Crystal Structure of Amine Oxidase from Bovine Serum Michele Lunelli 1 , Maria Luisa Di Paolo 1 , Marianna Biadene 2 Vito Calderone 2 , Roberto Battistutta 2,3 , Marina Scarpa 4 , Adelio Rigo 1 and Giuseppe Zanotti 2,3 * 1 Department of Biological Chemistry, University of Padua Viale G. Colombo 3 35121 Padua, Italy 2 Department of Chemistry University of Padua, and ICTB Section of Padua, Via Marzolo 1 35131 Padua, Italy 3 Venetian Institute of Molecular Medicine, Via Orus 2 35127 Padua, Italy 4 Department of Physics and INFM, University of Trento Via Sommarive 14 38050 Povo-Trento, Italy Copper-containing amine oxidase extracted from bovine serum (BSAO) was crystallized and its three-dimensional structure at 2.37 A ˚ resolution is described. The biological unit of BSAO is a homodimer, formed by two monomers related to each other by a non-crystallographic 2-fold axis. Each monomer is composed of three domains, similar to those of other amine oxidases from lower species. The two monomers are structurally equivalent, despite some minor differences at the two active sites. A large funnel allows access of substrates to the active-site; another cavity, accessible to the solvent, is also present between the two monomers; this second cavity could allow the entrance of molecular oxygen necessary for the oxidative reaction. Some sugar residues, bound to Asn, were still present and visible in the electron density map, in spite of the exhaustive deglycosylation necessary to grow the crystals. The comparison of the BSAO structure with those of other resolved AO structures shows strong dissimilarities in the architecture and charge distribution of the cavities leading to the active-site, possibly explaining the differences in substrate specificity. q 2005 Elsevier Ltd. All rights reserved. Keywords: copper-containing amine oxidase; CAO; topaquinone; primary amines; crystal structure *Corresponding author Introduction Copper-containing amine oxidases (CAOs; amine–oxygen oxidoreductase (deaminating) (cop- per/TPQ-containing); EC 1.4.3.6) are a class of ubiquitous enzymes that catalyze the oxidation of primary amines into corresponding aldehydes, with reduction of molecular oxygen to hydrogen peroxide and the release of ammonia: 1–3 R ÿ CH 2 K NH C 3 C O 2 C H 2 O ÿÿÿÿ / AO R ÿ CHO C NH C 4 C H 2 O 2 CAOs are found in a wide variety of organisms. In mammals, they are widely present in many organs and tissues, such as vascular smooth cells, adipo- cytes, placenta, kidney, liver spleen, and plasma, showing tissue specificity in different species. 4 Furthermore, two forms of CAOs are present: a membrane-bound form and a soluble form found in plasma. 5 The latter presumably arises from the proteolytic cleavage of the membrane-bound enzyme, 6 known also as vascular-adhesion protein- 1 (VAP-1). 7,8 CAOs are involved in many physiological pro- cesses and metabolic pathways related to the biological function of their natural substrates (amines) and reaction products (hydrogen peroxide and aldehydes). 9 In mammals, CAOs have been associated with cell proliferation, differentiation, development of cells, apoptosis, detoxification, and cell signaling. In particular, VAP-1 is involved in glucose uptake and lipolysis in adipocytes, as well as cell adhesion and granulocyte extravasation 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Present addresses: M. Biadene, Institute of Inorganic Chemistry, University ofGoettingen, Tammanstr. 4, D37077 Goettingen, Germany; V. Calderone, Department of Chemistry, University of Siena, Via Aldo Moro 1, 53100 Siena, Italy. Abbreviations used: AGAO, Arthrobacter globiformis amine oxidase; BSAO, bovine serum amine oxidase; CAO, copper amine oxidase; ECAO, Escherichia coli amine oxidase; HPAO, Hansenula polymorpha amine oxidase; NAcGlc, N-acetyl-D-glucosamine; PPLO, Pichia pastoris lysyl oxidase; PSAO, pea seedling amine oxidase; rmsd, root mean square deviation; TPQ, 2,4,5-trihydroxy- phenylalanine quinine; VAP-1, human vascular-adhesion protein 1. E-mail address of the corresponding author: giuseppe.zanotti@unipd.it doi:10.1016/j.jmb.2004.12.038 J. Mol. Biol. (2005) 346, 991–1004