Biology subjects

Courtade, G.

Publications and source records attributed to Courtade, G..

13 recordsLinked to original sources

bioRxiv2026

NMR assignment and functional characterization of biotechnologically modified xanthan

Xanthan gum is one of the most important industrial microbial polysaccharides, yet comprehensive molecular-level structural characterization remains limited because of its high molecular weight, conformational rigidity, and severe spectral overlap in nuclear magnetic resonance spectroscopy. Here, we present a near-complete 1H/13C NMR chemical shift assignment of the xanthan repeating unit by combining multidimensional NMR spectroscopy with targeted glycosyltransferase knockout strains and controlled enzymatic depolymerization. Comparative analysis of wild-type xanthan and{Delta} gumI and{Delta} gumK variants enabled the unambiguous assignment of {beta}-mannose and glucuronic acid resonances, and, by extension, of the remaining sugars, supporting future structural studies of engineered xanthan variants. In addition, we investigated how side-chain composition affects xanthan properties by analyzing genetically modified variants lacking specific acetyl, pyruvyl, or sugar residues. Temperature-dependent 1H NMR determined order-disorder transition temperatures and revealed that removing pyruvate or negatively charged side-chain components increased thermal stability, whereas losing the terminal {beta}-mannose destabilized the ordered conformation. Together, these results establish a robust NMR assignment for xanthan and provide new insights into how individual side-chain substituents influence the structural stability of this polysaccharide, facilitating the rational characterization of engineered xanthan variants and structurally closely related polysaccharides.

biochemistry↗

bioRxiv2026

Decoding CBM2 binding modes: how similar folds achieve distinct substrate preferences

Understanding the mechanisms underlying substrate recognition and selectivity is essential for developing new biotechnological applications of carbohydrate-binding modules (CBMs). Many members of CBM family 2 (CBM2) prefer binding to crystalline substrates and show diverse substrate specificities despite sharing similar folds. We show here that CBM2 specificity cannot be rationalized solely based on sequence and fold. Rather, specific binding of CBM2s to crystalline surfaces depends on a balance of topological complementarity, hydrophobic dehydration, and hydrogen-bonding interactions. We compare the binding modes of ScCBM2 and BaCBM2 on cellulose, chitin, and polyethylene terephthalate (PET) with respect to protein and substrate properties. Using MD simulations and NMR, we explore the CBMs' surface topology, hydration, solvent accessibility, and electrostatics. The results indicate that the two CBMs may bind to crystalline substrates in opposite orientations, and that the interplay between hydrophobic and {pi}-stacking interactions along the aromatic triad and hydrogen-bonding interactions on either side determines their orientation. We further show that higher water density at the aromatic triad correlates with higher substrate affinity. Lastly, we probe the interaction dynamics of BaCBM2 with PET nanoparticles and the monomer bis(2-hydroxyethyl) terephthalate (BHET) using NMR relaxation and solvent paramagnetic relaxation enhancement (sPRE) experiments, revealing a mixture of transient, low-population states involved in PET binding. These insights clarify the interplay among factors that drive CBM2 substrate recognition and highlight the importance of evaluating surface hydration and protein dynamics in future CBM2 studies.

biophysics↗

bioRxiv2026

Conformation-Dependent Donor Selectivity in the Xanthan Gum Glycosyltransferase GumK Revealed by AI-Based Docking

The interdomain flexibility of GT-B fold glycosyltransferases regulates substrate binding and catalysis, yet the role of local structural variations in donor substrate specificity remains unclear. GumK, a GT70 enzyme from Xanthomonas campestris, exhibits local plasticity within its donor-binding pocket. We classify this plasticity into two conformational states, defined by the presence (closed state) or absence (open state) of a conserved hydrophobic interaction stabilizing the pocket. Using the AI-enhanced docking approach GNINA, we investigated the relationship between these states and substrate specificity by comparing UDP-glucuronate with five acidic and neutral ligand analogs. While docking scores showed limited discrimination among ligands, distance-based analysis between the sugar C6 atom and Lys307 revealed conformation-dependent trends. In the open state, negatively charged sugars preferentially interact with Lys307 via their carboxylate groups. Conversely, the closed state favors interactions with the pyrophosphate moiety. To provide orthogonal validation, we generated ensembles of GumK-substrate complexes using AI-based cofolding methods (AlphaFold3 and Boltz). While Boltz-2 affinities correctly ranked substrate specificity, only the GNINA-generated ensembles were consistent with physics-based simulations in capturing the conformational basis of selectivity. These results suggest that donor specificity arises from the interplay between ligand chemistry and binding-site plasticity rather than from a single rigid binding mode, and highlight complementary strengths of docking and cofolding approaches for studying flexible CAZymes.

biophysics↗

bioRxiv2026

Molecular basis of protein-glycan cross-linking by CpCBM92A revealed by NMR spectroscopy

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.

biophysics↗

bioRxiv2026

Comparative study of two xanthan gum glycosyltransferases combining AI structure predictions and molecular modeling

Xanthan gum is a widely used industrial polysaccharide employed as a thickening and stabilizing agent in food, pharmaceutical, and technological applications. Its biosynthesis involves membrane-associated glycosyltransferases that assemble the repeating unit at the cytoplasmic side of the inner membrane. Among them, GumH and GumI catalyze consecutive reactions using the same donor substrate, guanosine 5-diphospho-alpha-D-mannose, but with opposite stereoselectivity. Despite their biochemical characterization, structural insights into their catalytic mechanisms and membrane interactions remain limited, hindering a detailed understanding of their function and future engineering efforts. In this work, we combined artificial intelligence-based structure prediction with atomistic molecular dynamics simulations to investigate the structural organization and substrate-binding modes of GumH (family GT4) and GumI (family GT94). The predicted apo structures exhibit a conserved GT-B fold but differ in interdomain flexibility and membrane-anchoring strategies. GumH displays a more structured interdomain linker and a defined clamp-like region in the acceptor-binding domain, consistent with stable membrane interaction, whereas GumI shows a more flexible linker and an open groove architecture. Modeling of the donor-bound complexes reveals distinct substrate-binding modes. In GumH, it adopts a geometry consistent with its retaining stereochemical outcome, positioning the sugar close to the conserved catalytic residue. In contrast, GumI exhibits a different donor orientation, lacking a clearly positioned catalytic base near the reactive center, suggesting a substrate-assisted catalytic mechanism. Although the predicted ternary complexes show limited stability in our simulations, they provide chemically reasonable conformations and offer structural insights into substrate recognition, membrane association, and stereochemical control in these two glycosyltransferase families. Significance statementXanthan gum is an industrially important polysaccharide widely used in food and other technological products. Although several enzymes in its biosynthetic pathway have been studied, structural information remains limited. Using AI-based structure predictions and molecular simulations, we revealed how these enzymes sit in the membrane and bind sugar substrates. These structural insights clarify xanthan biosynthesis and could help improve or engineer its production.

biophysics↗

bioRxiv2025

The C-terminal intrinsically disordered region of a fungal LPMO binds copper and displays anti-fungal properties

Lytic Polysaccharide Monooxygenases (LPMOs) are enzymes that play a crucial role in the degradation of complex polysaccharides such as cellulose and chitin. While LPMOs have attracted significant interest for industrial applications to convert biomass into biofuels, emerging evidence suggests alternative functions in fungal plant pathogenesis, microbial diseases and (micro)organism development. The AA14 LPMO family is widely distributed in filamentous fungi but remains enigmatic as its initially-suspected substrate specificity was recently challenged. In this study, we investigated the disordered C-terminal regions (dCTRs), found in more than half of AA14 family members and hypothesized to have functional relevance. Focusing on the Pycnoporus coccineus AA14A LPMO, we used small angle X-ray scattering (SAXS) and circular dichroism to show that its dCTR is highly disordered. It consists of a heavily glycosylated low complexity region followed by a charged C-terminal tail. We uncovered that the PcoAA14A dCTR binds copper ions through histidine residues located in the C-terminal tail. Using electron paramagnetic resonance (EPR), we further demonstrated that dimer formation occurs through an oxidative process involving copper and a redox-sensitive cysteine. Noting similarities between the last residues of the C-terminal tail and antimicrobial peptides, we investigated its potential antimicrobial function. We found that the positively charged region of the C-terminal tail selectively inhibits the growth of basidiomycete fungal spores. These data reveal that the dCTRs appended to AA14 LPMOs are functional regions that must be considered to unveil the role of these atypical LPMOs. Significance StatementLytic polysaccharide monooxygenases (LPMOs) degrade complex polysaccharides and are central to biomass conversion. Their biological relevance is expanding, with emerging roles in fungal development and host interactions. Despite this broad potential, research has largely focused on their catalytic domain. One area that remains poorly understood is the function of their C-terminal region, predicted to be intrinsically disordered (dCTR). The AA14 LPMO family, whose substrate specificity remains enigmatic, is enriched in dCTRs. This study provides the first experimental evidence of the disordered nature of the dCTR of an AA14 LPMO. Using multidisciplinary approaches, we demonstrate that the dCTR binds metals and exhibits antifungal activity. These findings establish dCTRs as functional regions, essential for understanding the biological roles of LPMOs.

biochemistry↗

bioRxiv2025

Computational characterization of the xanthan gum glycosyltransferase GumK

The activity of GT-B glycosyltransferases depends on their conformational flexibility and high substrate specificity, but the molecular basis of these features is still not well defined. The GT70 family contains a single well-characterized enzyme, GumK, a glucuronosyltransferase from Xanthomonas campestris required for xanthan gum biosynthesis. Here, we applied multiscale molecular simulations and sequence analysis to probe GumK dynamics and substrate specificity. We show that GumK undergoes twisting and bending motions constrained by interdomain contacts and modulated by membrane anchoring. Acceptor-substrate binding within an amphiphilic clamp promotes opening, whereas donor-substrate binding stabilizes closure, defining a substrate-dependent catalytic cycle. Specificity for UDP-glucuronate is mediated by a conserved electrostatic environment centered on Lys307 and a hydrophobic triad that orients the sugar moiety. On the acceptor side, the binding site selectively accommodates polyisoprenyl carriers up to three isoprene units in length and wraps around the substrate, constraining the trisaccharide moiety in a catalytically competent conformation. Comparative analysis highlights GumK-specific motifs that distinguish it from homologous GTs. This work provides mechanistic insight into the GT70 family and the dynamic behavior of GT-B enzymes, establishing principles for the rational engineering of GumK to modify the monosaccharide composition of xanthan gum. Author summaryGlycosyltransferases are enzymes that build many of the sugars and polysaccharides essential for life. One of them, called GumK, is responsible for a key step in producing xanthan gum -- a natural polymer widely used as a thickener in food and industrial materials. Despite its importance, how GumK works at the molecular level has remained unclear. In our study, we used multiscale computer simulation strategies to explore the dynamics of this enzyme and how it interacts with the cell membrane and its sugar substrates. We found that GumK acts like a flexible clamp that opens and closes as it binds two different sugar molecules through specific residues that may control the enzymes selectivity. We also discovered how the surrounding membrane helps the enzyme remain in the correct orientation to perform its function. By identifying the molecular details that determine GumKs selectivity and flexibility, our work provides a foundation for modifying this enzyme to produce xanthan gum with new properties. More broadly, our findings help explain how a large family of related enzymes controls sugar transfer in living organisms, with potential applications in biotechnology and materials science.

biophysics↗

bioRxiv2025

Development of a Vibrio natriegens-based plate-clearing assay for rapid screening of PET-hydrolyzing enzymes

Polyethylene terephthalate (PET) is a major contributor to plastic waste. Enzymatic PET degradation offers a sustainable recycling approach, but screening for effective enzymes remains challenging. Herein, we established an experimental plate-clearing assay using Vibrio natriegens, exploiting its protein secretion system to rapidly identify catalytic activity without time-consuming downstream processing. To validate the assays robustness, we tested mutants of Fusarium solani pisi cutinase (FsC) and found that one mutant, T45P, exhibited threefold higher activity and a TPA-to-MHET ratio than the wild-type. As a further test case, we screened an error-prone PCR library of FsC using the assay, obtaining a 25% hit rate after screening 150 colonies. This demonstrates the scalability of the method for screening PET-hydrolytic activity in a large number of enzymes.

biochemistry↗

bioRxiv2023

Experimental and computational insights into the functional and structural role of the linker region of a two-domain lytic polysaccharide monooxygenase

A considerable number of lytic polysaccharide monooxygenases (LPMOs) and other carbohydrate-active enzymes are modular, with catalytic domains being tethered to additional domains, such as carbohydrate-binding modules, by flexible linkers. While such linkers may affect the structure, function, and stability of the enzyme, their roles remain largely enigmatic, as do the reasons for natural variation in length and sequence. Here, we have explored linker functionality using the two-domain cellulose-active ScLPMO10C from Streptomyces coelicolor as a model system. In addition to investigating the wild-type enzyme, we engineered three linker variants to address the impact of both length and sequence and characterized these using SAXS, NMR, MD simulations, and functional assays. The resulting data revealed that, in the case of ScLPMO10C, linker length is the main determinant of linker conformation and enzyme performance. Both the wild-type and a serine-rich variant, which have the same linker length, demonstrated better performance compared to those with either a shorter or longer linker. A highlight of our findings was the substantial thermostability observed in the serine-rich variant. Importantly, the linker affects thermal unfolding behavior and enzyme stability. In particular, unfolding studies show that the two domains unfold independently when mixed, while the full-length enzyme shows one cooperative unfolding transition, meaning that the impact of linkers in biomass processing enzymes is more complex than mere structural tethering.

biochemistry↗

bioRxiv2023

Following the fate of lytic polysaccharide monooxygenases (LPMOs) under oxidative conditions by NMR spectroscopy

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO action is key to the efficient varlorization of biomass, but the instability of LPMOs in turnover conditions limits their efficiency. LPMO catalysis requires the presence of a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors.. While it is known that reduced LPMOs are prone to auto-catalytic oxidative damage due to off-pathway reactions with the oxygen co-substrate, little is known about the structural consequences of such damage. Here, we present atomic-level insight into how the structure of the chitin-active SmLPMO10A is affected by oxidative damage, using NMR and CD spectroscopy. Incubation with ascorbic acid, led to rearrangements of aromatic residues, followed by more profound structural changes near the copper active site and loss of activity. Longer incubation times induced changes in larger parts of the structure, indicative of progressing oxidative damage. Incubation with ascorbic acid in the presence of chitin led to similar changes in the observable (i.e., not substrate-bound) fraction of the enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals corresponding to seemingly intact SmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed that SmLPMO10A retained catalytic activity when pre-incubated with chitin before being subjected to conditions that induce oxidative damage. Overall, this study provides structural insights into the process of oxidative damage of SmLPMO10A and demonstrates the protective effect of the substrate. The impact of turnover conditions on aromatic residues in the core of the enzyme suggests a role for these residues in dealing with redox-active species generated in the copper center.

biochemistry↗

bioRxiv2022

Biochemical characterization and NMR study of a PET-hydrolyzing cutinase from Fusarium solani pisi

In recent years, the drawbacks of plastics have become evident, with plastic pollution becoming a major environmental issue. There is an urgent need to find solutions to efficiently manage plastic waste by using novel recycling methods. Biocatalytic recycling of plastics by using enzyme-catalyzed hydrolysis is one such solution that has gained interest, in particular for recycling polyethylene terephthalate (PET). To provide insights into PET hydrolysis by cutinases, we have here characterized the kinetics of a PET-hydrolyzing cutinase from Fusarium solani pisi (FsC) at different pH values, mapped the interaction between FsC and the PET analog BHET by using NMR spectroscopy, and monitored product release directly and in real time by using time-resolved NMR experiments. We found that primarily aliphatic side chains around the active site participate in the interaction with BHET, and that pH conditions and mutation around the active site (L182A) can be used to tune the relative amounts of degradation products. Moreover, we propose that the low catalytic performance of FsC on PET is caused by poor substrate binding combined with slow MHET hydrolysis. Overall, our results provide insights into obstacles that preclude efficient PET hydrolysis by FsC and suggest future approaches for overcoming these obstacles and generating efficient PET-hydrolyzing enzymes. TOC Graphic (For Table of Contents use only) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/514593v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1f59d35org.highwire.dtl.DTLVardef@242467org.highwire.dtl.DTLVardef@d2198eorg.highwire.dtl.DTLVardef@18bba0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Standard Intein Gene Expression Ramps (SIGER) for protein-independent expression control

Coordination of multi-gene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features of the 5UTR in combination with the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures. These features strongly influence translation yield and protein quality by regulating ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5UTRs in combination with different target genes leads to a wide variety of gene ramp compositions with irregular translation rates leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramps (SIGER) system for controlling protein expression. The SIGER system makes use of inteins to decouple the translation initiation features from the gene of a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways. As a proof of concept of the potential of the system, we also used a SIGER system to express two difficult-to-produce proteins, GumM and CBM73. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/471673v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1c759acorg.highwire.dtl.DTLVardef@d03c87org.highwire.dtl.DTLVardef@135d6f9org.highwire.dtl.DTLVardef@1b48eac_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

NMR structures and functional roles of two related chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus

Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. Still, structural and functional properties of some of these CBMs remain unknown and it is not clear why some LPMOs, like CjLPMO10A from Cellvibrio japonicus, have two CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on the functional variation, and determined the solution structures of both by NMR, which includes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the {micro}M range, CjCBM73 has higher affinity than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding to soluble chitin was detected for CjCBM73. These functional differences correlated with distinctly different architectures of the substrate-binding surfaces of the two CBMs. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.

biochemistry↗