Christopher M. Topham

The nociceptin (ORL1) receptor: molecular cloning and functional architecture

Nociceptin and the ORL1 receptor share high sequence similarity with opioid peptides, particularly dynorphin A, and their receptors. However, nociceptin and dynorphin A may use distinct molecular pathways to bind and activate their cognate receptors. Activation of the kappa-opioid receptor by dynorphin A is thought to require interactions of its N-terminal hydrophobic domain (Y(1)GGF) with the receptor opioid binding pocket, located within the transmembrane helix bundle, while activation of the ORL1 receptor appears to require interactions of the positively charged core (R(8)KSARK) of nociceptin with the negatively charged second extracellular receptor loop.

Role of glycoside phosphorylases in mannose foraging by human gut bacteria.

Background: The relations between the gut microbiota, food, and host play a crucial role in human health. Results: Prevalent bacterial glycoside phosphorylases are able to break down dietary carbohydrates and the N-glycans lining the intestinal epithelium. Conclusion: GH130 enzymes are new targets to study interactions between host and gut microbes. Significance: Glycoside phosphorylases are key enzymes of host glycan catabolism by gut bacteria. To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible modifier.

A generalized theoretical treatment of the kinetics of an enzyme-catalysed reaction in the presence of an unstable irreversible inhibitor (or activator) is presented. Analytical expressions describing the time-dependence of product formation have been derived in coefficient form amenable to non-linear regression analysis for two operationally distinct types of reaction mechanism dependent on whether the reaction of the unstable modifier (X) with either or both the free enzyme (E) and enzyme-substrate complex (ES) occurs as a simple bimolecular process, or proceeds through the intermediacy of either or both adsorptive enzyme-modifier (EX) and enzyme-modifier-substrate (EXS) complexes in what may be considered as an extension of the Botts-Morales general modifier mechanism for (stable) reversible enzyme inhibitors and activators. Special cases of both models are classified in an analogous way to the traditional naming of reversible enzyme modifications, and guidelines concerning tests of mechanism and determination of kinetic parameters are given. In particular, it has been shown that kinetic constants describing enzyme inactivation by an unstable site-specific inhibitor forming a reversible EX complex prior to covalent modification step may be determined from a single progress curve. Kinetic analysis of the extended Botts-Morales mechanism describing irreversible enzyme inactivation has demonstrated that analytical expressions describing the time-course of product formation may be derived for a stable modifier by retaining the usual steady-state assumptions regarding the fluxes around ES and EXS provided quasi-equilibrium modifier binding to E and ES is assumed, but for unstable modifiers all of the binding steps must be assumed to be at quasi-equilibrium in the steady-state, except under restrictive circumstances.

Direct Identification of a Peptide Binding Region in the Opioid Receptor-like 1 Receptor by Photoaffinity Labeling with [Bpa10,Tyr14]Nociceptin

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe10,Tyr14]nociceptin ([Bpa10,Tyr14]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa10,Tyr14]noc binds the receptor with high affinity (K i ∼0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC50 ∼0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa10,Tyr14]noc results in the irreversible labeling of a glycoprotein of ∼65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (∼6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of ∼3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu295 → Asp mutant receptor also yields a single labeled fragment of ∼6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296–302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa10]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile300 within the experimentally identified photoreactive region.

Direct identification of a peptide binding region in the ORL1 receptor by photo-affinity labelling with [Bpa10, Tyr14]nociceptin

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe10,Tyr14]nociceptin ([Bpa10,Tyr14]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa10,Tyr14]noc binds the receptor with high affinity (K i ∼0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC50 ∼0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa10,Tyr14]noc results in the irreversible labeling of a glycoprotein of ∼65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (∼6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of ∼3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu295 → Asp mutant receptor also yields a single labeled fragment of ∼6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296–302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa10]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile300 within the experimentally identified photoreactive region.

Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase

The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside‐hydrolases that naturally catalyzes the synthesis of α‐glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite −1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure‐based approach was followed to reshape the subsite −1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site‐directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8°C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite −1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.

Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyric acid

2,4-Dihydroxybutyric acid (DHB) is a molecule with considerable potential as a versatile chemical synthon. Notably, it may serve as a precursor for chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate, thus, targeting a considerable market in animal nutrition. However, no natural metabolic pathway exists for the biosynthesis of DHB. Here we have therefore conceived a three-step metabolic pathway for the synthesis of DHB starting from the natural metabolite malate. The pathway employs previously unreported malate kinase, malate semialdehyde dehydrogenase and malate semialdehyde reductase activities. The kinase and semialdehyde dehydrogenase activities were obtained by rational design based on structural and mechanistic knowledge of candidate enzymes acting on sterically cognate substrates. Malate semialdehyde reductase activity was identified from an initial screening of several natural enzymes, and was further improved by rational design. The pathway was expressed in a minimally engineered Escherichia coli strain and produces 1.8 g l−1 DHB with a molar yield of 0.15.

An Atomistic Statistically Effective Energy Function for Computational Protein Design.

Shortcomings in the definition of effective free-energy surfaces of proteins are recognized to be a major contributory factor responsible for the low success rates of existing automated methods for computational protein design (CPD). The formulation of an atomistic statistically effective energy function (SEEF) suitable for a wide range of CPD applications and its derivation from structural data extracted from protein domains and protein-ligand complexes are described here. The proposed energy function comprises nonlocal atom-based and local residue-based SEEFs, which are coupled using a novel atom connectivity number factor to scale short-range, pairwise, nonbonded atomic interaction energies and a surface-area-dependent cavity energy term. This energy function was used to derive additional SEEFs describing the unfolded-state ensemble of any given residue sequence based on computed average energies for partially or fully solvent-exposed fragments in regions of irregular structure in native proteins. Relative thermal stabilities of 97 T4 bacteriophage lysozyme mutants were predicted from calculated energy differences for folded and unfolded states with an average unsigned error (AUE) of 0.84 kcal mol(-1) when compared to experiment. To demonstrate the utility of the energy function for CPD, further validation was carried out in tests of its capacity to recover cognate protein sequences and to discriminate native and near-native protein folds, loop conformers, and small-molecule ligand binding poses from non-native benchmark decoys. Experimental ligand binding free energies for a diverse set of 80 protein complexes could be predicted with an AUE of 2.4 kcal mol(-1) using an additional energy term to account for the loss in ligand configurational entropy upon binding. The atomistic SEEF is expected to improve the accuracy of residue-based coarse-grained SEEFs currently used in CPD and to extend the range of applications of extant atom-based protein statistical potentials.

A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.