Patricia T. Borges

Galactose oxidases (GalOxs) are copper radical oxidases that catalyze the selective oxidation of primary alcohols and constitute attractive platforms for carbohydrate biocatalysis. Although fungal AA5_2 enzymes have been extensively characterized, bacterial representatives remain unexplored, and the contribution of appended carbohydrate-binding modules (CBMs) to redox enzyme function is poorly understood. Here, we report the first biochemical and structural characterization of a bacterial galactose oxidase, Ps GalOx from Pseudarthrobacter siccitolerans , revealing a previously unexplored bacterial architectural solution within the AA5_2 family. While the catalytic Cu(II)–tyrosyl radical center is conserved, Ps GalOx displays a divergent modular architecture comprising two tandem N-terminal CBM32 domains. Functional analyses reveal pronounced asymmetry between these duplicated modules: one enhances galactan binding but compromises solubility, whereas the second primarily contributes to structural stabilization. Removal of the N-terminal CBM markedly increases soluble expression without affecting intrinsic catalytic parameters toward D-galactose, demonstrating that enzyme performance can be tuned independently of the conserved catalytic core. Consistently, adaptive engineering trajectories preferentially target domain-scale rearrangements within the CBM region, indicating that selection initially acts on modular architecture to improve physicochemical properties such as solubility, rather than directly enhancing catalytic turnover. These findings expand the structural landscape of AA5_2 oxidases and establish domain architecture as a primary determinant of solubility, substrate recognition, and catalytic output in modular copper radical oxidases.

Shotgun metagenomic sequencing has emerged as a powerful tool for exploring microbial diversity and uncovering genes encoding novel biocatalysts from complex environments. Here, we report the discovery and characterization of a new FAD-dependent D-lactate dehydrogenase (PdG-D-LDH) from the gut microbiome of the isopod Porcellio dilatatus . The enzyme was identified through in silico screening using BLAST and AlphaFold3 and functionally characterized as a homodimeric, thermoactive, and thermostable protein, demonstrating the robustness required for biotechnological applications. PdG-D-LDH exhibits a strong catalytic preference toward D-lactate and preferentially reduces quinones over cytochrome c or molecular oxygen. X-ray crystallography revealed a VAO/PCMH-like fold with a solvent-accessible active site that harbors both a FAD cofactor and an Fe(II) ion. Molecular docking studies provided insights into the structural determinants of its stereoselective substrate recognition. Under mild conditions, the enzyme catalyzed the oxidation of D-lactate to pyruvate with a 90% yield after 24 h of reaction, using molecular oxygen as the electron acceptor. IMPORTANCE: This study illustrates how metagenomics, structural biology, and computational tools can jointly drive the discovery of new enzymes with valuable biotechnological applications aligned with circular economic principles. The newly identified D-lactate dehydrogenase, PdG-D-LDH, exhibits thermostability, stereoselectivity, and high catalytic efficiency, providing new insights into the structure-function relationships of lactate-metabolizing enzymes.

Stabilizing industrial enzymes is crucial for advancing environmentally responsible bioprocesses; however, the structural basis of thermostability remains incompletely understood. Here, we engineered thermostable variants of a tetrameric dye-decolorizing peroxidase (DyP) using two independent open-source design algorithms, yielding enzymes with significantly improved thermal performance and prolonged activity at elevated temperatures. Subsequent recombination strategies minimize the mutational burden while maintaining or enhancing stability. Structural and dynamic analyses of the thermostable variants revealed convergent features, including increased compactness, rigidity, and an enriched network of hydrogen bonds and hydrophobic interactions. Despite differing mutation profiles, stabilizing substitutions clustered in similar structural regions. Notably, the integration of dynamic modeling with protein correlation network analysis uncovered a previously unrecognized fingerprint of stabilization: highly connected structural networks characterized by denser and more persistent intra- and intermonomer interactions, greater internal cohesion, and enhanced cooperative dynamics. Tetramers exhibit long-range communication pathways and redundant routes, supporting coordinated motions that can hinder local unfolding and tetramer dissociation. These findings identify dynamic interaction networks as hypothetical new indicators of protein stability and offer a previously unexplored framework for rational enzyme design.

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O₂ to H₂O₂. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsG3Ox oxidizes at 50,000-fold higher specificity (k_cat/K_m) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsG3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities.

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme’s overall stability by 2 kcal mol⁻¹, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

Flavodiiron proteins have emerged in the last two decades as a newly discovered family of oxygen and/or nitric oxide reductases widespread in the three life domains, and present in both aerobic and anaerobic organisms. Herein we present the main features of these fascinating enzymes, with a particular emphasis on the metal sites, as more appropriate for this special issue in memory of the exceptional bioinorganic scientist R. J. P. Williams who pioneered the notion of (metal) element availability-driven evolution. We also compare the flavodiiron proteins with the other oxygen and nitric oxide reductases known until now, highlighting how throughout evolution Nature arrived at different solutions for similar functions, in some cases adding extra features, such as energy conservation. These enzymes are an example of the (bioinorganic) unpredictable diversity of the living world.