Biology subjects

Grandjean, C.

Publications and source records attributed to Grandjean, C..

6 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↗

bioRxiv2025

Synthetic pectin-cellulose nanofiber capsule provides minimal model capturing mechanics of a regenerating plant cell wall

Plant primary cell walls are dynamic supramolecular assemblies composed of layered cellulose, hemicellulose and pectin, progressively built through synthesis and secretion. However, the specific architectural features and structural components sufficient to endow the mechanical properties of the wall remain unclear. Here, we construct a minimal synthetic spherical shell and compare its structural and mechanical properties to those of a plant single-cell system. To eliminate complexities from intercellular connectivity and developmental history, we exploit the ability of plant protoplasts to regenerate cell walls de novo. Compression tests of regenerating protoplasts between parallel plates reveal that wall stiffness increases with wall thickening over time. Despite differences in assembly pathways, architecture, and composition, the synthetic shell exhibits a similar thickness-dependent modulus and similar material stiffness. The synthetic shell, composed of pectin and cellulose layers, mirrors the mechanical behavior of regenerating primary cell walls, suggesting that these components are sufficient to confer key viscoelastic properties in the limit of small deformations. Given the complexity of natural plant cell walls, the synthetic analogue offers a controllable platform to dissect the mechanical contributions of individual wall components.

plant biology↗

bioRxiv2024

Galacturonic acid oxidation: a radical way to stick together

The middle lamellae (ML) of plant cells, enriched in Homogalacturonan (HG) is considered to function as a crucial glue responsible for cell-cell cohesion (for review see 1). Mutants with defective HG content exhibit cell adhesion defects2,3. Despite advances, the mechanisms governing cell adhesion during plant development remain elusive. We previously hypothesized that cell-cell cohesion relies on cell wall integrity signaling, yet the specifics remain undefined4. OligoGalacturonans (OG), degradation products of HG, are prime candidates for informing cells about ML status and thereby influencing cell adhesion. This integrity signal is crucial for adhesion homeostasis 4. OGs serve as signaling molecules, recognized by membrane-bound cell wall receptors. Notably, restoring adhesion in qua2-1 mutants by modulating pectin response gene expression in esmd/qua2 mutants underscores potential OGs importance4-6. Deciphering the diversity and role of endogenous OGs is imperative for understanding cell adhesion modulation. Our study aims to identify compounds in this signalling pathway that regulate cell adhesion. We focused on characterizing HG degradation products in dark-grown hypocotyls. Our findings highlight various oligomers, along with two key monomers: galacturonic acid and its oxidized form, galactaric acid. These monomers appear to play a pivotal role in controlling cell adhesion by indirectly enhancing the crosslinking of extensin, a cell wall structural protein. This crosslinking leads to the densification of extensin-based cell wall networks, ultimately restoring cell adhesion in defective mutants. Our research sheds light on the intricate interplay between HG degradation product monomer and cell adhesion mechanisms.

plant biology↗

bioRxiv2023

Fine-Tuning and Remodelling of Pectins Play a Key Role in the Maintenance of Cell Adhesion

Plant cell adhesion is essential for development and stress response, mediated by pectin-rich middle lamella deposition between cell walls. However, the precise control mechanism of cell adhesion remains unclear. The qua2-1 and esmd1-1 mutants provide a better understanding of this process and suggest a signaling pathway triggering the loss and restoration of adhesion via cell wall modifications. This study attempts to characterize the potential regulatory role of endogenous oligogalacturonides (OGs) and pectin modifications in the control of cell adhesion in Arabidopsis. From dark-grown hypocotyls, our extraction revealed seven distinct endogenous OGs with varying polymerization and modifications. Abundance variations of OGs were observed among wild type, qua2-1, esmd1-1, and qua2-1/esmd1-1 mutants. The structure of homogalacturonans was analyzed by enzymatic fingerprint, in order to identify changes in esterification patterns. Expression analysis of pectin-modifying enzymes showed significant variations in PME, PMEI, and PAE genes. Gene expressions correlate with homogalacturonans modifications and cell adhesion phenotypes. This study enhances our understanding of a feedback loop between the endogenous OGs, homogalacturonans esterification fine tuning, and pectin remodeling enzymes expression in controlling cell adhesion.

plant biology↗

bioRxiv2022

Immunometabolic profiling of in vitro and ex vivo Leishmania-infected macrophages (LIMs) reveals unique polarization and bioenergetic signatures

Macrophages are the major host cells of the protozoan parasite Leishmania in mammalian infection. These key innate immune cells display remarkable phenotypic plasticity ranging from pro-inflammatory M1 to anti-inflammatory M2 macrophages that can control infection and tissue homeostasis, respectively. It has been recognized that Leishmania exploits macrophage phenotypic plasticity to establish chronic infection. However, the current notion that these parasites simply trigger an M2-like phenotype seems over-simplified considering the immunopathology observed during leishmaniasis - in particular in response to Leishmania amazonensis - which is often characterized by a mixed Th1/Th2 immune response. Here we combined a series of systems-level analyses to shed new light on the phenotype of Leishmania-infected macrophages (LIMs) during short- and long-term infection, in vitro and in vivo. Immuno-metabolic profiling by RNA-seq, RT-qPCR, cytokine immunoassays, and real-time bioenergetic flux analysis of L. amazonensis-infected bone marrow-derived macrophages (BMDMs) revealed a highly complex and unique phenotypic and bioenergetic signature. In vitro LIMs were characterized by co-expression of both M1 and M2 markers at RNA and protein levels and increased expression of glycolytic genes that matched a progressive metabolic switch from a M2-like respiratory to a M1-like glycolytic energy production observed for both long-term in vitro and in vivo infected macrophages. Unlike in M1 macrophages, glycolytic gene expression did not correlate with increased expression of its key regulatory HIF-1. In contrast, siRNA knock down experiments in primary BMDMs uncovered an essential role of the m6A reader protein IGF2BP2 in stabilizing m6A modified transcripts of the glycolytic pathway, contributing to HIF-1-independent induction of glycolysis. In conclusion, L. amazonensis establishes a complex and unique phenotypic shift in infected macrophages in vitro and in vivo that combines M1-like and M2-like immuno-metabolomic characteristics and implicates differential mRNA stability in induction of aerobic glycolysis. Our data thus uncover epi-transcriptomic regulation as a novel target for Leishmania immune subversion to establish a host cell phenotype beneficial for intracellular parasite development and chronic infection.

immunology↗