Biology subjects

Czjzek, M.

Publications and source records attributed to Czjzek, M..

9 recordsLinked to original sources

bioRxiv2026

Exploring the substrate specificity and catalytic mechanism in a PAPS independent ASST from Salmonella typhimurium

Sulfation plays an important role in many metabolic processes such as substrate detoxification, solubilization or activation. The enzymes responsible for these reactions, sulfotransferases, occur in all domains of life and can be classified in two major, evolutionary independent groups, PAPS dependent and PAPS independent or arysulfate sulfotransferases (ASST) enzymes. Besides the fact that they play key roles in biological functions, sulfotransferases can be interesting enzymatic tools to selectively sulfate specific positions of various metabolites. To this end we need to understand the relation between sequence, structure and function. Actually, eukaryotic PAPS dependent sulfotransferases have extensively been studied, while we largely lack knowledge about the diversity of bacterial PAPS-independent ASSTs. We have conducted a multidisciplinary study of a bacterial sulfotransferase from Salmonella typhimurium, revealing the importance of active site residues for activity and guiding substrate specificity. Based on the crystal structure in complex with substrate, we used molecular docking combined with Quantum Mechanics/Molecular Mechanics (QM/MM) free energy calculations to investigate the reaction catalytic mechanism with a focus on the role of specific active site residues in substrate specificity. Our results show that the catalytic mechanism involving three conserved histidines is common with previously determined ASSTs, and highlight a specific pair of aromatic residues, Phe174 and Tyr557, that play an important role in defining the substrate specificity in ASST clade 5. These findings enhance our general understanding of how substrate specificity is attained and has the potential to guide further enzyme engineering to expand the acceptor scope of ASSTs.

biochemistry↗

bioRxiv2026

Mechanistic Basis of Sulfo Transfer in a PAPS-Independent ArylSulfate SulfoTransferases Revealed by Crystallography and QM/MM Simulations

Enzymatic sulfation offers a green-chemistry route to the selective sulfation of phenolic and other hydroxylated compounds. Bacterial arylsulfate sulfotransferases (ASSTs) are particularly attractive biocatalysts because they use simple aryl sulfate donors, such as p-nitrophenyl sulfate (pNPS), instead of the complex and labile cofactor PAPS required by conventional sulfotransferases. However, their rational application and engineering remain limited because the molecular principles controlling substrate recognition, catalytically competent donor and acceptor positioning, covalent sulfo-enzyme formation, and sulfo transfer are not fully understood. In particular, it has remained unclear how conserved active-site histidines cooperate during catalysis and how active-site preorganization promotes efficient sulfo transfer. Here, we address these questions for the newly characterized ASST (ASTB) from Desulfitobacterium hafniense clade 6 (Dh_ASST_6) by combining X-ray crystallography, steady-state enzyme kinetics, and quantum mechanics/molecular mechanics (QM/MM) simulations. Crystal structures of Dh_ASST_6 in the apo state and donor-soaked state reveal a monomeric {beta}-propeller enzyme with a histidine-centered active site and directly identify His350 as the transient sulfo carrier. Kinetic analysis shows efficient turnover and supports a ping-pong bi-bi mechanism, consistent with formation of a covalent sulfo-enzyme intermediate. Steered QM/MM simulations of the second catalytic step show that sulfuryl transfer from sulfated His350 to the phenolic acceptor proceeds through a proton-coupled, multistep pathway in which His283 assists acceptor activation by proton transfer. Active-site preorganization precedes the main bond-reorganization event and generates a near-attack geometry in which acceptor alignment, proton transfer, and stabilization of the sulfo group by a conserved hydrogen-bonding network jointly promote sulfo transfer. Free-energy reconstruction gives a low barrier of approximately 9.6 kcal/ mol, in agreement with efficient enzymatic turnover. The structural, kinetic, and computational data support a complete catalytic cycle for Dh_ASST_6 based on a cooperative two-histidine mechanism. This model establishes catalytically competent acceptor positioning and preservation of the histidine-centered hydrogen-bonding network as key design principles for ASST engineering. These features provide concrete targets for expanding substrate scope and tuning donor or acceptor preference while maintaining efficient sulfuryl transfer.

biochemistry↗

bioRxiv2026

The cuticle lamellae responsible for structural coloration in red algae display common features with the metazoan extracellular matrix

Structural coloration is a physical phenomenon observed in many living organisms, including seaweeds, via the formation of highly organized nanostructures. Although the deposition of multilayer thin-film in the extracellular matrix (ECM) is a widespread characterized system, enabling light interference, to date such lamellae have only been described in the cuticle of red algae belonging to the Gigartinaceae family within macroalgae. Here, we determine the composition of the multilayered cuticle components in the model species Chondrus crispus (Rhodophyta), using genomic, transcriptomic, proteomic, metabolomic and analytical profiling of carbohydrates. The complex structural assembly revealed common features with the ECM of animals. The carbohydrate fraction consists of a complex mixture of ECM carrageenans and glycosaminoglycan-like structures. A major von Willerbrand factor A domain (VWA) protein plays a critical role for the adhesion of macromolecules, including protein-protein interactions and to sulfated polysaccharides. We propose the name of Lamellae Cohesive Protein (LCP) regarding the alternate distribution of chemically distinct constituents within the cuticle. For the first time, we identified major proteins of the algal cuticle, providing a framework for addressing the evolutionary origins of the algal cuticle biosynthesis and raise important questions regarding its role, particularly across the life cycle marked by major structural ECM modifications.

biochemistry↗

bioRxiv2025

The role of Akkermansia muciniphila sulfatases in colonic mucinutilisation

Akkermansia muciniphila, an obligate mucin degrader, is a major member of the human colonic microbiota and has been associated positive health outcomes. Mucins are complex glycoproteins that contain heavily sulfated O-glycans and form the protective colonic mucus layer. Bacterial carbohydrate sulfatases are required to metabolise these heavily sulfated mucin glycans and excessive bacterial foraging has been associated with several diseases. Sulfatases have been linked with inflammatory bowel disease, making these microbiota enzymes potential drug targets. A. muciniphila expresses carbohydrate sulfatases that can act on colonic mucins yet their roles in its metabolism remain opaque. Our data reveal that A. muciniphila requires glycopeptides/protein forms of colonic mucin for metabolism and its sulfatases have unique adaptations compared to Bacteroides species. Localisation studies reveal that desulfation of N-acetyl-D-glucosamine, but not D-galactose, is exclusively periplasmic. A cell surface sulfatase has a novel carbohydrate binding module that binds to colonic mucin. This paints a contrasting picture of sulfated mucin metabolism by Akkermansia muciniphila versus Bacteroides species. These data will be important for understanding the contexts for Akkermansia muciniphilas positive health correlations.

biochemistry↗

bioRxiv2024

SusC/D-like proteins in Gammaproteobacteria that utilize fructans

Fructans are ubiquitous in terrestrial ecosystems, however, these glycans are unexplored in the marine environment. We have discovered that the Antarctic gammaproteobacterium Pseudoalteromonas distincta is highly adapted to the degradation of fructose-containing substrates. This is enabled by proteins encoded in several genomic regions, including a fructan polysaccharide utilization locus (PUL). In addition to a glycoside hydrolase from family 32 (GH32), the fructan PUL encodes two proteins that have been described as specific for Bacteroidota and were previously unknown for Gammaproteobacteria: a glycan-binding SusD-like protein and a SusC-like TonB-dependent transporter (TBDT), which work as a complex in glycan import. Proteome analyses and biochemistry results suggest that the SusC/D-like proteins of P. distincta shuttle small-sized inulin-type fructans directly into the cell, where they are degraded by a periplasmic exo-active GH32. A SusD-like protein could provide a competitive adavantage in the absence of extracelluar endo-active inulinases. Comparative genomics identified further SusC/D-like proteins in Gammaproteobacteria, most of which are co-encoded with GH32s, indicative of fructan PULs, and are frequently associated with the marine habitat. Our study thus shows the first known exception to the paradigm that only Bacteroidota use SusC/D-like proteins. It further suggests that fructans contribute to the marine glycan pool.

molecular biology↗

bioRxiv2024

Evolutionary history of the main extracellular matrix polysaccharides in brown algae

Brown algae belong to the Stramenopiles phylum and are phylogenetically distant from plants and other multicellular organisms. This independent evolutionary history has shaped brown algae with numerous metabolic characteristics specific to this group, including the synthesis of peculiar polysaccharides contained in their extracellular matrix (ECM). Alginates and fucose-containing sulphated polysaccharides (FCSP), the latter including fucans, are the main components of ECMs. However, the metabolic pathways of these polysaccharides remain poorly described due to a lack of genomic data. An extensive genomic dataset has been recently released for brown algae and their close sister species. We performed an expert annotation of key genes involved in ECM-carbohydrate metabolisms, combined with comparative genomics, phylogenetics analyses, and protein modelling. Our analysis indicates that the gene families involved in both the synthesis and degradation of alginate were acquired by the common ancestor of brown algae and their closest sister species Schizocladia ischiensis, and subsequently expanded in brown algae. The pathway for the biosynthesis of fucans still remains biochemically unresolved and we identify the most likely fucosyltransferase genes that may harbour a fucan synthase activity in brown algae. Our analysis questions the possible occurrence of FCSPs outside brown algae, notably within their closest sister taxon.

evolutionary biology↗

bioRxiv2023

Novel laminarin-binding CBMs in multimodular proteins of marine Bacteroidota feature prominently in phytoplankton blooms

The {beta}-(1,3)-glucan laminarin functions as storage polysaccharide in marine stramenophiles such as diatoms. Laminarin is abundant, water-soluble and structured simply, making it an attractive substrate for marine bacteria. As a consequence, many marine bacteria have developed competitive strategies to scavenge and decompose laminarin, which involves carbohydrate-binding modules (CBMs) as key players. We therefore functionally and structurally characterized two yet unassigned domains as laminarin-binding CBMs in multimodular proteins from our model bacterium Christiangramia forsetii KT0803T, hereby unveiling the novel laminarin-binding CBM families CBMxx and CBMyy (official CAZy numbering will be provided upon acceptance of the manuscript in a peer-reviewed journal). We discovered four CBMxx repeats in a surface glycan-binding protein (SGBP) and a single CBMyy combined with a glycoside hydrolase module from family 16 (GH16_3). Our analyses revealed that both modular proteins have an elongated shape, and that the GH16_3 displayed a higher flexibility than the SGBP. While motility of both polypeptide chains may facilitate recognition and/or degradation of laminarin, constraints in the SGBP may support docking of laminarin onto the bacterial surface. The exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea revealed that both laminarin-binding CBM families are widely distributed among marine Bacteroidota, illustrating the high adaptability of modularity in sugar-binding and -degrading proteins. High expression of CBMxx- and CBMyy-containing proteins during phytoplankton blooms further underpins their importance in marine laminarin usage.

biochemistry↗

bioRxiv2021

Sulfated host glycan recognition by carbohydrate sulfatases of the human gut microbiota

The vast microbial community that resides in the human colon, termed the human gut microbiota, performs important roles in maintaining host health. Sulfated host glycans comprise both a major nutrient source and important colonisation factors for this community. Carbohydrate sulfatases remove sulfate groups from glycans and are essential in many bacteria for the utilisation of sulfated host glycans. Additionally, carbohydrate sulfatases are also implicated in numerous host diseases, but remain some of the most understudied carbohydrate active enzymes to date, especially at the structural and molecular level. In this work, we analyse 7 carbohydrate sulfatases, spanning 4 subfamilies, from the human gut symbiont Bacteroides thetaiotaomicron, a major utiliser of sulfated host glycans, correlating structural and functional data with phylogenetic and environmental analyses. Together, these data begin to fill the knowledge gaps in how carbohydrate sulfatases orchestrate sulfated glycan metabolism within their environment.

biochemistry↗

bioRxiv2020

A single bacterial sulfatase is required for metabolism of colonic mucin O-glycans and intestinal colonization by a symbiotic human gut bacterium

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

microbiology↗