Dean Rea

Structure-function properties of prolyl oligopeptidase family enzymes

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 α/β-hydrolase catalytic domain that is similar to lipases and esterases. An N-terminal β-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.

Arabidopsis thaliana Circadian clock is regulated by the small GTPase LIP1

BACKGROUND At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcriptional/translational negative-feedback loops and generate a basic approximately 24 hr oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of the organism only if it corresponds to the natural day/night cycles. Light is the most effective signal in synchronizing the oscillator to environmental cycles. RESULTS The lip1-1 (light insensitive period 1) mutant isolated from the model plant Arabidopsis thaliana displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. In wild-type plants, period length shortens with increasing light fluence rates and the phase of rhythms can be shifted by light pulses administered to dark-adapted plants. In contrast, in lip1-1, period length is nearly insensitive to light intensity and significantly larger phase shifts (delays) can be induced during the subjective night. The mutant also displays elevated photomorphogenic responses to red and blue light, which cannot be explained by the circadian defect, suggesting distinct functions for LIP1 in the circadian light input and photomorphogenesis. The LIP1 gene encodes a functional, plant-specific atypical small GTPase, and therefore we postulate that it acts similarly to ZEITLUPE at postranscriptional level. CONCLUSIONS LIP1 represents the first small GTPase implicated in the circadian system of plants. LIP1 plays a unique negative role in controlling circadian light input and is required for precise entrainment of the plant clock.

Selective inhibition of carotenoid cleavage dioxygenases : phenotypic effects on shoot branching

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 μm to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.

Identification of Manganese Superoxide Dismutase from Sphingobacterium Sp. T2 as a Novel Bacterial Enzyme for Lignin Oxidation.

The valorization of aromatic heteropolymer lignin is an important unsolved problem in the development of a biomass-based biorefinery, for which novel high-activity biocatalysts are needed. Sequencing of the genomic DNA of lignin-degrading bacterial strain Sphingobacterium sp. T2 revealed no matches to known lignin-degrading genes. Proteomic matches for two manganese superoxide dismutase proteins were found in partially purified extracellular fractions. Recombinant MnSOD1 and MnSOD2 were both found to show high activity for oxidation of Organosolv and Kraft lignin, and lignin model compounds, generating multiple oxidation products. Structure determination revealed that the products result from aryl-Cα and Cα-Cβ bond oxidative cleavage and O-demethylation. The crystal structure of MnSOD1 was determined to 1.35 Å resolution, revealing a typical MnSOD homodimer harboring a five-coordinate trigonal bipyramidal Mn(II) center ligated by three His, one Asp, and a water/hydroxide in each active site. We propose that the lignin oxidation reactivity of these enzymes is due to the production of a hydroxyl radical, a highly reactive oxidant. This is the first demonstration that MnSOD is a microbial lignin-oxidizing enzyme.

Structure of thermobifida fusca DyP-type peroxidase and activity towards kraft lignin and lignin model compounds

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 λmax 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a β-aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8 Å 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.

Crystal Structure and Functional Assignment of YfaU, a Metal Ion Dependent Class II Aldolase from Escherichia coli K12†‡

One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of the Escherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg (2+)-pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the homologous 2-dehydro-3-deoxygalactarate aldolase (DDGA). Enzyme kinetic measurements show activity with the probable physiological substrate 2-keto-3-deoxy- l-rhamnonate, supporting the functional assignment, as well as the structurally similar 2-keto-3-deoxy- l-mannonate and 2-keto-3-deoxy- l-lyxonate (see accompanying paper: Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Hubbard, B. K., Delli, J. D., Babbitt, P. C., Almo, S. C., and Gerlt, J. A. (2008) Biochemistry 47, 9944-9954). YfaU has similar activity toward the HpcH substrate 4-hydroxy-2-ketoheptane-1,7-dioate and synthetic substrates 4-hydroxy-2-ketopentanoic acid and 4-hydroxy-2-ketohexanoic acid. This indicates a relaxed substrate specificity that complicates the functional assignment of members of this enzyme superfamily. Crystal structures suggest these enzymes use an Asp-His intersubunit dyad to activate a metal-bound water or hydroxide for proton transfer during catalysis.

The Loops Facing the Active Site of Prolyl Oligopeptidase are Crucial Components in Substrate Gating and Specificity.

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.

Expression, purification and preliminary crystallographic analysis of oligopeptidase B from Trypanosoma brucei

Recombinant oligopeptidase B from T. brucei has been prepared and crystallized. Data were collected to 2.7 Å. Heavy-atom soaks and preparation of selenomethionine-substituted protein are in progress for structure determination by MAD or MIR.

Prolyl Oligopeptidase Structure and Dynamics

Prolyl oligopeptidase or prolyl endopeptidase (PREP; EC 3.4.21.26) is an atypical serine protease that hydrolyses peptides and peptide hormones after proline in peptides up to around 30 residues long. Evidence suggests an involvement in learning and memory, and the enzyme is implicated in diseases including amnesia and depression. The first crystal structures determined, of the porcine enzyme, provided direct insight into the mechanisms of substrate size selectivity, substrate specificity, and catalysis. However in these structural studies the enzyme is in a closed state, even in the absence of ligand, leaving questions as to how substrates and products can enter and exit the enclosed central cavity that houses the active site. More recent crystal structures of bacterial PREP have captured the enzyme in an open state, revealing the true extent and nature of the structural dynamics involved, and illuminating an induced fit mode of catalysis and regulation. Molecular modeling has further contributed to our understanding of the conformational changes that occur during catalysis. Here we review the data that has led to our current understanding of the structure and dynamics of this biologically and pharmaceutically important enzyme.

Crystal structures of Trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes.

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.