Showing papers by "Johan P. Turkenburg published in 2020"

Structure of a GH51 α-l-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi

Abstract: α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.

The efficiency of insulin production and its content in insulin-expressing model β-cells correlate with their Zn2+ levels

Abstract: Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.

Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis.

Abstract: Background Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site However, the functional importance of such dynamics is unknown Methods Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS) Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals Results The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process

Crystal structures of the GH18 domain of the bifunctional peroxiredoxin–chitinase CotE from Clostridium difficile

Abstract: CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349–712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.