A 30 Å Long U-Shaped Catalytic Tunnel in the Crystal Structure of Polyamine

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Research Article 265

A 30 Å long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase Claudia Binda1, Alessandro Coda1, Riccardo Angelini2, Rodolfo Federico2, Paolo Ascenzi2 and Andrea Mattevi1*

Background: Polyamines are essential for cell growth and differentiation; Addresses: 1Dipartimento di Genetica e compounds interfering with their metabolism are potential anticancer agents. Microbiologia, Università di Pavia, Via Abbiategrasso 207, I-27100 Pavia, Italy and Polyamine oxidase (PAO) plays a central role in polyamine homeostasis. The 2Dipartimento di Biologia, Università Roma Tre, enzyme utilises an FAD cofactor to catalyse the oxidation of the secondary Viale Guglielmo Marconi 446, I-00146 Roma, Italy. amino groups of spermine and spermidine. *Corresponding author. E-mail: [email protected] Results: The first crystal structure of a polyamine oxidase has been determined to a resolution of 1.9 Å. PAO from Zea mays contains two domains, which define Key words: enzyme catalysis, flavoenzymes, a remarkable 30 Å long U-shaped catalytic tunnel at their interface. The structure monoamine oxidase, polyamine oxidase, X-ray of PAO in complex with the inhibitor MDL72527 reveals the residues forming the crystallography catalytic machinery and unusual enzyme-inhibitor CH...O H bonds. A ring of Received: 19 October 1998 glutamate and aspartate residues surrounding one of the two tunnel openings Revisions requested: 3 December 1998 contributes to the steering of the substrate towards the inside of the tunnel. Revisions received: 16 December 1998 Accepted: 18 December 1998 Conclusions: PAO specifically oxidises substrates that have both primary and Published: 24 February 1999 secondary amino groups. The complex with MDL72527 shows that the primary amino groups are essential for the proper alignment of the substrate with Structure March 1999, 7:265–276 respect to the flavin. Conservation of an N-terminal sequence motif indicates http://biomednet.com/elecref/0969212600700265 that PAO is member of a novel family of flavoenzymes. Among these, © Elsevier Science Ltd ISSN 0969-2126 monoamine oxidase displays significant sequence homology with PAO, suggesting a similar overall folding topology.

Introduction Despite these differences, the plant, bacterial and animal Spermidine and spermine are polyamine molecules essen- polyamine oxidases essentially share identical molecular tial for cell proliferation and differentiation [1,2]. It is well properties. PAO is a monomeric soluble protein with a mol- known that polyamines bind DNA. Moreover, some pro- ecular mass of about 53 kDa and a non-covalently bound teins such as the insulin receptor, protein kinase CK2, FAD molecule as cofactor [8]. As typically observed in certain potassium channels and N-methyl-D-aspartate flavin-dependent oxidases, the overall reaction catalysed (NMDA) receptors possess a specific polyamine-binding by PAO can be divided into a reductive half reaction, in site, possibly having a regulatory function [3,4,5]. Growing which the flavin is reduced upon polyamine oxidation, and evidence arises indicating that polyamine catabolism in an oxidative half reaction, in which the reduced flavin is plants is associated with physiological events such as lig- reoxidised by molecular oxygen with the release of hydro- nification, cell-wall stiffening and cellular defence [6,7]. gen peroxide (Figure 1a). Polyamine oxidation results in Polyamine levels are controlled by a major regulatory the formation of an imino compound, which is hydrolysed system that includes pathways for polyamine biosynthesis to produce the final products (Figure 1a) [7,8]. and degradation as well as for their transport across the cell membrane. PAO is a key enzyme in the polyamine catabolic pathway. The interest in polyamine metabolism has Polyamine oxidase (PAO) catalyses the oxidation of the increased with the discovery that some polyamine ana- secondary amino groups of polyamines (Figure 1a). The logues show antitumoral effect on a number of cell lines exact nature of the reduction product depends on the [9]. Although the precise mechanism of the cytotoxic source of the enzyme. Animal PAO transforms spermidine activity is not known, it has been noticed that the accu- and spermine into putrescine (1,4-diaminobutane) and mulation of these analogues leads to DNA fragmentation spermidine, respectively, plus 3-amino-propionaldehyde. and cell death by apoptosis [10]. PAO plays a crucial role Conversely, plant and bacterial polyamine oxidases catal- in these processes. For instance, some of the polyamine yse the conversion of spermidine and spermine to 4-amino- analogues, such as N1-ethyl-N11-[(cyclopropyl)methyl]- butyraldehyde and 3-aminopropyl-4-aminobutyraldehyde, 4,8-diazaundecane, can induce programmed cell death respectively, plus 1,3-diaminopropane [7] (Figure 1b). by increasing polyamine catabolism and inducing the 266 Structure 1999, Vol 7 No 3

Figure 1

(a) + NH2(CH2)3NHCH2(CH2)3NH2 [NH2(CH2)3NH=CH(CH2)3NH2] NH2(CH2)3NH2 + NH2(CH2)3CHO

- FAD FADH H2O

H2O2 O2 (b) Spermidine A 1 2 3 4 5 6 7 8 9 10 NH2(CH2)2CHO + NH2(CH2)4NH2 + H2O2 NH2CH2CH2CH2NH CH2CH2CH2CH2NH2 + O2 + H2O

NH2(CH2)3CHO + NH2(CH2)3NH2 + H2O2 AB B

Spermine A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 NH2(CH2)2CHO + NH2(CH2)4NH(CH2)3NH2 + H2O2 NH2CH2CH2CH2NH CH2CH2CH2CH2NHCH2CH2CH2NH2 + O2 + H2O

NH2(CH2)3NH(CH2)3CHO + NH2(CH2)3NH2 + H2O2 B A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14

CH2 CCHNHCH2 CH2CH2CH2CH2 NHCH2 CH C CH2 Structure

Substrates and inhibitor of PAO. (a) Scheme of spermidine-oxidation indicate the site of enzymatic attack in the mammalian and plant/bacterial reaction catalysed by PAO from Zea mays. (b) Oxidation of spermine PAO, respectively. (c) Chemical structure and atom numbering of the and spermidine with their relative reaction products. Labels A and B inhibitor N,N’-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527).

consequent production of toxic H2O2 [11]. Progress in the and crystallisation experiments. Furthermore, the maize analysis of the metabolism of these antineoplastic com- enzyme represents the first PAO of known primary struc- pounds is a promising route for the design of new drugs ture [6,7]. The X-ray analysis reveals that the overall topol- for the treatment of cancer [2]. ogy of PAO resembles that of other flavoproteins and reveals a remarkable U-shaped catalytic tunnel, which In the framework of a project devoted to the analysis of the extends throughout the structure. functional properties of flavoenzymes [12,13], we have carried out the structure determination of PAO from Zea Results and discussion mays L. in the native state and in complex with MDL72527, Structure determination a widely used inhibitor of the enzyme (Figure 1c). Com- The X-ray crystal structure of native PAO from Zea mays pared to animal PAO, the maize protein offers the advan- L. has been determined at 1.9 Å resolution by multiple tage that it can be purified from plant leaves in sufficiently isomorphous replacement (MIR) combined with solvent large quantities to carry out enzymological characterisation flattening and threefold averaging. After refinement, the Research Article Structure of polyamine oxidase Binda et al. 267

Figure 2

Fobserved–Fcalculated omit map of the MDL72527 inhibitor. The map is contoured at the 3σ level.

Structure

final model has an R factor of 19.9% for 216,570 reflections Fβ13) flanked by a β meander (Fβ8, Fβ9, Fβ10 and Fβ12) to the resolution of 1.9 Å and a free R factor of 23.4% for and three α helices (Fα1, Fα8 and Fα4). This arrangement 2000 randomly chosen reflections (see Figure 2 and corresponds to the classical folding topology observed in Table 1). The Ramachandran plot for the three crystallo- several dinucleotide-binding enzymes [14]. The substrate- graphically independent subunits shows that 90.3% of the binding domain is composed of two fragments (residues residues are in the most favoured regions and 9.4% in additionally allowed regions. Only Asp351, which belongs Table 1 to a type II′ turn connecting Sβ5 to Sα4 (Figure 3a), is outside the allowed regions, the conformation of this Refinement statistics. residue being unambiguously defined in the electron- density map. The native structure has been used as an Native MDL72527 initial model for the refinement of the PAO–MDL72527 Resolution range (Å)* 100.0–1.9 100.0–1.9 complex, which has been refined to an R factor of 19.3% Number of protein atoms† for 211,025 reflections up to 1.9 Å resolution and a free R subunit A 3766 3780 factor of 22.9% (Table 1). subunit B 3797 3811 subunit C 3821 3835 Rmsd for NCS restraints (Å)‡ The asymmetric unit of the crystals contains three PAO subunit A–subunit B 0.29 0.29 subunits (named A, B and C). In the description of the subunit B–subunit C 0.31 0.28 structure we refer to subunit C, as it displays a slightly subunit A–subunit C 0.21 0.20 2 Number of water molecules 678 629 lower average B factor for the protein atoms (14.7 Å ) in R factor (%)§ 19.9 19.3 comparison with the other two subunits (17.6 Å2 and Rfree (%; 2000 reflections) 23.4 22.9 15.7 Å2 for subunits A and B, respectively). However, the Rmsd from ideal values‡ three subunits are essentially identical, with root mean bond lengths (Å) 0.013 0.011 bond angles (°) 2.252 2.262 square (rms) deviations between subunits A and C and trigonal atoms (Å) 0.013 0.014 subunits B and C of 0.21 Å and 0.31 Å, respectively, for planar group (Å) 0.015 0.018 459 Cα atom pairs. non-bonded atoms (Å) 0.106 0.136

*All measured reflections were used for refinement (no σ cut-off). †Out The overall structure of a total of 472 amino acids for each monomer, subunit A comprises Maize PAO is a 53 kDa monomeric enzyme [7] consisting residues 5–463, whereas subunits B and C include residues 5–466. of 13 α helices and 19 β strands, which fold to form two ‡Root mean square deviations calculated for 459 Cα atom pairs. The well-defined domains (Figures 3a, 3b and 4). The FAD- rmsds from ideal values were calculated using the program TNT [39]. NCS restraints were applied to the three subunits during refinement. binding domain comprises three fragments (residues 7–87, § Σ Σ R factor = || Fobs |–|Fcalc || / |Fobs |. The indicated value refers to 187–292 and 412–466) whose main structural elements are the final R factor as calculated for all reflections (including those β β β β a central parallel sheet (strands F 1, F 2, F 11 and omitted for Rfree calculation). 268 Structure 1999, Vol 7 No 3

Figure 3

(a) (b)

380 380 Substrate-binding domain Sα5 100 100 Sβ1 340 340 Sβ3 Fβ4 Sβ4 Sβ6 320 320 Sα1 Sβ5 Fβ6 200 Sα3 160 360 160 360 200 Sβ2 Fβ7 300 300 Fβ3 Sα4 60 60 Fβ5 80 120 120 80 Fα2 Sα2 140 140 Fα3 400 440 40 220 400 440 40 220 Fα4 180 180 Fα5 Fα1 Fα8 20 20 Fβ2 Fα7 Fβ1 240 420240 β 420 β Fβ13 F 11 α F 12 F 6 Fβ10 280 260 280 260 Fβ9 Fβ8 460 460 C N FAD-binding domain Structure

The overall structure of PAO. (a) MOLSCRIPT [42] diagram of the β strands of the substrate-binding domain are indicated by the letter S, maize PAO subunit. The secondary structure elements of the FAD- followed by sequential numbers. Labels N and C indicate the binding domain are labelled with the letter F followed by numbers, N terminus and C terminus, respectively. (b) Stereo diagram of the Cα according to their order in the sequence. Similarly, helices and trace with every twentieth residue labelled.

88–186 and 293–411) and is characterised by a six-stranded less well-defined and only two N-acetyl-D-glucosamine mixed β sheet flanked by five α helices. The two domains residues could be modelled. So far, the role of glycosylation create a tunnel that defines the enzyme active centre at in PAO has not been established, although the glycosidic their interface (Figures 5a and 5b). residues are likely to represent a secretion signal related to the apoplastic localisation of the enzyme [7]. The folding topology of PAO resembles that of other flavoenzymes such as p-hydroxybenzoate hydroxylase The FAD-binding site [15,16], cholesterol oxidase [17], glucose oxidase [18] and The FAD prosthetic group is non-covalently bound to D amino acid oxidase [19,20]. The most closely related the protein and is deeply embedded within the structure structure is that of p-hydroxybenzoate hydroxylase, which (Figures 3b and 5a). All FAD atoms are solvent-inacces- displays an rms deviation from PAO of 3.9 Å for 217 Cα sible, with the exception of the flavin C5a, N5 and C4a atom pairs (the sequence identity for the equivalent atoms that line the active centre. The binding mode and residues is 15%). The similarity is particularly evident in the conformation of the ADP-ribityl fragment of the the FAD-binding domain, the sole divergence being due cofactor is very similar to that observed in the enzymes to residues 47–57 that in PAO form an antiparallel β sheet sharing the dinucleotide-binding fold [14]. The main (Fβ3 and Fβ4, Figures 3a and 4) absent in p-hydroxyben- features are (Figure 6): the H bonds between the N1 and zoate hydroxylase. More consistent structural variations N6 atoms of the adenine ring with the backbone atoms occur at the substrate-binding domain. The β sheet at the of Val237, at the beginning of Fβ8; the interactions of heart of this domain is essentially conserved but the sec- Glu35 and Tyr399 sidechains with the hydroxyl groups ondary structures around it are quite different. In particu- of the ribose; the compensation of the pyrophosphate lar, the major deviation involves residues 95–192 and negative charges by the backbone nitrogen and sidechain 313–330, which form five α helices (Sα1, Sα2, Sα3, Sα4 of Arg43, the mainchain nitrogen of Glu430 and the N and Sα5) that surround one side of the catalytic tunnel terminus of helix Fα1 (Ser15); and the involvement in (Figures 3a and 5a). the ADP-ribityl binding of seven ordered water molecules, present in the three crystallographically indepen- PAO is a glycosylated enzyme [21]; the electron-density dent subunits of both native and PAO–MDL72527 map shows that the glycosylation site is Asn77. In subunit complex structures. C, a branched chain of five ordered sugars, two N-acetyl-D- glucosamine residues as well as a fucose and two mannose The isoalloxazine ring is located at the interface of the residues have been modelled (not shown). The electron- two domains, with the re side facing the inner part of the density map at the glycosylation site of subunits A and B is substrate-binding site (Figures 5a and 7). The flavin Research Article Structure of polyamine oxidase Binda et al. 269

Figure 4

Fβ1 Fα1 Fβ2 Fβ3 Fβ4 Fβ5 Fα2 Fβ6 eeee hhhhhhhhhhhh eeee eeeee eeeeee eeee hhhhh eeee PAO ------ATVGPRVIVVGAGMSGISAAKRLSEAGITDLLILEATDHIGGRMHKTNFA-GINVELGANWVEGVNGGKMNPIWPIVNSTLKLRNFRSD 88 MAO-A MENQEKASIAGHMFDVVVIGGGISGLSAAKLLTEYGVS-VLVLEARDRVGGRTYTIRNEHVDYVDVGGAYVGPTQNRILRLSKELGIETYKVNVSERL * * * * * ** **** * * * * *** * *** * * * * * UUUUGAGUXGLXXAXXLXXXXXXXUXLUEXXXXUGGXXXXXXXXXGXXXEXG

Sβ1 Sα1 Sα2 Sα3 eee hhhhhhhhhhhhhhhhhhhhhh hhhhhhhhh hhhhhhhhhhhhhhh PAO FDYLAQNVYKEDGG---VYDEDYVQKRIELADSVEEMGEKLS--ATLHASGRDDMSILAMQRLNEHQPNGPATPVDMVVDYYKFDYEFAEPPRVTSLQ 181 MAO-A VQYVKGKTYPFRGAFPPVWNPIAYLDYNNLWRTIDNMGKEIPTDAPWEAQHADKWDKMTMKELID------KICWTKTARRFAYLFVNI-NVTSEP * * * * * ** * * * * * * * * ***

Fα3 Fβ7 Fα4 F β8 Fβ9 Fβ10 Fβ11 Fα5 hhhhhh eeeeee hhhhhhhh eeeeeee eeeeee eeee eee hhhhh PAO NTVPLATFSDF-----GDDVYFVADQRGYEAVVYYLAGQYLKTDDKSGKIVDPRLQLNKVVREIKYSPGGVTVKTEDNSVYSADYVMVSASLGVLQSD 274 MAO-A HEVSALWFLWYVKQCGGTTRIFSVTNGGQERKFVGGSGQ---VSERIMDLLGDQVKLNHPVTHVDQSSDNIIIETLNHEHYECKYVIN-AIPPTLTAK * * * * * * ** ** * * * * ** * *

Fβ12 Fα6 Sβ2 Sβ3 Sβ4 Sβ5 Sα4 Sα5 eee hhhhhhhhh eeeeeee eeeee eeee eeee hhhhhhhhh hhhhhhhhh PAO LIQFKPKLPTWKVRAIYQFDMAVYTKIFLKFPRKFWPEGKGREFFLYASSRRGYYGVWQEFEKQYPDANV--LLVTVTDEESRRIEQQSDEQTKAEIM 370 MAO-A -IHFRPELPAERNQLIQRLPMGAVIKCMMYYKEAFW---KKKDYCGCMIIEDEDAPISITLDDTKPDGSLPAIMGFILARKADRLAKLHKEIRKKKIC * * * ** * * * ** * ** * * * *

S β6 Fα7 Fβ13 Fα8 hhhhhh eee hhhhhhh eee hhhhhhhhhhhhhhhhhhh PAO QVLRKMFPGKDVPDATDILVPRWWSDRFYKGTFSNW-PVGVNRYEYDQL-RAPVGRVYFTGEHTSEHYNGYVHGAYLSGIDSAEILINCAQKKMCKYH 466 MAO-A ELYAKVLGSQEALHPVHYEEKNWCEEQYSGGCYTAYFPPGI-MTQYGRVIRQPVGRIFFAGTETATKWSGYMEGAVEAGERAAREVLNGLGKVTEKDI * * *| * * * * **** * * * ** ** * * * * * flavin C8α

PAO VQGKYD------472 MAO-A WVQEPESKDVPAVEITHTFWERNLPSVSGLLKIIGFSTSVTALGFVLYKYKLLPRS Structure

Structure-based sequence alignment of maize PAO and human flavoprotein family identified by Dailey & Dailey [29] is shown, where U monoamine oxidase A (MAO-A; accession code from the sequence is for hydrophobic residues and X is any residue. The label ‘flavin C8α’ data base P21397), which is taken as a representative protein of outlines the position of residue Cys452, which in MAO-A is covalently monoamine oxidases. The fingerprint motif characterising the bound to the flavin C8α atom.

dimethylbenzene ring is held in position by extensive van The active centre der Waals contacts with the sidechains of Trp393 and The PAO active centre consists of a remarkable ‘U-shaped’ Thr402, whereas the pyrimidine ring is engaged in four H tunnel, which passes through the protein structure at the bonds with backbone atoms (Figure 6). Moreover, the N5 interface between the substrate- and FAD-binding atom is H-bonded with a tightly bound water molecule, domains (Figures 5a and 5b). The tunnel extends for a which is kept in position by an additional interaction with length of about 30 Å but the U shape brings its two open- the ε-amino group of Lys300 and with the carbonyl ings onto the same side of the protein surface, at a distance oxygen of Gly57. An intriguing feature of the isoallox- of 16 Å from each other (measured by referring to Asp194 azine binding concerns the evident deviation from pla- and Asn437). Furthermore, the width of the tunnel is fairly narity displayed by the flavin, which adopts a sort of constant along its path, with a diameter of 3.8–4.3 Å, as cal- twisted conformation in both the native protein and in the culated from the van der Waals surface of the protein complex with MDL72527, with a 30° angle between the atoms lining the active centre. pyrimidine and the dimethylbenzene rings (Figure 7). A nonplanar conformation of the oxidised flavin is not The present description of the catalytic tunnel begins at unprecedented and has been observed in other oxidases the entrance shown on the right-hand side of Figures 5a, such as cholesterol oxidase [17] and NADH oxidase [22]. 5b and 8. This opening is framed by several solvent-acces- Interestingly, in the case of PAO, the distortion of the sible glutamate and aspartate sidechains (Asp117, Glu120, flavin makes the N5 atom point in the direction of the Glu121 and Glu124 on Sα1, as well as Asp194 and Asp195 substrate-binding site in the active-site tunnel (Figure 7). on loop Fα3–Fβ7), which form a sort of ‘carboxylate ring’ This feature suggests that the nonplanar conformation at the entrance of the tunnel (Figure 5b). Subsequently, might be useful in precisely aligning the flavin N5 atom the tunnel, which is surrounded by several aromatic with respect to the substrate. residues, goes deep into the protein, leading to the core of 270 Structure 1999, Vol 7 No 3

Figure 5

The catalytic U-shaped tunnel of PAO. (a) The (a) (b) Tunnel openings drawing illustrates the solvent-accessible surface of the tunnel, shown in chicken-wire representation (red). The MDL72527 inhibitor is depicted as a CPK model. The PAO subunit has been rotated by 70° about the vertical axis with respect to the orientation of Figure 3a. (b) Electrostatic potential surface of PAO. The molecule is approximately represented in the same orientation as in (a), so that the two entrances of the U-shaped tunnel are in evidence. Positive and negative potentials are represented in blue and red, respectively. The scale of the surface display ranges from –25 to +21 kT/e. The figure was produced using the program GRASP [44].

Structure

the catalytic site where the flavin ring is located. The 25.0°C; RF, unpublished results). The electron-density sidechains of Glu62 and Glu170 protrude in front of the map unambiguously reveals the position of the inhibitor flavin, forming the turning point around which the tunnel (Figure 2). Ligand binding does not cause any significant sharply bends and reverses its direction. These two car- conformational change of the enzyme, with a rms deviation boxylate groups are within H-bonding distance of one of 0.11 Å from all Cα atoms of the native structure. another (Figures 7 and 8), suggesting that the Glu62– MDL72527 binds in the central part of the catalytic tunnel, Glu170 pair is protonated. Another crucial element charac- adopting a conformation that closely matches the shape of terising the innermost section of the catalytic tunnel is the tunnel. This type of ‘lock-and-key’ binding mode built up by Phe403 and Tyr439. These two residues are (Figure 5a) makes the inhibitor molecule completely positioned parallel to each other and flank the tunnel on solvent-inaccessible. The bound inhibitor is involved in opposite sides. In this way, they define a kind of ‘aromatic extensive H-bond and van der Waals contacts with the sur- sandwich’, which embraces the section of the active centre rounding protein atoms (Figures 7 and 8). Moreover, it located immediately after the tunnel U-turn (Figures 7 interacts with the flavin ring, the shortest contact being the and 8). Finally, after Phe403 and Tyr439, the tunnel points 3.8 Å distance between the flavin N5 and the inhibitor C8. towards the protein surface (left-hand side in Figures 5a In fact, as a result of the tight packing between the cofactor and 8), being lined mainly by the carbonyl oxygen atoms of ring and the central diaminobutane unit of MDL72527 several backbone peptide bonds in this region. (Figure 1c), the flavin C4a–N5–C5a locus (Figure 6), which in the ligand-free enzyme is solvent-accessible, There is a marked contrast in the chemical nature of the two becomes shielded from the solvent upon inhibitor binding. arms of the U-shaped active centre. The right-hand-side arm (Figures 5b and 8) is lined mainly by aromatic residues The N5 and N10 secondary amino groups of MDL72527 are and it opens to the outside like a funnel with several acidic anchored to the protein by H bonds (Figure 7 and 8): N10 is sidechains on its rim (Figure 5b). In contrast, the other arm H-bonded to the sidechain of Tyr298, whereas N5 is within presents mostly oxygen atoms on its surface and it displays a H-bonding distance of the mainchain oxygen of Gly438. narrow entrance. This asymmetry in the chemical environ- Furthermore, the inhibitor N5 atom is positioned inside the ment of the active centre suggests that the substrate might Tyr439–Phe403 aromatic sandwich and is thus able to estab- be admitted into its binding site preferentially through only lish favourable interactions with the electron-rich aromatic one of the two tunnel openings. In this respect, the ring of rings [23]. A striking feature of the MDL72527 binding Asp and Glu residues, located on the right-hand-side mode is that six of the twelve inhibitor carbons are located entrance (Figures 5b and 8), seems to be suited to fulfilling within 3.4 Å distance of protein oxygens. These belong to the role of guiding the polyamine substrate into the tunnel. either mainchain carbonyls (Phe171, Glu170, Ser404 and Gly438) or sidechains (Asn405, Tyr439 and Glu62). With the Inhibitor binding exception of C1, the carbon atoms involved in the these con- The substrate analogue MDL72527 (Figure 1c) competi- tacts are either covalently bound (C4, C6, C9, C11, Figures 7 × –7 tively inhibits PAO with Ki = 5.5 10 M (pH = 6.5, and 8) or separated by only two covalent bonds (C7) from Research Article Structure of polyamine oxidase Binda et al. 271

Figure 6

Schematic drawing of the interactions between FAD and the protein. Hydrogen Lys300

bonds are indicated by dashed lines. The Gly57 Asn59 NH O interatomic distances are in angstroms. W Trp393 2 NH indicates ordered water molecules. 2.8 3.0 2.9 W 2.9

O Trp60

N 3.1 HN N 2.9 6 5a 4a 4 OC 7 5 3NH

8 2

N 10N 1 2.7

9 9a 10a W HO O

2.7 Thr402 HN HO Val440

3.1 OH O W 2.8 HO Tyr439 2.6 3.0 W

2.9 O W 2.6 NH

2.8 HN 3.0 O P O Ser15 2.8 Glu430 HO C O O 3.0 NH2 NH2 O P O 3.0 Arg43

2.8 HN NH O 2.8 Arg43 O N N W

3.0 OH OH N NH2 3.0 W 2.7 2.8 3.0 Val237 N OC 3.0

OH 2.9 HN NH O O Ala36

Glu35

Tyr399 Structure

the inhibitor N5 and N10 amino groups. These and deoxyhypusine synthase [27], the only other spermi- PAO–inhibitor interactions (see Figures 7 and 8) appear to dine-binding proteins for which the crystal structure has have the stereochemical characteristics of the so-called been determined. In these two proteins, the spermidine CH...O H bonds (see [24] and references therein). This kind primary amino groups are bound by acidic residues and no of H bond has only recently been recognised as being of sig- interactions of the CH...O type have been described. nificance in satisfying hydrogen-bonding potentials in proteins and for stabilisation of DNA–protein interactions. In Implications for the catalytic mechanism particular, the ability of a methylene to act as a H-bond Polarised absorption microscopic studies have shown that donor is enhanced by the proximity to a polar or charged PAO crystals can be reduced by spermine and reoxidised atom such as an amino group [25]. by air, thereby demonstrating that crystalline PAO is enzy- matically active (A Mozzarelli, personal communication). The substrate-binding site of PAO is quite dissimilar from The precise mechanism by which the enzyme catalyses that observed in the spermidine transporter protein [26] the oxidation of the polyamine substrate is still unknown. 272 Structure 1999, Vol 7 No 3

Figure 7

Stereoview of the mode of binding of MDL72527 in the PAO active site. The inhibitor is shown in ball-and-stick representation. H bonds are represented by Tyr169 121314 Tyr169 121314 dashed lines, whereas CH...O H bonds are

11 11 represented by dotted lines. Furthermore, the W W contact between the MDL72527 C8 and the 10 10 flavin N5 is indicated by a dotted line. 9 Glu62 9 Glu62 Glu170 Glu170 8 8 Tyr298 7 Tyr298 7 6 6

4 5 4 5 3 3 2 2 1 Phe403 Tyr439 1 Phe403 Tyr439

Gly438 Gly438 Asn405 Asn405

Ser404 Ser404 Structure

In this respect, the three-dimensional structure of PAO is unlikely to induce large conformational changes. reveals several relevant features. Firstly, the reaction takes Thirdly, the water molecule that is H bonded to the flavin place in the solvent-protected environment provided by N5 atom (Figures 6 and 7) seems well positioned to the catalytic tunnel. Shielding the substrate from the perform the hydrolytic attack on the imino compound solvent is a feature shared by several flavin-dependent resulting from polyamine oxidation to produce the final oxidases and dehydrogenases [13]. Secondly, the structure aldehyde product (Figure 1a). Finally, the three-dimen- of the PAO–MDL72527 complex is essentially identical to sional structure shows that MDL72527 is a non-covalent that of the native enzyme, implying that substrate binding inhibitor of maize PAO. In this respect, the plant enzyme

Figure 8

Schematic representation of the catalytic tunnel and inhibitor binding. The H bonds NN O 10 between the inhibitor secondary amino groups and the protein are represented by dashed ... Phe403 5 Tyr 298 lines, whereas the CH O H bonds are 4a N represented by dotted lines. The 3.8 Å contact between the inhibitor C8 and the Gly438 3.8

O flavin N5 is outlined. The distances shown are O 3.4 7 2.9 2.9 6 8 9 HO in angstroms. HN3.0 NH 5 2.8 O O 2.9 10 O Glu170 2.9 3.3 O 3 4 11 12 Tyr169 OH 2.7 2.6 Ser404 O 2 O O 13 2.8 Glu62 1 Tyr 169 H2N O 3.0 14 Tyr 439 Tr p 6 0 Glu170 Phe89 O Asn405 Tyr 165 Phe189 Phe171

Asp117 Glu124 Asp194 Glu121 Asp195 Structure Glu120 Research Article Structure of polyamine oxidase Binda et al. 273

differs from the mammalian PAO, which is covalently and interest because its homology with PAO extends beyond irreversibly inactivated by MDL72527 [28]. This differ- the fingerprint motif [6] (Figure 4). In fact, in addition to ence in the mechanism of inhibition may be related to the the amino acids involved in ADP-ribityl binding (Ser15, different specificities displayed by mammalian and plant Glu35, Ala36 and Arg43), several residues interacting with proteins (Figure 1b). An in-depth analysis of these obser- the flavin (Lys300, Trp393 and residues on Fα8) are also vations must, however, await the determination of the conserved among PAO and MAO primary sequences primary sequence of a mammalian PAO. (Figure 6). Furthermore, Thr402, which in PAO interacts with the flavin C8α, is homologous to Cys452, which in MDL72527 differs from spermine in that the two sper- human MAO-A is located in the proximity of the flavin, mine primary amino groups are replaced by a diene unit being covalently bound to the flavin C8α atom (Figure 4). (Figures 1b and 1c). Thus, the substrate primary amino Taken together, these data suggest that PAO and MAO groups are strictly required by the enzyme in order to are likely to share a similar overall folding topology and catalyse substrate oxidation. The substrate primary nitro- FAD-binding site. However, on the basis of sequence gen atoms are likely to bear a positive charge, which may homology, this similarity does not extend to the substrate- affect the protonation state and pKa value of the surround- binding site. In fact, a survey of the PAO catalytic tunnel ing catalytic residues. Similarly, the positive charge of the reveals that some homology is detectable only in the aro- primary amino groups may influence the pKa value of the matic-sandwich region surrounding the MDL72527 N5 substrate secondary amino groups, as suggested by the atom (Figure 8): Gly438 and Tyr439 are conserved, observation that, in the PAO–MDL72527 complex, the whereas Phe403 is replaced by Tyr in MAO. Such a low inhibitor N5 and N10 are at 4.6 Å and 4.0 Å distance from similarity in the substrate-binding region is not surprising; the terminal C1 and C14 atoms, respectively (Figure 7). it reflects the different substrate specificities of the two Furthermore, the primary amino groups are likely to affect enzymes. In this respect, it will be interesting to see the binding mode of the substrate. In the complex with whether the predicted partial structural similarity between MDL72527, the flavin N5–C4a locus is at a distance of PAO and MAO reflects a common catalytic mechanism. more than 4.5 Å from the N5, C6, C9 and N10 atoms of the inhibitor diaminobutane unit (Figures 7 and 8). Biological implications Regardless of the type of mechanism, such a distance is During recent years interest has been growing in natu- too long to allow oxidation by the flavin. Therefore the rally occurring polyamines. These are linear molecules position of the substrate within the catalytic tunnel is with two (putrescine, NH2-(CH2)3-NH2), three (spermi- probably shifted with respect to that of the inhibitor, dine, NH2-(CH2)3-NH-(CH2)4-NH2) or four (spermine, allowing a closer approach between the flavin and the sub- NH2-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2) positively strate C–N bond, which is oxidized by the enzyme. charged nitrogen atoms. Polyamines are involved in a variety of biological activities, ranging from the scaveng- Similarity between PAO and monoamine oxidase ing of free radicals to the specific regulation of proteins Very recently, a new family of FAD-dependent proteins such as certain protein kinases and DNA topoiso- has been identified by sequence alignment, which has merases. In plants, polyamines are also involved in cell- revealed a 50-residue long signature motif [29] (Figure 4). wall differentiation and in defence responses. The This family encompasses oxidoreductases catalysing a polyamine intracellular level is finely controlled through a broad spectrum of reactions, such as protoporphyrinogen network of pathways for the biosynthesis, degradation oxidase, phytoene desaturase and monoamine oxidases. and transport of these compounds. The discovery that Analysis of the amino acid sequence shows that PAO is also polyamine analogues have strong anticancer activity indi- a member of the family, as residues 7–57 match the family cates that enzymes involved in polyamine metabolism signature motif. Particularly, the PAO three-dimensional may represent attractive targets for new anticancer drugs. structure reveals that motif residues are involved in binding the ADP-ribityl moiety of FAD. These observations imply The FAD-dependent polyamine oxidase (PAO) plays a that members of the family possess a similar ADP-ribityl- central role in several physiological processes, including binding site, leading to the prediction that these proteins polyamine homeostasis and cell death [8,11]. The share a topologically similar FAD-binding domain. enzyme catalyses the degradative oxidation of the secondary amino groups of spermine and spermidine. Here, Monoamine oxidase (MAO) is a thoroughly investigated we report the first crystal structure of PAO, which flavoenzyme that binds to the outer mitochondrial mem- reveals the remarkable architecture of enzyme active brane and catalyses the oxidation of primary aromatic site. PAO from Zea mays consists of two domains that amines (see [30] and references therein). The enzyme is define a 30 Å long U-shaped tunnel at their interface. the target of drugs employed in the treatment of depres- The complex with the inhibitor MDL72527 reveals that sive disorders and Parkinson’s disease [31]. Among the the innermost segment of the tunnel forms the enzyme members of the flavoenzyme family, MAO is of particular active site. The walls of this catalytic part of the tunnel 274 Structure 1999, Vol 7 No 3

are decorated by carbonyl oxygens, which extensively (pH 6.0). The best crystals were obtained in two weeks at 293K by interact with the snugly fitting inhibitor through unusual mixing equal volumes of protein and reservoir solution, the latter con- ... taining 42–48% saturated ammonium sulphate, 0.02% NaN3 and CH O H bonds. 100 mM sodium acetate buffer pH 4.6. Crystals belong to space group

P6522 with cell parameters a = b = 185 Å and c = 281 Å. MDL72527 differs from the spermine substrate only in the absence of the primary amino groups. The three- Two heavy-atom derivatives were obtained by soaking the crystal in dimensional structure indicates that MDL72527 adopts a 1 mM Pt(NH3)2Cl2 for 6 hours and in a UO2(CH3COO)2-saturated mother liquor for 24 hours, respectively. Binding studies were per- kind of ‘out-of-register’ binding mode, in which the formed by soaking the crystals in a solution consisting of 60% satu- inhibitor secondary nitrogen atoms are not properly rated ammonium sulphate in 100 mM acetate buffer (pH 4.6) plus aligned with respect to the flavin in order for oxidation 10 mM MDL72527 inhibitor. by the cofactor to be possible. This feature provides the basis for understanding the intriguing ability of PAO to Data collection specifically oxidise a polyamine molecule that possesses Data were collected at 100K [32] using cryocrystallographic techniques. The native data used for phasing (native I in Table 2), the platinum and both primary and secondary amino groups. uranyl derivative data were collected using a MarResearch imaging plate detector at the X-ray diffraction beamline of ELETTRA (Trieste, Italy) and The primary sequence of PAO shares homology with at the X11 beamline of DESY/EMBL (Hamburg, Germany), respectively. members of a family of FAD-dependent oxidoreductases, The native data used for refinement (native II) and the data for the MDL72527 complex were measured at the ID14-EH3 beamline of ESRF which includes protophorphirinogen oxidase and (Grenoble, France) using a MarCCD detector. In all cases, the images monoamine oxidase. It can be predicted that members of were processed with MOSFLM (by AGW Leslie) and data reduction was this family share a folding topology resembling that of carried out using the CCP4 suite of programs [33]. PAO. In particular, monoamine oxidase displays homology to PAO throughout the sequence, implying that the MIR phasing MIR phases were derived from the platinum and uranyl derivatives in two enzymes are likely to have a similar FAD-binding site combination with the native I data set (Table 2). The Patterson super- and some conserved features in the active centre. These position option of SHELXS-97 [34] was used to identify four heavy- observations provide the framework for protein engineer- atom sites for the uranyl derivative, which were then refined with the ing studies aimed at the characterisation of the catalytic program MLPHARE of the CCP4 package [33]. The resulting single isomorphous replacement phases were used to calculate difference mechanism of these flavin-dependent amine oxidases. Fourier maps, which provided four additional uranyl sites and revealed the presence of four heavy-atom sites in the platinum derivative. The Materials and methods platinum and uranyl sites were then refined together, resulting in MIR Crystallisation and crystal soaking phases having an overall figure of merit of 0.47 to 3.0 Å resolution. The

The purification and crystallisation protocols of maize PAO have been choice between the enantiomorphic P6122 and P6522 space-group described [32]. Briefly, the enzyme was crystallised by the hanging- pairs and the assignment of the sign of the heavy-atom coordinates drop vapour-diffusion method, with the protein solution consisting of was based on heavy-atom refinement statistics and the handedness of 5 mg enzyme/ml in 300 mM NaCl and 50 mM sodium phosphate buffer the α helices in the electron-density map.

Table 2

Data collection and phasing statistics.

Data set Native I Native II Pt(NH3)2Cl2 UO2(CH3COO)2 MDL72527

Cell axes a = b, c (Å) 184.7, 281.7 184.8, 282.2 184.8, 280.7 184.8, 297.8 184.6, 281.5 Number of crystals 11111 Beamline ELETTRA ESFRF, ID14-EH3 ELETTRA DESY/EMBL, X11 ESRF, ID14-EH3 Detector Mar image plate Mar CCD Mar image plate Mar image plate Mar CCD Maximum resolution (Å) 3.0 1.9 2.7 3.0 1.9 I/σ* 6.1 (5.8) 9.0 (2.6) 12.6 (9.4) 13.6 (9.9) 9.8 (3.5) Observations 262,167 785,004 218,260 455,820 704,940 Unique reflections 55,695 216,570 74,309 50,430 211,025 Completeness (%)* 97.6 (88.9) 98.1 (88.4) 93.3 (78.2) 99.7 (96.8) 96.6 (95.2) † Rmerge (%) * 9.4 (20.2) 8.2 (33.5) 4.2 (7.3) 5.9 (11.2) 8.8 (17.3) Number of sites ––58– ‡ Rderiv (%) – – 15.5 22.5 18.2 § Rcullis (%) – – 85 68 – Phasing power# – – 0.54 1.13 –

§ Σ ± Σ ± *The values in brackets refer to the highest resolution shell. factor of the native l crystal. Rcullis = || FPH FP |–FH |/ |FPH FP | † Σ < > Σ< > Rmerge = | Ij – Ij |/ Ij , where Ij is the intensity of an observation where FPH and FP are defined as above and FH is the calculated heavy- < > of reflection j and Ij is the average intensity for reflection j. atom structure factor. Centric reflections only were included for this ‡ Σ Σ # Rderiv = || FPH |–|FP || / |FP | where FPH is the structure factor for calculation. Phasing power = the rms heavy-atom structure factor the derivative or MDL72527-bound crystal and FP is the structure amplitudes divided by the lack of closure. Research Article Structure of polyamine oxidase Binda et al. 275

Density averaging and model tracing Italiana (N° ARS96-191). The MDL72527 inhibitor was a generous gift Considering the volume of the unit cell, the number of molecules in the from Dr M De Agazio (Consiglio Nazionale delle Ricerche). asymmetric unit could range from three to six, corresponding to a solvent content of 71% to 42%, respectively. Various self rotation func- References tion calculations were performed using data at different resolution limits 1. Tabor, C.W. & Tabor, H. (1984). Polyamines. Annu. Rev. Biochem. and several values for the integration radius. However, a clear peak did 53, 749-790. 2. Marton, L.J. & Pegg, A.E. (1995). Polyamines as targets for not appear in any of these calculations. For this reason, once the MIR therapeutic intervention. Annu. Rev. Pharmacol. Toxicol. 35, 55-91. phases became available, solvent flattening was performed with the 3. 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