L’importante représentation des amines chirales dans les molécules biologiquement actives a entrainé une forte dynamique de recherche autour de leur synthèse stéréosélective. A l’heure où l’impact environnemental est de plus en plus considéré, les procédés biocatalytiques sont apparus comme une alternative aux synthèses chimiques, utilisatrices de métaux de transitions et ligands chiraux difficiles à éliminer. Ces voies enzymatiques d’obtention d’amines chirales optiquement pures font majoritairement appel à des lipases (résolution cinétique) et des transaminases mais ces méthodes souffrent de certains désavantages comme de la perte du co-produit de réaction (transaminase). La synthèse enzymatique d’amines chirales à partir de composés cétones et d’ammoniac comme seule source d’azote s’est finalement présentée comme un réel défi pour l’industrie en 2007. En 2012, les premières amines déshydrogénases catalysant cette réaction d’amination réductrice ont été obtenues par ingénierie protéique, mais aucun représentant naturel n’a encore été identifié et caractérisé.Les travaux effectués au cours de cette thèse ont consisté tout d’abord à explorer la biodiversité afin de découvrir des gènes codant des enzymes natives catalysant cette réaction. Par approche génomique basée sur la comparaison de séquence, plusieurs AmDHs ont été identifiées. Trois d’entre elles ont fait l’objet d’études plus approfondies : DH2B1 issue de Petrotoga mobilis, DH13B5 venant de Mycobacterium smegmatis et DH13F8 de Cystobacter fuscus. Grâce à la résolution de leur structure 3D par nos collaborateurs, mais aussi par l’établissement de leur spectre de substrat, de la création de mutant impliquant les résidus du site actif et de la détermination des constantes cinétiques, un mécanisme réactionnel a pu être proposé. Les conditions réactionnelles d’utilisation de DH2B1, DH13B5 et DH13F8 ont ensuite été optimisées pour les synthèses du (4S)-4-aminopentanoate, cis-(1S,2R)-2-méthylcyclohexyl- amine, cis-(1S,3R)-3-méthylcyclohexylamine et (2S)-pentan-2-amine, et ont permis l’obtention de ces produits avec des rendements de 35-88% et de très bons excès énantiomériques (>97%). La découverte et la caractérisation de ces enzymes ouvrent des perspectives pour la synthèse biocatalysée d’amines chirales variées par des homologues natifs ou fruit d'un travail d'ingénierie. / Widely widespread in small biologically active molecules, chiral amines have given rise to an increasing amount of research directed towards the development of asymmetric synthesis. As the environmental impact is increasingly taken into account, biocatalytic routes appear as a benefitial alternative to chemical synthesis that uses expensive transition metals and chiral ligands that are difficult to remove. Among these eco-friendly processes to obtain pure chiral amines, lipases (dynamic resolution) and transaminases are the most common ones, but suffer from drawbacks, particularly the loss of the amine donor in case of transaminases. The enzymatic synthesis of chiral amines starting from ketones and ammonia, as the only nitrogen source, appeared as a real challenge for the industry. In 2012, the first amine dehydrogenases, catalyzing this reductive amination reaction, were obtained by protein engineering and no native representatives had yet been identified.These research studies have firstly consisted in exploring the biodiversity in order to discover gene coding for native enzymes capable to catalyze this reductive amination. By pairwise sequence alignment-based approach, several AmDHs were identified and three of them were more extensively studied: DH2B1 from Petrotoga mobilis, DH13B5 from Mycobacterium smegmatis and DH13F8 from Cystobacter fuscus. Thanks to their external collaborative 3D structural resolution, substrate scope studies, active site mutant engineering and the kinetic constants determination, a mechanism was postulated. The reaction conditions of DH2B1, DH13B5 and DH13F8 were then optimized for the synthesis of (4S)-4-aminopentanoate, cis-(1S,2R)-2-methylcyclohexyl- amine, cis-(1S,3R)-3-methylcyclohexylamine and (2S)-pentan-2-amine obtained with 35 to 88 % yield and excellent enantiomeric/diastereosiomeric excesses (>97 %).The discovery and characterization of these native amine dehydrogenases offer new perspectives for the biocatalytic synthesis of various chiral amines by native or engineered homologs.

Th.--Sci. phys.--Grenoble--I.N.P., 1983. N°: D.E 150.

No description available.

Thesis (Ph. D.)--California Institute of Technology, 1996. UM #9617425. / Advisor names found in the Acknowledgments pages of the thesis. Title from home page. Viewed 01/19/2010. Includes bibliographical references.

Lipoprotein(a) (Lp(a)) has been identified as an emerging risk factor for the development of vascular diseases. The Lp(a) particle is assembled in a 2-step process upon secretion of the LDL and apo(a) components from hepatocytes. Work done by the Koschinsky group has identified an oxidase-like activity present in the conditioned medium (CM) harvested from human hepatoma (HepG2), as well as HEK 293 (human endothelian kidney) cells that catalyzes the rate of covalent Lp(a) formation. We have taken a candidate enzyme approach to identifying this oxidase activity. Specifically, we have proposed that the QSOX (Quiescin/sulfhydryl oxidase) is responsible for catalysis of covalent Lp(a) assembly. An oxidase activity assay developed by Dr. Thorpe (University of Delaware) was used to detect QSOX1 in CM harvested from cultured cell lines that catalyze covalent Lp(a) assembly. In addition, the QSOX1 transcript was identified in each cell line and quantified with the use of Real-Time RT-PCR. Quantitative assays of covalent Lp(a) assembly were performed to study some characteristics of the unkwown oxidase activity. First, conditioned medium was dialyzed through a 5 kDa cutoff, as this has previously been shown to reduce the aforementioned oxidase activity. Purified QSOX was then added back to the reaction and the rate of catalysis was observed. The addition of QSOX appeared to enhance the rate of covalent Lp(a) assembly in a dose-dependent manner. Additional covalent Lp(a) assembly assays were performed where various chemicals were added to determine whether Lp(a) assembly was affected. The addition of EDTA did not affect covalent assembly, suggesting that the oxidase activity may not be metallo-dependent. Moreover, dose-dependent addition of Calcium, DTT, Copper and glutathione to dialyzed medium also did not affect the rate of Lp(a) assembly. Taken together, these studies will aid in identifying the nature of the oxidase activity that catalyzes covalent Lp(a) assembly. This will provide us with valuable information on how Lp(a) particles are assembled, and may lead to the development of drugs inhibiting Lp(a) formation. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-29 12:13:24.786

No description available.

Thesis (M.S.)--University of Wisconsin--Madison, 1984. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 70-75).

No description available.

No description available.

Texte remanié de: Thèse--Sc. nat.--Aix-Marseille II, 1973. N°: 38. / C.N.R.S. A.O. 7027. L'article principal recouvrant en partie la thèse, extrait de Biochimie. T. 54, N° 9, 1972, pp. 1217-1219, est conservé sous la cote: [4° THS. Pièce. 661.