Biology subjects

Cordara, G.

Publications and source records attributed to Cordara, G..

4 recordsLinked to original sources

bioRxiv2023

Calcium binding site in AA10 LPMO from Vibrio cholerae suggests modulating effects during environment survival and infection

Despite major efforts towards its eradication, cholera remains a major health and economic burden in many developing countries. Between outbreaks, the bacterium responsible for the disease, Vibrio cholerae, survives in aquatic environmental reservoirs, where it commonly forms biofilms, e.g., on zooplankton. N-acetyl glucosamine binding protein A (GbpA) is an adhesin that binds to the chitinaceous surface of zooplankton and breaks its dense crystalline packing thanks to its lytic polysaccharide monooxygenase (LPMO) activity, which provides V. cholerae with nutrients. In addition, GbpA is an important colonization factor associated with bacterial pathogenicity, allowing the binding to mucins in the host intestine. Here, we report the discovery of a cation-binding site in proximity of the GbpA active site, which allows Ca2+, Mg2+ or K+ to bind close to its carbohydrate-binding surface. In addition to the X-ray crystal structures, we explored how the presence of ions affects the stability of the protein, compared the new GbpA LPMO structures to those of other LPMOs, and discussed the relevance of our discovery for bacterial survival. Calcium ions, abundant in natural sources of chitin, have been found to have the strongest effect on GbpA stability. Our findings suggest a V. cholerae-specific cation-binding site in GbpA that may fine-tune activity and binding to the different substrates during environmental survival and host infection.

biochemistry↗

bioRxiv2023

Using Vibrio natriegens for high-yield production of challenging expression targets and for protein deuteration

Production of soluble proteins is essential for structure/function studies, however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens appeared as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (VmaxTM X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted, and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein deuteration using deuterated minimal media with deuterium oxide as solvent, and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news since isotopic labeling is expensive and often ineffective, but represents a necessary prerequisite for some structural techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein deuteration in amounts suitable for structural biology studies.

biochemistry↗

bioRxiv2023

Novel exported bifunctional fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: shikimate pathway enzymes teamed up in no man's land

Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of phenylalanine. Surprisingly, exported CMs and CDTs exist in bacterial pathogens. Here, we report the discovery of novel and extremely rare exported bifunctional fusion enzymes, consisting of fused CM and CDT domains. Such enzymes were found in only nine bacterial species belonging to non-pathogenic {gamma}- or {beta}-proteobacteria. In {gamma}-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas in {beta}-proteobacteria the order is inversed. The CM domains share 15-20% sequence identity with the AroQ{gamma} class CM holotype of Mycobacterium tuberculosis (*MtCM), and the CDT domains 40-60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 {micro}M, much lower than for *MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathways end product Phe, and no catalytic benefit of the domain fusion compared to engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 [A] resolution, respectively. Neither the crystal structures nor size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.

biochemistry↗

bioRxiv2022

What drives chorismate mutase to top performance? Insights from a combined in silico and in vitro study

Unlike typical chorismate mutases, the enzyme from Mycobacterium tuberculosis (MtCM) has only low activity on its own. Remarkably, its catalytic efficiency kcat/Km can be boosted more than 100-fold by complex formation with a partner enzyme. Recently, an autonomously fully active MtCM variant was generated using directed evolution, and its structure solved by X-ray crystallography. However, key residues were involved in crystal contacts, challenging the functional interpretation of the structural changes. Here, we address these challenges by microsecond molecular dynamics simulations, followed up by additional kinetic and structural analyses of selected sets of specifically engineered enzyme variants. A comparison of wild-type MtCM with naturally and artificially activated MtCMs revealed the overall dynamic profiles of these enzymes as well as key interactions between the C-terminus and the active site loop. In the artificially evolved variant of this model enzyme, this loop is pre-organized and stabilized by Pro52 and Asp55, two highly conserved residues in typical, highly active chorismate mutases. Asp55 stretches across the active site and helps to appropriately position active site residues Arg18 and Arg46 for catalysis. The role of Asp55 can be taken over by another acidic residue, if introduced at position 88 close to the C-terminus of MtCM, as suggested by MD simulations and confirmed by kinetic investigations of engineered variants.

biochemistry↗