Biology subjects

Stepnov, A. A.

Publications and source records attributed to Stepnov, A. A..

5 recordsLinked to original sources

bioRxiv2025

Discovery and characterization of bacterial unspecific peroxygenase-like heme-thiolate enzymes

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 (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, 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.

biochemistry↗

bioRxiv2025

Microbial degradation of a widely used model polyethylene is restricted to medium- and long-chain alkanes and their oxidized derivatives

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 (Mw) [~]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.

microbiology↗

bioRxiv2025

Production and evaluation of fluorophore-doped polymer substrates to screen for plastic-degrading enzymes

Fast and sensitive analytical methods are the key to efficient screening of plastic-degrading enzymes. While liquid and gas chromatography with mass spectrometry detection offer great power, the cost of the equipment is high, and the methods may not be straightforward when studying crude environmental samples. Here, we present a streamlined and affordable approach to assess the enzymatic deconstruction of insoluble synthetic polymers by blending them with a fluorescent probe, 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 engineered 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.

biochemistry↗

bioRxiv2025

Discovery and characterization of a copper-binding carbohydrate-binding module (CBM) regulating the activity of lytic polysaccharide monooxygenases

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.

biochemistry↗

bioRxiv2024

Revisiting the activity of two poly(vinyl chloride)- and polyethylene-degrading enzymes

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. We attempted 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 with the recombinant proteins, we did 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, leading to the erroneous claim that enzymatic deconstruction of polyethylene and poly(vinyl chloride) had occurred. Our results should lead to caution when interpreting the growing amount of literature claiming enzymatic degradation of non-hydrolyzable plastics.

biochemistry↗