MOESM1 of The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail

Additional file 1. The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail. Figure S1. Stability of Glc4gemGlc and its impact on the results presented in this paper. Figure S2. Degradation of Glc4gemGlc during incubation under conditions similar to those during biomass saccharification in bioreactors

How a Lytic Polysaccharide Monooxygenase Binds Crystalline Chitin

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from <i>Serratia marcescens Sm</i>AA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme–substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H₂O, O₂, and H₂O₂ can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of <i>Sm</i>AA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies

How a Lytic Polysaccharide Monooxygenase Binds Crystalline Chitin

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from <i>Serratia marcescens Sm</i>AA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme–substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the copper site, through which only small molecules such as H₂O, O₂, and H₂O₂ can diffuse. Furthermore, MD simulations, quantum mechanics/molecular mechanics calculations, and electron paramagnetic resonance spectroscopy demonstrate that rearrangement of Cu-coordinating water molecules is necessary when binding the substrate and also provide a rationale for the experimentally observed C1 oxidative regiospecificity of <i>Sm</i>AA10A. This study provides a first, experimentally supported, atomistic view of the interactions between an LPMO and crystalline chitin. The confinement of the catalytic center is likely crucially important for controlling the oxidative chemistry performed by LPMOs and will help guide future mechanistic studies

Molecular Design of Non-Leloir Furanose-Transferring Enzymes from an α‑l‑Arabinofuranosidase: A Rationale for the Engineering of Evolved Transglycosylases

The vast biodiversity of glycoside hydrolases (GHs) constitutes a reservoir of readily available carbohydrate-acting enzymes that employ simple substrates and hold the potential to perform highly stereopecific and regioselective glycosynthetic reactions. However, most GHs preferentially hydrolyze glycosidic bonds and are thus characterized by a hydrolysis/transglycosylation partition in favor of hydrolysis. Unfortunately, current knowledge is insufficient to rationally modify this partition, specifically mutating key molecular determinants to tip the balance toward transglycosylation. In this study, in the absence of precise knowledge concerning the hydrolysis/transglycosylation partition in a hydrolytic GH51 α-l-arabinofuranosidase, we describe how an iterative protein engineering approach has been used to create the first “non-Leloir” transarabinofuranosylases. In the first step, random mutagenesis yielded a point mutation (R69H) at a position that is highly conserved in clan GH-A. Characterization of R69H revealed that this enzyme displays high transglycosylation activity but severely reduced (61-fold) activity on <i>p</i>NP-α-l-arabinofuranoside. Upon recombination of R69H with other point mutations selected using semirational or <i>in silico</i> approaches, transfer rates close to 100% and transarabinofuranosylation yields of the main (1→2)-linked oligosaccharide product of 80% (vs 11% for the wild-type) were obtained. Combining data presented here with knowledge drawn from the literature, we suggest that the creation of non-Leloir transglycosylases necessarily involves the destabilization of the highly developed transition states that characterize the predominantly hydrolytic <i>exo</i>-acting GHs; this is an efficient way to prevent ubiquitous water molecules from performing the deglycosylation step

Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive Off-Pathway Processes

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the degradation of polysaccharides in biomass by catalyzing powerful oxidative chemistry using only a single copper ion as a cofactor. Despite the natural abundance and importance of these powerful monocopper enzymes, the structural determinants of their functionality have remained largely unknown. We have used site-directed mutagenesis to probe the roles of 13 conserved amino acids located on the flat substrate-binding surface of CBP21, a chitin-active family AA10 LPMO from <i>Serratia marcescens</i>, also known as <i>Sm</i>LPMO10A. Single mutations of residues that do not interact with the catalytic copper site, but rather are involved in substrate binding had remarkably strong effects on overall enzyme performance. Analysis of product formation over time showed that these mutations primarily affected enzyme stability. Investigation of protein integrity using proteomics technologies showed that loss of activity was caused by oxidation of essential residues in the enzyme active site. For most enzyme variants, reduced enzyme stability correlated with a reduced level of binding to chitin, suggesting that adhesion to the substrate prevents oxidative off-pathway processes that lead to enzyme inactivation. Thus, the extended and highly evolvable surfaces of LPMOs are tailored for precise multipoint substrate binding, which provides the confinement that is needed to harness and control the remarkable oxidative power of these enzymes. These findings are important for the optimized industrial use of LPMOs as well as the design of LPMO-inspired catalysts