Crystallographic and mechanistic studies on selected enzymes

Enzyme sind Biomoleküle, die eine Vielzahl verschiedener biochemischer Prozesse katalysieren. Ihrer Bedeutung für lebende Systeme wird dadurch sichtabr, dass fast alle physiologischen Prozesse in unserem Körper direkt oder indirekt von einer enzymatischen Reaktion abhängen. In letzter Zeit werden Enzyme außerdem auch in vielen biotechnologischen Prozessen angewendet (z.B. in der Lebensmittelindustrie, in der Lederherstellung, zur Synthese organischer Moleküle und für neuartige medikamentöse Therapien). Studien zur Struktur und zum Mechanismus von Enzymen werfen Licht auf die molekularen Ereignisse während der Katalyse. Diese Dissertation handelt von kristallographischen Studien an DPP III (Di peptidyl Peptidase III) aus Saccharomyces cerevisiae und an DAAO (D-Aminosäure-Oxidase) aus Trigonopsis variabilis. In einem weiteren Abschnitt werden werden auch experimentelle und rechnerische Studien zu Hydroxynitrillyasen beschrieben. Die dreidimensionale Struktur von DPP III aus der Bäckerhefe zeigte eine neuartige Proteinfaltung. Im vergleich mit homologen Enzymen aus Säugetieren weist das Hefeenzym eine besondere Sequenzähnlichkeit rund um das aktive Zentrum des Enzyms auf. Die Struktur erlaubte auch eine Analyse des Reaktionsmechanismus und der Substratselektivität des Enzyms. Sie bildet außerdem die Grundlage für die rationale Entwicklung von Arzneimitteln für die Schmerz-Therapie.Die D-Aminosäure-Oxidase aus Trigonopsis wurde als ein wichtiger Biokatalysator, vor allem in der industriellen Produktion von Cephalosporin-Antibiotika, verwendet. Kristallographische Studien an diesem Enzym und an Komplexen mit Cephalosporin und syn-(D)-phenylserin lieferte Erklärungen von Eigenschaften bestimmter Enzymvarianten im industriellen Einsatz.Enzymes are biomolecules, which catalyze various biochemical processes. Their importance to life can be understood by the fact that almost every physiological process in our body is linked directly or indirectly to an enzymatic action. Apart from this, enzymes are also extensively used in many biotechnological applications including food processing, leather manufacturing, organic molecule synthesis and novel drug therapy to name a few. Structural and mechanistic studies on enzymes throw light on the molecular events during catalysis. This thesis constitutes crystallographic studies of DPP III (Di peptidyl peptidase III) from Saccharomyces cerevisiae and DAAO (D-amino acid oxidase) from Trigonopsis variabilis. In the later part, computational as well as experiment mechanistic studies on hydroxynitrile lyase enzymes are also performed.The structure of yeast DPP III exhibits a novel fold. Sequence conservation to mammalian homologs is maximal around the active site cleft. The structure was analyzed with respect to the reaction mechanism of the enzyme and its substrate selectivity. It also provides the basis for a rational drug design in pain therapy.The D-amino acid oxidase from Trigonopsis has been used as an important industrial biocatalyst, mainly in the cephalosporin antibiotic production. Crystallographic studies involving this enzyme as well as complexes with cephalosporin and syn-(D)-phenyl serine help to rationalize site-specific mutations designed to improve its industrial use.In the mechanistic studies on several, different hydroxynitrile lyases, a positive potential is calculated in all active sites, irrespective of the different structural fold. Based on our mechanistic findings we tried to graft this active site on a homologous esterase structure through structure-guided approach.Pravas Kumar BaralAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung dt. u. engl.Graz, Univ., Diss., 2008(VLID)20124

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion

Conversion of the cellular prion protein PrPC into its pathogenic isoform PrPSc is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti‐prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis‐folding events

Structure-Function Correlations of Two Highly Conserved Motifs in Saccharomyces cerevisiae Squalene Epoxidase▿

Saccharomyces cerevisiae squalene epoxidase contains two highly conserved motifs, 1 and 2, of unknown function. Amino acid substitutions in both regions reduce enzyme activity and/or alter allylamine sensitivity. In the homology model, these motifs flank the flavin adenine dinucleotide cofactor and form part of the interface between cofactor and substrate binding domains

The crystal structure of an octapeptide repeat of the Prion protein in complex with a fab fragment of the POM2 antibody

Prion diseases are progressive, infectious neurodegenerative disorders caused primarily by the misfolding of the cellular prion protein, PrP(c) , into an insoluble, protease-resistant, aggregated isoform termed PrP(sc) . In native conditions, PrP(c) has a structured C-terminal domain and a highly flexible N-terminal domain. A part of this N-terminal domain consists of 4-5 repeats of an unusual glycine rich, eight amino acids long peptide known as the octapeptide repeat (OR) domain. In this paper, we successfully report the first crystal structure of an octapeptide repeat of PrP(c) bound to the POM2 Fab antibody fragment. The structure was solved at a resolution of 2.3 Å by molecular replacement. While several studies have previously predicted a β-turn like structure of the unbound octapeptide repeats, our structure shows an extended conformation of the octapeptide repeat when bound to a molecule of the POM2 Fab indicating that the bound Fab disrupts any putative native β turn conformation of the octapeptide repeats. Encouraging results from several recent studies have shown that administering small molecule ligands or antibodies targeting the OR domain of PrP result in arresting the progress of peripheral prion infections both in ex vivo and in in vivo models. This makes the structural study of the interactions of POM2 Fab with the OR domain very important as it would help us to design smaller and tighter binding OR ligands

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G(25)S, D(335)X (W, F, P), and G(210)A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G(30)S and L(37)P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R(269)G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines

Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody

Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°

Phosphorylation Status of 72 kDa MMP-2 Determines Its Structure and Activity in Response to Peroxynitrite

<div><p>Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO⁻) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following <i>in vitro</i> treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO⁻ treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1–1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO⁻. Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO⁻ activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2.</p></div

Effect of MMP-2 phosphorylation status in regard to activity changes following ONOO^- treatment using troponin I (TnI) as a known intracellular substrate.

<p>(A) Representative time-dependent troponin I (TnI) hydrolysis by 0.3 µM ONOO⁻ - treated, native (upper panel) or dephosphorylated (lower panel) 72 kDa MMP-2, in the presence of 30 µM GSH, following different incubation times (30, 60, or 120 min) at 37°C with TnI. Representative Coomassie blue stained SDS-PAGE gels. (B) Quantitative analysis of TnI hydrolysis by native (left) or dephosphorylated (right) 72 kDa MMP-2, treated with different concentrations of ONOO⁻ (0–0.3 µM) or DPN, in the presence of 30 µM GSH. Incubation for 120 min at 37°C. Mean ± SEM, N = 4–7/group. * p<0.05 compared with control (C, TnI alone). DPN, decomposed peroxynitrite.</p