Browsing by Author "Guixe, Victoria"

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    An Evolutionary Marker of the Ribokinase Superfamily Is Responsible for Zinc-Mediated Regulation of Human Pyridoxal Kinase
    (2020) Ramirez-Sarmiento, Cesar A.; Engelberger, Felipe; Guixe, Victoria
    The ribokinase superfamily catalyzes the phosphorylation of a vast diversity of substrates, and its members are characterized by the conservation of a common structural fold along with highly conserved sequence motifs responsible for phosphoryl transfer (GXGD) and stabilization of the metal-nucleotide complex (NXXE). Recently, a third motif (HXE) exclusive from ADP-dependent enzymes was identified, with its glutamic acid participating in water-mediated interactions with the metal-nucleotide complex and in stabilization of the ternary complex during catalysis. In this work, we bioinformatically determine that the aspartic acid of another motif (DPV), exclusively found in hydroxyethyl thiazole (THZK), hydroxymethyl pyrimidine (HMPK) and pyridoxal kinases (PLK), is structurally equivalent to the acidic residue in the HXE motif. Moreover, this residue is highly conserved among all ribokinase superfamily members. To determine whether the functional role of the DPV motif is similar to the HXE motif, we employed molecular dynamics simulations using crystal structures of phosphoryl donor substrate-complexed THZK and PLK, showing that its aspartic acid participated in water-mediated or direct interactions with the divalent metal of the metal-nucleotide complex. Lastly, enzyme kinetic assays on human PLK, an enzyme that utilizes zinc, showed that site-directed mutagenesis of the aspartic acid from the DPV motif abolishes the inhibition of this enzyme by increasing free zinc concentrations. Altogether, our results highlight that the DPV and HXE motifs are evolutionary markers of the functional and structural divergence of the ribokinase superfamily and evidence the role of the DPV motif in the interaction with both free and nucleotide-complexed divalent metals in the binding site of these enzymes.
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    Antarctic Polyester Hydrolases Degrade Aliphatic and Aromatic Polyesters at Moderate Temperatures
    (2022) Blazquez-Sanchez, Paula; Engelberger, Felipe; Cifuentes-Anticevic, Jeronimo; Sonnendecker, Christian; Grinen, Aransa; Reyes, Javiera; Diez, Beatriz; Guixe, Victoria; Richter, P. Konstantin; Zimmermann, Wolfgang; Ramirez-Sarmiento, Cesar A.
    Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in the packaging industry, and consequently has become one of the main components of plastic waste found in the environment. However, several microorganisms have been described to encode enzymes that catalyze the depolymerization of PET. While most known PET hydrolases are thermophilic and require reaction temperatures between 60 degrees C and 70 degrees C for an efficient hydrolysis of PET, a partial hydrolysis of amorphous PET at lower temperatures by the polyester hydrolase IsPETase from the mesophilic bacterium Ideonella sakaiensis has also been reported. We show that polyester hydrolases from the Antarctic bacteria Moraxella sp. strain TA144 (Mors1) and Oleispira antarctica RB-8 (OaCut) were able to hydrolyze the aliphatic polyester polycaprolactone as well as the aromatic polyester PET at a reaction temperature of 25 degrees C. Mors1 caused a weight loss of amorphous PET films and thus constitutes a PET-degrading psychrophilic enzyme. Comparative modeling of Mors1 showed that the amino acid composition of its active site resembled both thermophilic and mesophilic PET hydrolases. Lastly, bioinformatic analysis of Antarctic metagenomic samples demonstrated that members of the Moraxellaceae family carry candidate genes coding for further potential psychrophilic PET hydrolases.
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    Structural insights into a functional unit from an immunogenic mollusk hemocyanin
    (2024) Munoz, Sebastian M.; Vallejos-Baccelliere, Gabriel; Manubens, Augusto; Salazar, Michelle L.; Nascimento, Andrey F. Z.; Tapia-Reyes, Patricio; Meneses, Claudio; Ambrosio, Andre L. B.; Becker, Maria Ines; Guixe, Victoria; Castro-Fernandez, Victor
    Mollusk hemocyanins, among the largest known proteins, are used as immunostimulants in biomedical and clinical applications. The hemocyanin of the Chilean gastropod Concholepas concholepas (CCH) exhibits unique properties, which makes it safe and effective for human immunotherapy, as observed in animal models of bladder cancer and melanoma, and dendritical cell vaccine trials. Despite its potential, the structure and amino acid sequence of CCH remain unknown. This study reports two sequence fragments of CCH, representing three complete functional units (FUs). We also determined the high -resolution (1.5 A & ring; ) X-ray crystal structure of an "FU -g type"from the CCHB subunit. This structure enables in-depth analysis of chemical interactions at the copper -binding center and unveils an unusual, truncated N-glycosylation pattern. These features are linked to eliciting more robust immunological responses in animals, offering insights into CCH's enhanced immunostimulatory properties and opening new avenues for its potential applications in biomedical research and therapies.