Biology subjects

Overkleeft, H. S.

Publications and source records attributed to Overkleeft, H. S..

8 recordsLinked to original sources

bioRxiv2026

Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures

Mycobacteria synthesise the unusual glycan [x1D05]-arabinan as a major component of the cell wall glycoconjugates arabinogalactan (AG) and lipoarabinomannan (LAM). We previously identified Dysgonomonas gadei, a member of the Bacteroidota, as capable of complete [x1D05]-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo--[x1D05]-arabinofuranosidase activity against AG and LAM, although their linkage specificities were unknown. Here, using defined synthetic substrates, we show that the three enzymes possess distinct linkage preferences. We also develop -[x1D05]-arabinofuranosyl cyclophellitol aziridines as covalent inhibitors and activity-based probes for GH172 enzymes. X-ray crystallography and cryo-EM to reveal strikingly different quaternary assemblies across the three homologues, while a 1.5 [A] cryo-EM structure of dodecameric Dg67 covalently modified by an aziridine inhibitor identifies the catalytic nucleophile and provides direct structural support for a retaining mechanism. A BODIPY-tagged aziridine probe selectively labelled the three GH172 enzymes in D. gadei cell lysates. Together, these findings define functional and structural diversity within GH172 and establish chemical probes for profiling -[x1D05]-arabinofuranosidase activity in complex biological samples.

biochemistry↗

bioRxiv2026

Glycosylation of anandamide and other bioactive N-acylethanolamines in mammalian cells and tissues

N-acylethanolamines (NAEs), including the endocannabinoid anandamide, are bioactive fatty acid amides that are normally hydrolyzed by fatty acid amide hydrolase (FAAH) or N-acyl acid amidohydrolase (NAAA). Strikingly, when canonical NAE degradation is blocked, NAE levels do not increase indefinitely but instead reach a plateau. This apparent metabolic ceiling suggests that additional, underexplored pathways contribute to NAE homeostasis. Identifying these pathways is essential to determine whether NAEs are converted into inactive metabolites or products with distinct biological properties. Here, we identify NAE glycosylation as a metabolic pathway that links endocannabinoid-related lipid metabolism to glycosphingolipid turnover. We synthesized glycosylated NAEs and their isotope-encoded standards and developed targeted LC-MS/MS assays to monitor their enzymatic processing and quantify their abundance in mouse and human cells, tissues, and plasma. We show that non-lysosomal glucosylceramidase GBA2 transfers glucose or galactose to anandamide, N-oleoylethanolamine and N-palmitoylethanolamine, and lysosomal glucosylceramidase GCase hydrolyses {beta}-Glycosylated-NAEs ({beta}-Glyco-NAE) back to their parent NAEs. {beta}-Glyco-NAEs occur endogenously in macrophages and neuronal cells, increase when canonical NAE degradation is impaired, and accumulate in human samples with GCase deficiency, including Gaucher disease and GBA1-associated Parkinsons disease. {beta}-Glyco-NAEs do not engage the cannabinoid receptors, TRPV1, or PPAR, and potentiate inflammatory cytokine release, including IL6 and TNF, from microglia. Based on these findings, we pose that GBA2-dependent NAE glycosylation may constitute an overflow lipid-remodeling pathway that connects NAE metabolism to lysosomal dysfunction, inflammation and neurodegeneration.

biochemistry↗

bioRxiv2022

Modularly designed protein fragments combine into thousands of active and structurally diverse enzymes

Design of structurally diverse enzymes is constrained by long-range interactions that are needed for accurate folding. We introduce an atomistic and machine-learning strategy for Combinatorial Assembly and Design of ENZymes, CADENZ, to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of active and structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a tenfold improved hit rate and >10,000 active enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.

bioengineering↗

bioRxiv2020

Tuning the transglycosylation reaction of a GH11 xylanase by a delicate enhancement of its thumb flexibility

Glycoside hydrolases (GH) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GH can perform transglycosylation reaction under specific conditions. In nature, oligosaccharides synthesis is performed by glycosyltransferase (GT). However, the industrial utilization of GT is limited by their instability in solution. A key difference between GT and GH is the flexibility of their binding sites architecture. In this report, we used the xylanase from Bacillus circulans (BCX) to study the interplay between active site flexibility and the transglycosylation reaction. Residues of the BCX thumb were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodeling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.

biochemistry↗

bioRxiv2019

Chemical genetics strategy to profile kinase target engagement reveals role of FES in neutrophil phagocytosis via SYK activation

Chemical tools and methods that report on target engagement of endogenously expressed protein kinases by small molecules in human cells are highly desirable. Here, we describe a chemical genetics strategy that allows the study of non-receptor tyrosine kinase FES, a promising therapeutic target for cancer and immune disorders. Precise gene editing was used in combination with a rationally designed, complementary fluorescent probe to visualize endogenous FES kinase in HL-60 cells. We replaced a single oxygen atom by a sulphur in a serine residue at the DFG-1 position of the ATP-binding pocket in an endogenously expressed kinase, thereby sensitizing the engineered protein towards covalent labeling and inactivation by a fluorescent probe. The temporal control offered by this strategy allows acute inactivation of FES activity both during myeloid differentiation and in terminally differentiated neutrophils. Our results show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis by activation of SYK kinase, a central regulator of immune function in neutrophils. This strategy holds promise as a target validation method for kinases.

physiology↗

bioRxiv2019

Catalysis by a rigid enzyme

Many enzymes are dynamic entities, sampling conformational states that are relevant for catalytic activity. Crystal structures of catalytic intermediates suggest, however, that not all enzymes require structural changes for activity. The single-domain enzyme xylanase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose. We demonstrate that BCX in solution undergoes minimal structural changes during catalysis. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow, enzyme induced substrate distortion. Therefore, we propose a model in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates catalysis.\n\nOne Sentence SummaryThe rigid matrix of BCX uses substrate flexibility in Michaelis complex formation.

biochemistry↗

bioRxiv2019

Progranulin deficiency leads to reduced glucocerebrosidase activity

Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GBA), and is essential for proper GBA activity. GBA activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.

neuroscience↗

bioRxiv2017

Qinichelins, novel catecholate-hydroxamate siderophores synthesized via a multiplexed convergent biosynthesis pathway

The explosive increase in genome sequencing and the advances in bioinformatic tools have revolutionized the rationale for natural product discovery from actinomycetes. In particular, this has revealed that actinomycete genomes contain numerous orphan gene clusters that have the potential to specify many yet unknown bioactive specialized metabolites, representing a huge unexploited pool of chemical diversity. Here, we describe the discovery of a novel group of catecholate-hydroxamate siderophores termed qinichelins (2-5) from Streptomyces sp. MBT76. Correlation between the metabolite levels and the protein expression profiles identified the biosynthetic gene cluster (BGC; named qch) most likely responsible for qinichelin biosynthesis. The structure of the molecules was elucidated by bioinformatics, mass spectrometry and NMR. Synthesis of the qinichelins requires the interplay between four gene clusters, for its synthesis and for precursor supply. This biosynthetic complexity provides new insights into the challenges scientists face when applying synthetic biology approaches for natural product discovery.\n\nPride repository reviewer account details:\n\nURL: https://www.ebi.ac.uk/pride/archive/login\n\nProject accession: PXD006577\n\nUsername: reviewer35793@ebi.ac.uk\n\nPassword: 3H0iM1FK

microbiology↗