Biology subjects

Melse, O.

Publications and source records attributed to Melse, O..

3 recordsLinked to original sources

bioRxiv2026

Unraveling Xanthomonas acetyltransferase GumG: decoding catalytic promiscuity, understanding the mechanism, and enhancing enzymatic versatility

Xanthan is a structurally complex exopolysaccharide produced by Xanthomonas campestris and one of the most extensively studied microbial biopolymers. As a sustainable alternative to petroleum-based polymers, its broader application requires precise control of polysaccharide decoration, yet the enzymatic basis of these modifications remains incompletely understood. Here, we characterise the activity and substrate scope of GumG, an AT-3 domain-containing membrane-bound acetyltransferase responsible for xanthan O-acetylation. Using mass spectrometry in combination with in vitro and in vivo assays, we show that GumG mediates non-specific acetylation of the outer mannose residue and displays pronounced substrate promiscuity. GumG also exhibits limited propionyltransferase activity, enabling the biosynthesis of hybrid acetylated-propionylated xanthan at an 8.27:1 ratio. Molecular docking and analysis of 31 xanthan variants identify a cytoplasmic substrate-binding pocket defined by Val67 and Phe71 that governs donor specificity, and an engineered GumG variant (F71L) shows enhanced propionyltransferase activity. In addition, a periplasmic His40-Trp143-Asp246-His297 motif is proposed to constitute the catalytic center. Together, these findings provide mechanistic insight into GumG multifunctionality and establish a framework for engineering xanthan derivatives with tailored physicochemical properties.

microbiology↗

bioRxiv2021

Assessment of Molecular Mechanics-based Zn2+ Models in Mono- and Bimetallic Ligand Binding Sites

Zn2+ ions play an important role in biology, but accurate sampling of metalloproteins using Molecular Mechanics remains challenging. Several models have been proposed to describe Zn2+ in biomolecular simulations, ranging from nonbonded models, employing classical 12-6 Lennard-Jones (LJ) potentials or extended LJ-potentials, to dummy-atom models and bonded models. We evaluated the performance of a large variety of these Zn2+ models in two challenging environments for which little is known about the performance of these methods, namely in a monometallic (Carbonic Anhydrase II) and a bimetallic ligand binding site (metallo-{beta}-lactamase VIM-2). We focused on properties which are important for a stable, correct binding site description during molecular dynamics (MD) simulations, because a proper treatment of the metal coordination and forces are here essential. We observed that the strongest difference in performance of these Zn2+ models can be found in the description of interactions between Zn2+ and non-charged ligating atoms, such as the imidazole nitrogen in histidine residues. We further show that the nonbonded (12-6 LJ) models struggle most in the description of Zn2+-biomolecule interactions, while the inclusion of ion-induced dipole effects strongly improves the description between Zn2+ and non-charged ligating atoms. The octahedral dummy-atom models result in highly stable simulations and correct Zn2+ coordination, and are therefore highly suitable for binding sites containing an octahedral coordinated Zn2+ ion. The results from this evaluation study in ligand binding sites can guide structural studies of Zn2+ containing proteins, such as MD-refinement of docked ligand poses and long-term MD simulations.

bioinformatics↗

bioRxiv2019

Broad spectrum antibiotic-degrading metallo-β-lactamases are phylogenetically diverse and widespread in the environment.

Antibiotic resistance has emerged as a major global health threat. The Zn2+-dependent metallo-{beta}-lactamases (MBLs) are of particular concern as they act on the most widely prescribed class of antibiotics, the {beta}-lactams, and are largely unaffected by commonly used {beta}-lactamase antagonists such as clavulanic acid. MBLs are subdivided into three groups (B1 to B3); despite low overall sequence similarity, their catalytic centers are conserved with two closely spaced Zn2+ binding sites ( and {beta} site). We recovered almost 1500 B3 MBLs from >100,000 public microbial genomes representing a wide range of habitats including pristine sites not impacted by human activity. Although homologs were predominantly identified in members of the bacterial phylum Proteobacteria, the recovered B3 MBLs represent a much broader phylogenetic diversity than is currently appreciated based on the study of model pathogens. This includes three active site variants inferred to have arisen from the ancestral B3 enzyme. One of these variants, B3-RQK, is noteworthy for being broadly sensitive to clavulanic acid. Through targeted mutations we demonstrate that the presence of a lysine residue (Lys263) in the {beta} site of the catalytic center of this variant confers sensitivity to this compound. Replacing this lysine with the canonical histidine (His263) found in all other MBLs restored resistance. Crystallographic and computational data reveal that clavulanic acid inhibits B3-RQK MBLs by displacing the Zn2+ ion in the {beta} site. Therefore, modifying clavulanic acid to effectively interact with His263 may increase the therapeutic range of this widely used antibiotic resistance drug.\n\nSignificanceThis study surveys the environmental and phylogenetic diversity of the B3 subgroup of antibiotic-degrading metallo-{beta}-lactamases (MBLs). B3-like MBLs are more widespread in the environment than previously appreciated suggesting multiple unrecognized reservoirs of antibiotic resistance. Three variants of the canonical active site were identified, including B3-RQK, which amongst the B3 MBLs is uniquely inhibited by the antibiotic resistance drug clavulanic acid. We demonstrate that the mode of inhibition involves the displacement of a catalytically essential Zn2+ ion from the active site. It may thus be possible to modify clavulanic acid so that it can compete with the Zn2+ ions in other MBLs as well, increasing the therapeutic range of this compound.

biochemistry↗