Structure and Function of Diamine Oxidase

Diamine oxidase (DAO) catalyzes the oxidative deamination of histamine and other diamines. The enzyme is a member of the class of copper-containing amine oxidases that are characterized by possessing the active site cofactor topa quinone. Previous studies of mammalian DAO proteins revealed their basic structural and enzymatic properties, the tissue-specific expression pattern, and the cellular and subcellular localisation. In order to learn more about the structure and function of DAO, these studies will be extended by carefully analyzing the enzymatic properties of mammalian DAO enzymes and by studying their interaction with other proteins and with the glycosaminoglycan heparin. Additionally, it will be attempted to solve the crystal structure of porcine DAO. The, genes encoding mammalian DAO proteins will be further characterized and the regulation of their expression will be studied in cultured cells. In order to clarify the in vivo function of the enzyme, DAO knockout mice will be produced and characterized especially with regard to alterations of histamine and polyamine degradation. Besides, the intracellular processing of DAO and substrate conversion by DAO will be studied in a cell culture model. Changes of the expression and activity of DAO and other histamine degrading enzymes will be analyzed in patients with inflammatory and neoplastic intestinal diseases to assess the importance of altered enzyme function for disease progression. Patients will also be analyzed for DAO and histamine N- methyltransferase gene polymorphisms to evaluate a possible genetic predisposition for developing these lesions.

Histamine is a small mediator formed from the amino acid histidine that induces smooth muscle contraction and stimulates gastric acid secretion but also acts as a neuromodulator and mediates inflammatory and allergic reactions. The aims of the project were the structural and functional characterization of the histamine inactivating enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HNMT). Native DAO and HNMT proteins were purified from various tissues and recombinant forms from bacteria and their biochemical and enzymatic properties were determined. Genes and cDNAs encoding HNMT, DAO and homologous copper amine oxidases were characterized and their expression was studied in tissues and in cultured cells. DAO turned out to be a secretory protein expressed in a limited number of tissues such as intestine and kidney that is likely responsible for the inactivation of extracellular histamine. In contrast, HNMT as a cytosolic protein inactivates intracellular histamine and was found in all tissues analysed. The presence of a constitutive histamine inactivation capacity implies that histamine has a more diverse signalling function than previously thought.