Modular polyketide synthases and nonribosomal peptide synthetases are molecular assembly lines consisting of several multienzyme subunits that undergo dynamic self-assembly to form a functional mega-complex. N-and C-terminal docking domains are usually responsible for Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at max 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a -aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS.
Prolyl oligopeptidase family enzymes regulate the activity of biologically active peptides and peptide hormones, and they are implicated in diseases, including amnesia, depression, diabetes, and trypanosomiasis. Distinctively, these enzymes hydrolyze only relatively short peptide substrates, while large structured peptides and proteins are not usually cleaved. Prolyl oligopeptidase has a C-terminal alpha/beta-hydrolase catalytic domain that is similar to lipases and esterases. An N-terminal beta-propeller domain regulates access to the buried active site, explaining the observed oligopeptidase activity. The catalytic and regulatory mechanisms have been investigated using a combination of X-ray crystallography, site-directed mutagenesis, and enzyme kinetic measurements. Crystal structures have now been determined for representative members of three of the four subfamilies and are facilitating a better understanding of the structure-function properties of these physiologically and pharmaceutically important enzymes.
D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure.
Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 M to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.
Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp 641 residue of the catalytic triad (Ser 554 , Asp 641 , His 680 ) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k cat /K m for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k cat /K m . This indicates that the contribution of Asp 641 is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His 680 with Z-Gly-Pro-CH 2 Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His 680 is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.