BsdA(Bsd2) -dependent vacuolar turnover of a misfolded version of the UapA transporter along the secretory pathway: prominent role of selective autophagy.

Transmembrane proteins translocate cotranslationally in the endoplasmic reticulum (ER) membrane and traffic as vesicular cargoes, via the Golgi, in their final membrane destination. Misfolding in the ER leads to protein degradation basically through the ERAD/proteasome system. Here, we use a mutant version of the purine transporter UapA (ΔR481) to show that specific misfolded versions of plasma membrane cargoes undergo vacuolar turnover prior to localization in the plasma membrane. We show that non-endocytic vacuolar turnover of ΔR481 is dependent on BsdA(Bsd2) , an ER transmembrane adaptor of HulA(Rsp5) ubiquitin ligase. We obtain in vivo evidence that BsdA(Bsd2) interacts with HulA(Rsp5) and ΔR481, primarily in the ER. Importantly, accumulation of ΔR481 in the ER triggers delivery of the selective autophagy marker Atg8 in vacuoles along with ΔR481. Genetic block of autophagy (atg9Δ, rabO(ts) ) reduces, but does not abolish, sorting of ΔR481 in the vacuoles, suggesting that a fraction of the misfolded transporter might be redirected for vacuolar degradation via the Golgi. Our results support that multiple routes along the secretory pathway operate for the detoxification of Aspergillus nidulans cells from misfolded membrane proteins and that BsdA is a key factor for marking specific misfolded cargoes.

Assessing the role of redox partners in TthLPMO9G and its mutants: focus on H2O2 production and interaction with cellulose.

BACKGROUND: The field of enzymology has been profoundly transformed by the discovery of lytic polysaccharide monooxygenases (LPMOs). LPMOs hold a unique role in the natural breakdown of recalcitrant polymers like cellulose and chitin. They are characterized by a "histidine brace" in their active site, known to operate via an O2/H2O2 mechanism and require an electron source for catalytic activity. Although significant research has been conducted in the field, the relationship between these enzymes, their electron donors, and H2O2 production remains complex and multifaceted. RESULTS: This study examines TthLPMO9G activity, focusing on its interactions with various electron donors, H2O2, and cellulose substrate interactions. Moreover, the introduction of catalase effectively eliminates H2O2 interference, enabling an accurate evaluation of each donor's efficacy based on electron delivery to the LPMO active site. The introduction of catalase enhances TthLPMO9G's catalytic efficiency, leading to increased cellulose oxidation. The current study provides deeper insights into specific point mutations, illuminating the crucial role of the second coordination sphere histidine at position 140. Significantly, the H140A mutation not only impacted the enzyme's ability to oxidize cellulose, but also altered its interaction with H2O2. This change was manifested in the observed decrease in both oxidase and peroxidase activities. Furthermore, the S28A substitution, selected for potential engagement within the His1-electron donor-cellulose interaction triad, displayed electron donor-dependent alterations in cellulose product patterns. CONCLUSION: The interaction of an LPMO with H2O2, electron donors, and cellulose substrate, alongside the impact of catalase, offers deep insights into the intricate interactions occurring at the molecular level within the enzyme. Through rational alterations and substitutions that affect both the first and second coordination spheres of the active site, this study illuminates the enzyme's function. These insights enhance our understanding of the enzyme's mechanisms, providing valuable guidance for future research and potential applications in enzymology and biochemistry.

Characterization of a novel AA16 lytic polysaccharide monooxygenase from Thermothelomyces thermophilus and comparison of biochemical properties with an LPMO from AA9 family.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which are categorized in the CAZy database under auxiliary activities families AA9-11, 13, 14-17. Secreted by various microorganisms, they play a crucial role in carbon recycling, particularly in fungal saprotrophs. LPMOs oxidize polysaccharides through monooxygenase/peroxygenase activities and exhibit peroxidase and oxidase activities, with variations among different families. AA16, a newly identified LPMO family, is noteworthy due to limited studies on its members, thus rendering the characterization of AA16 enzymes vital for addressing controversies around their functions. This study focused on heterologous expression and biochemical study of an AA16 LPMO from Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila), namely MtLPMO16A. Substrate specificity evaluation of MtLPMO16A showed oxidative cleavage of hemicellulosic substrates and no activity on cellulose, accompanied by a strong oxidase activity. A comparative analysis with an LPMO from AA9 family explored correlations between these families, while MtLPMO16A was shown to boost the activity of some AA9 family LPMOs. The results offer new insights into the AA16 family's action mode and microbial hemicellulose decomposition mechanisms in nature.

Lytic polysaccharide monooxygenases as powerful tools in enzymatically assisted preparation of nano-scaled cellulose from lignocellulose: A review.

Nanocellulose, either in the form of fibers or crystals, constitutes a renewable, biobased, biocompatible material with advantageous mechanical properties that can be isolated from lignocellulosic biomass. Enzyme-assisted isolation of nanocellulose is an attractive, environmentally friendly approach that leads to products of higher quality compared to their chemically prepared counterparts. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively cleave the ß-1,4-glycosidic bond of polysaccharides upon activation of O2 or H2O2 and presence of an electron donor. Their use for treatment of cellulose fibers towards the preparation of nano-scaled cellulose is related to the ability of LPMOs to create nicking points on the fiber surface, thus facilitating fiber disruption and separation. The aim of this review is to describe the mode of action of LPMOs on cellulose fibers towards the isolation of nanostructures, thus highlighting their great potential for the production of nanocellulose as a novel value added product from lignocellulose.

ß-Glucosidase and ß-Galactosidase-Mediated Transglycosylation of Steviol Glycosides Utilizing Industrial Byproducts.

Stevia rebaudiana Bertoni is a plant cultivated worldwide due to its use as a sweetener. The sweet taste of stevia is attributed to its numerous steviol glycosides, however, their use is still limited, due to their bitter aftertaste. The transglycosylation of steviol glycosides, aiming at the improvement of their taste, has been reported for many enzymes, however, glycosyl hydrolases are not extensively studied in this respect. In the present study, a ß-glucosidase, MtBgl3a, and a ß-galactosidase, TtbGal1, have been applied in the transglycosylation of two steviol glycosides, stevioside and rebaudioside A. The maximum conversion yields were 34.6 and 33.1% for stevioside, while 25.6 and 37.6% were obtained for rebaudioside A conversion by MtBgl3a and TtbGal1, respectively. Low-cost industrial byproducts were employed as sugar donors, such as cellulose hydrolyzate and acid whey for TtbGal1- and MtBgl3a- mediated bioconversion, respectively. LC-HRMS analysis identified the formation of mono- and di- glycosylated products from stevioside and rebaudioside A. Overall, the results of the present work indicate that both biocatalysts can be exploited for the design of a cost-effective process for the modification of steviol glycosides.