Esteban Lopez-Tavera

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds because of their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have been reported in prokaryotic organisms. Here, we describe the discovery of a diverse family of bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial proteins, termed bacterial UPOs (BUPOs), display clear structural homology to family I (short) fungal UPOs despite low sequence identity (HydBUPO) in its native host (Hydrogenophaga sp. A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in Escherichia coli. In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, as well as two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol, and 16-hydroxypalmitic acid, using hydrogen peroxide as a cosubstrate. These enzymes thus represent a previously unknown group of bacterial heme-thiolate peroxygenases that share key structural and functional features with eukaryotic UPOs, offering potential candidates for the development of industrially relevant biocatalysts.

Fast and sensitive analytical methods are the key to efficient screening of plastic-degrading enzymes. Here, we present a streamlined and affordable approach to assess the enzymatic deconstruction of insoluble synthetic polymers by blending them with a fluorescent dye, rhodamine 6G, and we evaluate this screening method using poly­(ethylene terephthalate) (PET) as a model material. Our results indicate that enzymatic depolymerization of the rhodamine-doped PET can be observed in a high-throughput fashion by following release of the fluorophore. The fluorescence data obtained during the hydrolysis of rhodamine-doped PET by 14 PET hydrolases, produced with a robotic platform, correlated with the quantitative chromatographic analysis of PET degradation products. Remarkably, the use of the rhodamine-loaded PET substrate resulted in negligibly low background signals even when detecting PETase activity in crude cell lysates, suggesting suitability for screening of a wide variety of samples. Encouraged by these results, we next produced a selection of polyethylene- and nylon-based materials loaded with rhodamine 6G. While rapid leaching of fluorophore observed with nylon substrates limits the utility of the method for detecting nylonase activity, the rhodamine-loaded polyethylene showed promising performance in passive diffusion tests, indicating that this latter substrate may be used to screen for polyolefin-degrading enzymes.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Like other redox enzymes, LPMOs face challenges in handling reactive oxygen species (ROS) generated at their active site, which must be controlled to prevent damaging off-pathway reactions. In the case of copper catalysts, oxidative damage can become self-reinforcing, as released copper from damaged catalytic centers promotes additional ROS formation via abiotic redox cycling. Here we show that a subgroup of carbohydrate-binding modules (CBM2s), exclusively tethered to cellulose-active LPMOs, has evolved the ability to bind copper. The copper site, found on the opposite side of the CBM from the cellulose-binding surface, confers redox protection through two distinct mechanisms: preferential binding of free Cu(I) and direct interaction with the reduced catalytic copper site of the LPMO. Together, these mechanisms prevent off-pathway reactions in the absence of substrate. The protective role of these copper-binding CBM2s is demonstrated by thorough characterization of the kinetics and redox stability of a series of engineered LPMO variants. Predicted models of CBM2-LPMO interactions revealed an interdomain copper-site resembling the Cu(B) site in particulate methane monooxygenase. Notably, this spatial arrangement enables substrate-dependent regulation of copper site reactivity: the CBM2-LPMO interactions that inhibit damaging redox chemistry in the absence of substrate are relieved upon cellulose binding. This work shows a road toward improved redox stability and tunable reactivity of copper catalysts.

Unspecific peroxygenases (UPOs, EC. 1.11.2.1) are promising biocatalysts for the oxyfunctionalization of organic molecules and the synthesis of industrially relevant compounds due to their vast repertoire of catalyzed reactions. To date, thousands of putative UPO genes have been identified in eukaryotic genomes, most of them in the Ascomycota and Basidiomycota phyla, and several UPOs have been characterized. Remarkably, no related enzymes have ever been reported in prokaryotic organisms. Here, we describe the discovery of a novel family of diverse bacterial heme-thiolate peroxygenases through structure database mining, followed by functional characterization of selected representatives. The bacterial UPO-like proteins (BUPOs) are structurally analogous to family I (“short”) fungal UPOs, despite having sequence similarity below 20%. Expression of one of these proteins ( Hyd BUPO) in its native host ( Hydrogenophaga sp. A37) was confirmed by proteomics. Several BUPOs were cloned and expressed in Escherichia coli . In biochemical assays, the BUPOs were able to catalyze one-electron oxidation (peroxidase activity) of ABTS and 2,6-dimethoxyphenol, and two-electron oxidation (peroxygenase activity) of naphthalene, indole, 3-phenyl-1-propanol and 16-hydroxypalmitic acid, using hydrogen peroxide as co-substrate. These enzymes thus represent a new family of bacterial heme-thiolate peroxygenases that share structural and functional features with eukaryotic UPOs, offering new potential candidates for developing industrially relevant biocatalysts.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that hydroxylate recalcitrant polysaccharides such as cellulose. Like other redox enzymes, LPMOs face challenges in handling the reactive oxygen species generated at their active site, which must be controlled to prevent off-pathway reactions that lead to enzyme inactivation. In the case of LPMOs, oxidative damage may be self-reinforcing because free copper released from damaged catalytic centers will promote abiotic redox reactions that generate reactive oxygen species. Here we show that some members of a widely spread family of carbohydrate-binding modules (CBM2s) have evolved the ability to bind copper and that this ability is exclusively found in CBM2s that are appended to LPMOs. We show that the copper site in these CBM2s protects the LPMO from inactivation both by scavenging free copper, preferably Cu(I), and by interacting directly with the reduced catalytic copper site of the LPMO, thus preventing the enzyme from engaging in off-pathway reactions. These effects are demonstrated by studies on the redox stability of a series of engineered LPMO variants as well as AlphaFold3 models of the CBM2-containing enzymes. Interestingly, the copper site and the cellulose-binding surface are located on different sides of these CBM2s, enabling a mode of action in which the CBM inhibits potentially damaging LPMO activity in the absence of substrate, while such inhibition would be relieved upon binding of the CBM2 to cellulose. These findings show that CBMs have biologically relevant functions beyond carbohydrate-binding and reveal a mechanism for substrate-dependent regulation of LPMO reactivity.

Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. Here, we attempt to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called “Ceres”) or a bacterial catalase-peroxidase from Klebsiella sp., respectively. Reproducing previously described experiments, we do not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted.