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Copper Amine Oxidase from Hansenula Polymorpha

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Copper Amine Oxidase from Hansenula Polymorpha Research Article 293 Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 Å resolution reveals the active conformation Rongbao Li1†, Judith P Klinman2* and F Scott Mathews1* Background: Copper-containing amine oxidases (CAOs) are widespread in Addresses: 1Department of Biochemistry and nature. These enzymes oxidize primary amine substrates to the aldehyde Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA and product, reducing molecular oxygen to hydrogen peroxide in the process. 2Departments of Chemistry and Molecular and Cell CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified Biology, University of California, Berkeley, CA tyrosine sidechain utilized as a redox cofactor. The methylamine oxidase from 94720, USA. the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a † preference for small aliphatic amine or phenethylamine substrates. The enzyme Present address: Biomolecular Structure Center, University of Washington, Seattle, WA 98195, is dimeric with a subunit molecular weight of 78 kDa. Structural studies are USA. directed at understanding the basis for cofactor biogenesis and catalytic efficiency. *Corresponding authors. E-mail: [email protected] [email protected] Results: The X-ray crystal structure of HPAO has been solved at 2.4 Å resolution by a combination of molecular replacement and single isomorphous Key words: amine oxidase, enzyme mechanism, replacement followed by refinement using sixfold symmetry averaging. The topaquinone, type II copper protein, X-ray electron density at the catalytic site shows that the TPQ conformation crystallography corresponds to that of the active form of the enzyme. Two channels, one on Received: 10 November 1997 either side of TPQ, are observed in the structure that provide access between Revisions requested: 18 December 1997 the active site and the bulk solvent. Revisions received: 12 January 1998 Accepted: 12 January 1998 Conclusions: The structure shows TPQ in a position poised for catalysis. This Structure 15 March 1998, 6:293–307 is the first active CAO structure to reveal this conformation and may help further http://biomednet.com/elecref/0969212600600293 our understanding of the catalytic mechanism. On the substrate side of TPQ a water-containing channel leading to the protein surface can serve as an © Current Biology Ltd ISSN 0969-2126 entrance or exit for substrate and product. On the opposite side of TPQ there is direct access from the bulk solvent of the dimer interface by which molecular oxygen may enter and hydrogen peroxide depart. In addition, a network of conserved water molecules has been identified which may function in the catalytic mechanism. Introduction RNA. Therefore, the CAOs may regulate fundamental cel- Copper-containing amine oxidases (CAO; E.C. 1.4.3.6) are lular processes such as tissue differentiation, tumor growth ubiquitous quinoenzymes which catalyze the oxidation of and programmed cell death. One of the reaction products primary amines by molecular oxygen to the corresponding of CAOs, hydrogen peroxide, may also play a role in cell aldehydes and hydrogen peroxide (Equation 1) [1–3]. regulation by acting as an intracellular second messenger. It has recently been shown that hydrogen peroxide can act + → + RCH2NH3 +O2+H2O RCHO + NH4 +H2O2 (1) as a signal transducing molecule in vascular smooth muscle, evoking responses including tyrosine phosphoryla- CAOs belong to the biologically important class of ‘type 2’, tion, mitogen-activated protein (MAP) kinase stimulation or ‘non-blue’, copper proteins and utilize topaquinone and DNA synthesis [4]. Another important CAO, lysyl (TPQ), a modified tyrosine sidechain, as a redox cofactor oxidase, catalyzes connective tissue maturation by cross- to achieve deamination. In microorganisms, these enzymes linking elastin and collagen. The redox cofactor of lysyl enable the amine substrate to be used as a source of carbon oxidase has recently been identified as lysine tyro- and nitrogen during growth. In higher organisms, the func- sylquinone, a derivative of TPQ in which the O2 atom is tion of CAOs is not well understood, but they appear to be displaced by a lysine sidechain, resulting in an intra- involved in physiologically important processes related to molecular cross-bridge [5]. the biological function of their natural substrates. For example, polyamines are important for transcription and For TPQ-containing enzymes, the precursor tyrosine translation of proteins and the synthesis of DNA and residue (Tyr‡) has been located in a highly conserved 294 Structure 1998, Vol 6 No 3 Figure 1 Proposed reaction scheme for the oxidation of + RCH2NH3 H2O amines by copper amine oxidase (CAO) and 2+ TPQ 2+ TPQ the roles for the cofactor topaquinone (TPQ) O Cu Cu O and the active-site base. The enzymatic OH OH 2 O 2 reaction consists of reductive and oxidative –O + N H –O – – O C-Asp O H half-reactions. During the reductive half- O CH C-Asp O reaction (right-hand side of the diagram), the R amine substrate is oxidized to the aldehyde I Oxidized TPQ II Substrate Schiff base product and the enzyme becomes reduced through the formation of two Schiff base intermediates (II and III); proton abstraction is catalyzed by the active-site base identified as NH + 4 aspartic acid (Asp319 for HPAO). During the + Proton abstraction H3O oxidative half-reaction (left-hand side of the diagram), the reduced enzyme can undergo electron and proton transfer to molecular oxygen to form hydrogen peroxide and 2+ TPQ TPQ O 2+ Cu OH species V. Release of an ammonium ion yields Cu – the starting enzyme (I). OH – + – OH NH O + – – 2 N H HO O C-Asp O O CH C-Asp O R V Iminoquinone intermediate III Product Schiff base H2O O2 H2O2 RCHO TPQ 2+ OH Cu – OH + – NH3 O HO C-Asp O IV Reduced TPQ Structure ‡ + → consensus sequence, Thr-X-X-Asn-Tyr -Asp/Glu-Tyr. Eox + RNH3 RCHO + Ered (2a) The second tyrosine is present in all CAOs, with the → + exception of the plant enzymes that contain an asparagine Ered +O2 Eox +H2O2+NH4 (2b) residue in its place [6]. There is evidence that the forma- tion of the TPQ cofactor occurs via a self-catalytic mecha- During the reductive half-reaction, the amine is oxidized nism, in which the Tyr‡ is oxidized by molecular oxygen to the aldehyde and the enzyme becomes reduced in a reaction catalyzed by protein-bound copper, rather through the formation of two separate Schiff base interme- than in a specific enzyme-catalyzed reaction [7,8]. In the diates (Figure 1; steps II and III) [1,10]. Schiff base forma- light of the ‘self processing’ of the precursor tyrosine to tion has been shown to occur at the O5 position of the TPQ, the conserved active site consensus sequence must TPQ ring [11]. During the oxidative half-reaction (Equa- play a role different from one of recognition by a substrate tion 2b; Figure 1 steps IV to V), the enzyme reacts with biogenetic enzyme system. From recent mutagenesis oxygen and ammonium ion is released. Turowski et al. studies by Cai et al. [9], at least one residue in the consen- [12] have proposed that the oxidative half-reaction occurs sus sequence (the asparagine that precedes TPQ) has by way of the intermediacy of the semiquinone of TPQ been proposed to play a role in the proper orientation of and reduced Cu+. the mature TPQ ring. CAO from the methylotrophic yeast Hansenula polymorpha Amine oxidation occurs via a ping pong mechanism is localized in the peroxisomes [13]. At least two isoforms (Figure 1), consisting of two half-reactions [1,10], a reduc- of CAO are present: one with preference for aromatic tive half-reaction (Equation 2a) and an oxidative half-reac- amines (benzylamine oxidase) and the other with prefer- tion (Equation 2b). ence for small aliphatic amines or phenethylamines Research Article Copper amine oxidase Li, Klinman and Mathews 295 (methylamine oxidase). The methylamine oxidase isoform structure (Figure 2c) and with TPQ in an alternative con- (HPAO) has been overexpressed in Saccharomyces cerevisiae formation (Figure 2d). The results of these tests conclu- [14]. Like other CAOs, HPAO is dimeric in solution; it has sively showed the SIR-derived TPQ orientation had been a subunit molecular weight of 78 kDa and is about 2% gly- correctly identified. cosylated. The amino acid sequence of HPAO is approxi- mately 35% identical to Arthrobacter CAOs, 28% identical The refined model of HPAO has good geometry to Escherichia coli amine oxidase (ECAO) and about 23% (Table 1). The Ramachandran plot indicates that 83.9% of identical to other eukaryotic CAOs [15]. A considerable the non-glycine and non-proline residues are in the most amount of spectroscopic and mechanistic data exist for favored region and 15.6% in the additionally allowed wild-type HPAO [14] and for a number of site-specific regions [21]. Only six residues, Ala403 in each of the six mutants [9,16]. The crystallization and preliminary struc- subunits, are in a disallowed region of conformational ture analysis of HPAO have been reported [17]. space. The correlation of the R factor and the resolution [22] indicates an approximate value of 0.25 Å for the Here we describe the crystal structure of HPAO which has overall error in atomic coordinates. The presence of six been determined to a resolution of 2.4 Å. Each subunit identical copies of the HPAO monomer in the asymmetric contains a large β-sandwich domain and two small α/β unit leads to an improved accuracy of the structure, as the domains, one TPQ and one copper atom, the latter being ratio of observations to parameters in the refinement using coordinated by three histidine sidechains and two water NCS restraints is effectively increased. This is reflected in molecules. The structure shows the TPQ in a conforma- the relatively small deviation (~4%) between the R factor tion poised for catalysis with the O5 atom situated near and Rfree.
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