Anton A. Stepnov

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.

Bacterial two-domain multicopper oxidases (2dMCOs) represent a structurally distinct class of trimeric multicopper oxidases. Differing considerably from well-characterized monomeric three-domain laccases, type B 2dMCOs have a T1 active site positioned in a tunnel in the trimer center. The biochemical properties and roles in lignin conversion of 2dMCOs remain poorly understood. Here, we present a comprehensive biochemical characterization of a type B 2dMCO from Cellvibrio japonicus (CjMCO) and discuss links between its structural organization and activity. The T1 copper of CjMCO had a redox potential of 537 mV, but the turnover number (0.4 s^-1) was ∼1000-fold lower than high-turnover fungal laccases. Stopped-flow UV-vis spectroscopy indicated that this low turnover likely reflects slow reoxidation of the enzyme by O₂, which has not been previously reported for laccases. Despite the steric constraints imposed by its trimeric structure, CjMCO oxidized the lignin model compound guaiacylglycerol-β-guaiacyl ether, resulting in both oxidative coupling and bond cleavage, and CjMCO was able to act on oligomeric birch organosolv lignin, promoting net oxidative polymerization. Interestingly, 2,6-dimethoxyphenol oxidation kinetics and the product profile for guaiacylglycerol-β-guaiacyl ether oxidation by CjMCO were influenced by pH, buffer composition, and ionic strength, suggesting a potential strategy for tailoring product profiles. Together, these findings demonstrated that CjMCO functions as a laccase and oxidizes phenolic lignin moieties, but its slow rates and trimeric architecture indicate that it is unlikely to efficiently degrade lignin polymers in vivo. This study expands the current understanding of bacterial laccase diversity and provides a foundation for exploring other physiological roles of type B 2dMCOs beyond lignin degradation.

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.

Plastics are widely used materials, yet their chemical stability hinders biodegradation, exacerbating pollution on a global scale. Soils contaminated with plastic may foster microbes adapted to degrade plastics or plastic derivatives, and these organisms and their enzymes offer promising avenues for the development of biotechnological recycling strategies. Here, two microbial communities originating from soil collected at a plastic-contaminated landfill in Norway were enriched to select for bacteria involved in the decomposition of a commonly used, model polyethylene (PE; weight average molecular weight (M _w ) ∼4000 g/mol). We leveraged genome-resolved metatranscriptomics to identify active population affiliated with Acinetobacter guillouiae and Pseudomonas sp., showing a suite of upregulated genes (including those encoding alkane 1-monooxygenases, flavin-containing monooxygenases FMOs, cytochrome P450 monooxygenases) with functions compatible with degradation of oxidized products as well as medium- and long-chain hydrocarbons. Strikingly, spectroscopic, spectrometric and chromatographic analyses revealed the unexpected presence of medium- and long-chain alkanes and 2-ketones in the model PE substrate, preventing the erroneous conclusion that the community was interacting with the polymeric component. Consistently, only alkanes and 2-ketones with chain length of 10-35 were selectively degraded by an A. guillouiae isolate, as confirmed by proteomics analyses and substrate characterization following bacterial growth. Besides extending the knowledge on the enzymatic basis for degradation of PE-derivatives in soil-associated microbial systems, our results provide an advanced compositional characterization of a widely used model “PE” material, while offering valuable insight to support future studies aimed at unequivocally identifying organisms and their enzymes implicated in PE transformation.

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.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are mono‐copper polysaccharide‐degrading peroxygenases that engage in several on‐ and off‐pathway redox reactions involving O₂ and H₂O₂. Herein, we show that the known metalloenzyme inhibitor cyanide inhibits reductive activation of LPMOs by binding to the LPMO‐Cu(II) state and that the degree of inhibition depends on the concentrations of the polysaccharide substrate, the reductant and H₂O₂. Importantly, this analysis revealed differences between fungal NcAA9C and bacterial SmAA10A, which have different secondary copper coordination spheres. These differences were also highlighted by the observation that phosphate, a commonly used buffer ion, strongly inhibits NcAA9C while not affecting reactions with SmAA10A. The results provide insight into LPMO inhibition and catalysis and highlight pitfalls in the analysis thereof.

Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose‐xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re‐evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose‐associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.