://cbmdb.glycopedia.eu/ and https://cbmcarb.webhost.fct.unl.pt.

Filipa Trovão at the UCIBIO BioSeminars!

Today, the star of the UCIBIO BioSeminars was our most recent PhD, Filipa Trovão, shining bright in her presentation en

Filipa Engrola has presented the PhD Thesis Plan

Today, our PhD student Filipa Engrola, supervised by Teresa Santos Silva, from XTAL, and Aldino Viegas, from

Such a great achievement, Filipa Engrola!

Congratulations to Filipa Engrola for the best flash presentation award at the 4th Meeting of Portuguese Young Biophysicists (
Vilela-Alves, G, Manuel RR, Pedrosa N, Cardoso Pereira IA, Romão MJ, Mota C.  2024.  {Structural and biochemical characterization of the M405S variant of ıt Desulfovibrio vulgaris} formate dehydrogenase}, May. Acta Crystallographica Section F. 80:98–106., Number 5 AbstractWebsite

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO${\sb 2}$ to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of ıt Desulfovibrio vulgaris} formate dehydrogenase AB (ıt Dv}FdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine S${\sp {$δ$}}$ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO${\sb 2}$ reduction, together with an increased sensitivity to oxygen inactivation.

PhD student Guilherme Alves was selected for oral presentation at the Instruct-ERIC biennial meeting

Today, at the Instruct-ERIC biennial meeting, in Cascais, our PhD student Guilherme Alves presented his research work entitled "Unveiling O2 damage on Mo/W Formate dehydrogenases and their innate protection mechanism", supervised by Cristiano Mota and Maria João Romão.
Congratulations to the full team!

loading