• Structural basis of terephthalate recognition by solute binding protein TphC

      Gautom, Trishnamoni; orcid: 0000-0001-7602-1228; Dheeman, Dharmendra; orcid: 0000-0002-8933-954X; Levy, Colin; Butterfield, Thomas; Alvarez Gonzalez, Guadalupe; orcid: 0000-0003-4387-2231; Le Roy, Philip; orcid: 0000-0002-5496-0272; Caiger, Lewis; orcid: 0000-0001-5156-9684; Fisher, Karl; orcid: 0000-0003-3539-8939; Johannissen, Linus; orcid: 0000-0002-0916-9094; Dixon, Neil; orcid: 0000-0001-9065-6764; email: neil.dixon@manchester.ac.uk (Nature Publishing Group UK, 2021-10-29)
      Abstract: Biological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC. Here, we report the biochemical and structural characterisation of TphC in both open and TPA-bound closed conformations. This analysis demonstrates the narrow ligand specificity of TphC towards aromatic para-substituted dicarboxylates, such as TPA and closely related analogues. Further phylogenetic and genomic context analysis of the tph genes reveals homologous operons as a genetic resource for future biotechnological and metabolic engineering efforts towards circular plastic bio-economy solutions.
    • The lexicon of antimicrobial peptides: a complete set of arginine and tryptophan sequences

      Clark, Sam; orcid: 0000-0002-6865-4452; Jowitt, Thomas A.; Harris, Lynda K.; Knight, Christopher G.; orcid: 0000-0001-9815-4267; Dobson, Curtis B.; orcid: 0000-0002-6483-4608; email: curtis.dobson@manchester.ac.uk (Nature Publishing Group UK, 2021-05-21)
      Abstract: Our understanding of the activity of cationic antimicrobial peptides (AMPs) has focused on well-characterized natural sequences, or limited sets of synthetic peptides designed de novo. We have undertaken a comprehensive investigation of the underlying primary structural features that give rise to the development of activity in AMPs. We consider a complete set of all possible peptides, up to 7 residues long, composed of positively charged arginine (R) and / or hydrophobic tryptophan (W), two features most commonly associated with activity. We found the shortest active peptides were 4 or 5 residues in length, and the overall landscapes of activity against gram-positive and gram-negative bacteria and a yeast were positively correlated. For all three organisms we found a single activity peak corresponding to sequences with around 40% R; the presence of adjacent W duplets and triplets also conferred greater activity. The mechanistic basis of these activities comprises a combination of lipid binding, particularly to negatively charged membranes, and additionally peptide aggregation, a mode of action previously uninvestigated for such peptides. The maximum specific antimicrobial activity appeared to occur in peptides of around 10 residues, suggesting ‘diminishing returns’ for developing larger peptides, when activity is considered per residue of peptide.
    • UbiD domain dynamics underpins aromatic decarboxylation

      Marshall, Stephen A.; orcid: 0000-0001-7678-4989; email: stephen.marshall@chem.ox.ac.uk; Payne, Karl A. P.; orcid: 0000-0002-6331-6374; Fisher, Karl; orcid: 0000-0003-3539-8939; Titchiner, Gabriel R.; orcid: 0000-0002-6378-9884; Levy, Colin; Hay, Sam; orcid: 0000-0003-3274-0938; Leys, David; orcid: 0000-0003-4845-8443; email: david.leys@manchester.ac.uk (Nature Publishing Group UK, 2021-08-20)
      Abstract: The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.