The ‘gating’ residues Ile199 and Tyr326 in human B function in substrate and inhibitor recognition Erika M. Milczek1,*, Claudia Binda2, Stefano Rovida2, Andrea Mattevi2 and Dale E. Edmondson1

1 Departments of Chemistry and Biochemistry, Emory University, Atlanta, Georgia, USA 2 Department of Genetics and Microbiology, University of Pavia, Italy

Keywords The major structural difference between human monoamine oxidases A dipartite to monopartite cavity conversion; (MAO A) and B (MAO B) is that MAO A has a monopartite substrate inhibitor specificity; monoamine oxidase B; cavity of  550 A˚3 volume and MAO B contains a dipartite cavity struc- mutations of gating residues; structure of ture with volumes of  290 A˚3 (entrance cavity) and  400 A˚3 (substrate complex cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO Correspondence B. To probe the function of these gating residues, Ile199Ala and Ile199Ala- D. E. Edmondson, Department of Tyr326Ala mutant forms of MAO B were investigated. Structural data on Biochemistry, Emory University, 1510 the Ile199Ala MAO B mutant show no alterations in active site geometries Clifton Road, Atlanta, GA 30322, USA compared with wild-type enzyme while the Ile199Ala-Tyr326Ala MAO B Fax: +1 404 727 2738 mutant exhibits alterations in residues 100–103 which are part of the loop Tel: +1 404 727 5972 gating the entrance to the active site. Both mutant enzymes exhibit catalytic E-mail: [email protected] properties with increased amine KM but unaltered kcat values. The altered *Present address KM values on mutation are attributed to the influence of the cavity struc- Department of Chemistry, Princeton ture in the binding and subsequent deprotonation of the amine substrate. University, Princeton, NJ 08544, USA. Both mutant enzymes exhibit weaker binding affinities relative to wild-type enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an (Received 23 August 2011, revised 29 increase in binding affinity for reversible MAO B specific inhibitors which September 2011, accepted 30 September bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits 2011) inhibitor binding properties more similar to those of MAO A than to doi:10.1111/j.1742-4658.2011.08386.x MAO B. These results demonstrate that the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provide insights into specific reversible inhibitor design for these membrane-bound enzymes.

Database The atomic coordinates and structural factors of the structure of human MAO B Ile199Ala- Tyr326Ala double mutant have been deposited in the Protein Data Bank under the accession number 3zyx.

Introduction Monoamine oxidases (EC 1.4.3.4) (MAOs) are mine and epinephrine) [1]. They exist in two isoforms mitochondrial outer membrane-bound flavoenzymes in mammals (MAO A and MAO B) as separately that catalyze the oxidative deamination of biogenic encoded X-linked gene products with amino acid amines and amine neurotransmitters (, dopa- sequences that are  70% identical [2]. Age-related

Abbreviations CHES, 2-(cyclohexylamino)-ethane sulfonic acid; CSC, 8-(3-chlorostyryl)caffeine; DPB, 1,4-diphenyl-2-butene; DPBD, 1,4-diphenyl-1,3-butadiene; MAO A, ; MAO B, monoamine oxidase B; WT, wild-type.

4860 FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS E. M. Milczek et al. Gating residue function in human MAO B increases in MAO B levels in neuronal tissues and insights into the gating function of the protein loop resulting catalytic production of H2O2 (leading to guarding the opening to the entrance cavity. reactive oxygen species) is thought to contribute to cel- lular apoptosis and subsequent neurodegenerative dis- Results eases [3]. Therefore, specific inhibitors of MAO B function as neuro-protectants. Structural determination of the Previous work has shown that long, planar com- Ile199Ala-Tyr326Ala mutant form of MAO B pounds with extended p-conjugation, like 8-(3-chloro- styryl)caffeine (CSC) [4] and trans,trans-farnesol [5], Previous structural work from this laboratory has exhibit high specificities for reversible inhibition of defined the 1.65 A˚structure of wild-type (WT) recom- human MAO B and do not bind to human MAO A. binant human MAO B [7] and the 2.0 A˚structure of Investigations of the structures of human MAO B and the Ile199Ala mutant enzyme [9]. These data show that MAO A show the main difference is the dipartite active replacing the isopropyl side chain of Ile199 with a site cavity in MAO B and monopartite active site cavity methyl group has no deleterious influence on the struc- in MAO A to explain this differential binding behavior tural integrity of the enzyme’s active site. In designing [6]. Ile199 of human MAO B functions as a conforma- crystallization trials with the Ile199Ala-Tyr326Ala tional gate between the two cavities and is substituted by double MAO B, preliminary experiments showed that a Phe in bovine MAO B [5]. Of interest, bovine MAO B methylene blue, a strong MAO A inhibitor [10], binds does not bind the above-mentioned compounds. to the double mutant with an affinity higher than that High-resolution crystal structures of human MAO B observed with WT MAO B. As will be discussed show that Ile199 adopts distinct conformations below, the increase in size of the active site cavity and depending on the nature of the inhibitor bound [7]. its conversion from a dipartite to monopartite struc- When small inhibitors are bound within the substrate ture are responsible for this increase in affinity. The cavity, the side chain of Ile199 rotates into a closed methylene-blue-inhibited Ile199Ala-Tyr326Ala MAO B conformation which creates the bipartite active site. mutant readily formed crystals which diffract to 2.2 A˚ With larger inhibitors such as trans,trans-farnesol resolution. The structure was solved by molecular bound in the active site, the side chain of Ile199 occu- replacement. Crystallographic data statistics are shown pies an open conformation resulting in the fusion of in Table 1 and the structure is shown in Fig. 1A–C. the two cavities to one of  700 A˚3. These observa- tions provide the groundwork for the suggestion that Table 1. Data collection and refinement statistics for the structure Ile199 serves as a structural determinant for substrate of human MAO B Ileu199Ala-Tyr326Ala double mutant complex and inhibitor recognition [5]. Such a ‘gating’ function with methylene blue. is not observed with MAO A since it contains a mono- Space group C222 partite active site cavity. Unit cell axes (A˚ ) a = 131.9, b = 225.0, In addition to conformational alterations of the c = 86.6 Ile199 ‘gating’ residue, the side chain of Tyr326 exhib- Resolution (A˚ ) 2.2 a,b its modest conformational changes on inhibitor bind- Rsym (%) 11.0 (33.0) Completenessb (%) 98.2 (89.8) ing to human MAO B [8]. Tyr326 and Ile199 thus Unique reflections 64 424 serve to separate these two cavities in human MAO Redundancy 4.4 (2.5) B. To probe the respective roles of Ile199 and Tyr326 I ⁄ rb 10.8 (2.8) in the maintenance of the active site geometry as well No. of atoms for 7894 ⁄ 2 · 53 ⁄ 2 · 20 ⁄ 534 as function in substrate and inhibitor recognition, the protein ⁄ FAD ⁄ ligand ⁄ water 2 Ile199Ala and Ile199Ala-Tyr326Ala mutant forms of Average B value for ligand atoms (A˚ ) 59.2 b,c human MAO B were constructed, expressed and puri- Rcryst (%) 19.0 (24.1) R b,c (%) 24.4 (32.2) fied. An Ile199Ala mutation would permanently free Rms bond length (A˚ ) 0.013 ‘open’ the gate with unknown functional conse- Rms bond angles () 1.38 quences. A double mutant involving Ile199 and P P a ) Tyr326 is proposed to create a large monopartite Rsym = |Ii ÆIæ| ⁄ Ii, where Ii is the intensity of i th observation b active site in MAO B which should dramatically alter and ÆI æ is the mean intensity of the reflection. Values in parenthe- ses areP for reflectionsP in the highest resolution shell. its substrate and inhibitor binding specificities. As c Rcryst = |Fobs ) Fcalc| ⁄ |Fobs| where Fobs and Fcalc are the shown in this paper, these alterations of MAO B con- observed and calculated structure factor amplitudes, respectively. vert the enzyme to one with functional properties Rcryst and Rfree were calculated using the working and test sets, more similar to those of MAO A and provide new respectively.

FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS 4861 Gating residue function in human MAO B E. M. Milczek et al.

A B

C

Fig. 1. (A) Ribbon overall structure of the human MAO B monomer (Ile199Ala-Tyr326Ala double mutant) in complex with methylene blue. The FAD cofactor is represented in yellow, whereas the methylene blue inhibitor bound to the active site cavity (semitransparent gray sur- face) is in blue. (B) Active site residues of human MAO B Ile199Ala-Tyr326Ala double mutant in complex with methylene blue. Nitrogen atoms are blue, oxygens are red, sulfurs are yellow and carbon atoms are gray. FAD and methylene blue carbons are in yellow and blue, respectively. Water molecules are shown as red spheres. Unbiased electron density map (contoured at 1.2r) is shown for the inhibitor, the FAD cofactor and the double mutation sites. (C) Zoomed view of the double mutant MAO B structure (in blue, same orientation as in A) superposed to the WT protein bound to safinamide (in cyan; PDB code 2v5z). The sites of mutation (Ile199Ala and Tyr326Ala) are shown. Phe103 is also drawn to highlight the different conformation of the side chain and of the cavity gating loop (residues 99–104). Previous human MAO B structures have demonstrated that the conformation of this loop depends on the dipartite nature of the active site cavity which in turn is related to the Ile199 conformation and the inhibitor bound.

4862 FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS E. M. Milczek et al. Gating residue function in human MAO B

These data show that replacement of the isopropyl and (Table 2). The alteration in KM values with the sub- phenolic side chains of Ile199 and Tyr326 with the strates tested result in catalytic efficiencies (kcat ⁄ KM) methyl groups of alanyl residues have no major effect values that are between 1% and 27% of those on the structure of the enzyme or on the conforma- observed with WT enzyme. Benzylamine is a poor sub- tions of other residues about the active site. The major strate for the double mutant with a KM value too high effect of these mutations is to alter the dipartite cavity to measure at pH 7.5. The rates of oxidation catalyzed structure of MAO B to one that is monopartite with a by the double mutant are linearly proportional to the calculated volume of 732 A˚3. Notably, the loop and concentration of benzylamine in the assay (Fig. 2). especially Phe103 shift by about 1 A˚toward the side Increasing the pH of the assay medium to a value of chain at position 199. In this regard, the structure of 9.3 results in a decrease in the KM value the double mutant exhibits dual structural conse- (KM = 866 lM) suggesting that the reason for non-sat- quences as a single cavity is observed (as in the com- uration with this substrate at lower pH is due to the plexes with large inhibitors such as safinamide [11]) inability of the double mutant enzyme either to bind and Phe103 adopts a conformation similar to that the protonated form of the substrate or to facilitate found in the MAO B structures in complexes with deprotonation of the amine moiety in the enzyme–sub- small inhibitors where the Ile199 side chain gate is in strate complex for catalysis to efficiently proceed. its ‘closed’ position [7]. These results suggested a role for these gating residues in presenting the deprotonated amine substrate for the reductive half-reaction with the flavin cofactor. Catalytic properties of the mutant forms of To provide more in-depth insights into the pH- MAO B dependent behavior of MAO B and its mutant forms, To examine the effects of both single and double the rates of oxidation of the substrates benzylamine mutations on the catalytic properties, the kinetic prop- (pKa = 9.33) and p-CF3-benzylamine (pKa = 8.75) erties for the oxidation of the substrates kynuramine, were determined in the pH range 7.0–9.5. These two benzylamine and several phenylalkylamines were deter- substrates have amine pKa values differing by 0.58 pKa mined (Table 2). Kynuramine is oxidized by either units which is enough to be observable on comparison MAO A or MAO B and the various phenylalkylam- of pH-dependent kinetic parameters for WT and ines allow comparisons of side chain length on cata- mutant enzymes. Plots of V ⁄ K versus pH provide esti- lytic turnover. Similar turnover numbers (kcat) are mates of pKa values of groups important in catalysis observed for WT and the two mutant enzymes; how- for the free enzyme and the ionization form amenable ever, the KM values observed for the mutant enzymes for catalysis for the free substrate. Jones et al. [12] are found to be considerably higher than those for WT found pKa values of 7.1 for MAO B indicating an enzyme. , a traditional MAO B sub- enzyme group deprotonation to enhance catalytic strate, exhibits a four-fold increase in KM with the sin- activity and a value of 9.97 for the pKa of a group gle mutant while the double mutant exhibits a KM where deprotonation diminishes catalytic activity in value 75-fold higher than that found with WT enzyme plots of the pH dependence of V ⁄ K for the MAO B

Table 2. Comparisons of substrate specificities of WT and mutant forms of MAO B at pH 7.5.

)1 )1 )1 Substrate Enzyme kcat (min ) KM (lM) kcat ⁄ KM (min ÆlM )

Kynuramine WT MAO B 96 ± 1 27 ± 2 3.55 ± 0.27 Ile199Ala 88 ± 2 94 ± 8 0.936 ± 0.082 Ileu199Ala-Tyr326Ala 64 ± 2 289 ± 23 0.22 ± 0.02 Benzylamine WT MAO B 300 ± 8 150 ± 27 2.0 ± 0.4 Ile199Ala 228 ± 13 2133 ± 409 0.107 ± 0.021 a )1 )1 Ileu199Ala-Tyr326Ala 2nd order rate =: 0.0106 ± 0.0002 lM Æmin Phenylethylamine WT MAO B 172 ± 3 9.4 ± 0.9 18 ± 2 Ile199Ala 123 ± 2 40 ± 2 3.1 ± 0.2 Ileu199Ala-Tyr326Ala 110 ± 2 703 ± 58 0.16 ± 0.01 Phenylbutylamine WT MAO B 110 ± 5 19 ± 3 5.8 ± 0.3 Ile199Ala 135 ± 3 8.9 ± 0.7 15.2 ± 0.6 Ileu199Ala-Tyr326Ala 142 ± 1 55 ± 2 2.6 ± 0.1 a )1 The kinetic values were determined to be kcat = 283 ± 4 min , KM = 866 ± 34 lM in 50 mM CHES at pH 9.3 with 0.5% (w ⁄ v) reduced Triton X-100 buffer in the activity assay.

FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS 4863 Gating residue function in human MAO B E. M. Milczek et al.

A the observed pKa with the known pKa of phenethyl- amine.

Using two substrates with differing pKa values for their respective amine moieties (see above), we find that WT MAO B and the two gate mutants exhibit ascend- ing catalytic activities with pH using either benzylamine

or p-CF3-benzylamine as substrate (Fig. 2B,C). Analysis of the V ⁄ K data using the method outlined by Fersht [13] shows identical values of 8.7 ± 0.1 using either

benzylamine or p-CF3-benzylamine for WT MAO B (Fig. S2 and Table 3). The identities observed demon-

strate that the assignment of these pKa values is due to a catalytically essential group on the free enzyme spe-

cies. If these pKa values were due to the ionization of B the free substrate, then a differential pKa ( 0.6) would be observed on comparison of benzylamine and p-CF3- benzylamine data. Examination of the Ile199Ala

mutant shows pKa values of 8.8 ± 0.2 and 8.6 ± 0.3 for the free enzyme and the same substrates (Figs 2B, S2, and Table 3). Thus, little or no perturbations of the

pKa value of this catalytically essential group in the free form of MAO B is observed on mutation of the Ile199 gate. The pH dependence of V ⁄ K with the double mutant shows a value of 8.7 ± 0.2 with benzylamine

and a value of 8.2 ± 0.3 with p-CF3-benzylamine. The C pKa values for the single and double mutant form have more uncertainty due to the lower levels of V ⁄ K values exhibited for each mutant. Extrapolation of V ⁄ K for the fully deprotonated free enzyme forms shows, with benzylamine, that the single mutant is 42% and the

double mutant is  5% of WT activity. Using p-CF3- benzylamine as substrate, the single mutant exhibits 33% and the double mutant 24% WT activity. These

data demonstrate that the presence of a p-CF3 substitu- ent on the substrate enhances catalytic turnover relative to the unsubstituted ring of benzylamine in the double Fig. 2. Comparisons of catalytic behaviors of WT MAO B ( ), mutant. Whether this is due to electronic effects of the Ile199Ala MAO B ( ) and Ile199Ala-Tyr326Ala MAO B (•). (A) WT electron withdrawing substituent or to the H-bonding MAO B and Ile199Ala-Tyr326Ala velocities in response to benzyl- capability of the substituent is not known. amine concentrations in their respective assays. (B) Effect of pH on The observed pKa values of the free enzyme species kcat ⁄ KM of WT MAO B, Ile199Ala MAO B and Ile199Ala-Tyr326Ala MAO B using benzylamine as substrate. (C) Effect of pH on with WT and mutant enzymes ( 8.6) are quite differ- ent from that observed with human granulocyte MAO B kcat ⁄ KM of WT MAO B, Ile199Ala MAO B and Ile199Ala-Tyr326Ala

MAO B using p-CF3-benzylamine as substrate. (pKa = 7.1) [12] using phenethylamine as substrate. The main difference in experimental protocols is that Jones catalyzed oxidation of phenethylamine. These results et al. [12] used membrane preparations of MAO B were interpreted [12] to suggest that deprotonation of while in this work we used purified preparations of a group with a pKa of 7.1 on the free enzyme is recombinant human MAO B. Thus, we propose that required for activity and that deprotonation of the the differences in observed values may reflect the influ- amine (pKa = 9.97) of the substrate diminishes cata- ence of the membrane environment on the pKa of this lytic function, suggesting that the protonated form of residue whose deprotonation is required for optimal the substrate is bound in the active site. It should be activity. There is no obvious residue in the substrate pointed out that assignment of higher pKa to the free cavity other than Cys172 that could ionize in this pH form of the substrate was based on the similarity of range; however, previous mutagenesis studies [14] have

4864 FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS E. M. Milczek et al. Gating residue function in human MAO B

Table 3. Comparisons of pKa values for deprotonation of a catalyti- Table 4. Comparisons of inhibition constants for non-specific MAO cally essential group in WT and mutant forms of MAO B with inhibitors with WT MAO B, WT MAO A and mutant forms of MAO benzylamine and p-CF3-benzylamine as substrates. B. Data were collected at pH 7.5 for WT and the single mutant. Double mutant data were collected at pH 9.3. kcat ⁄ KM pKa kcat ⁄ KM pKa values with values with Isatin

Enzyme benzylamine p-CF3-benzylamine Enzyme Ki (lM) Ki (lM)

WT MAO B 8.6 ± 0.1 8.7 ± 0.1 WT MAO B 3a 16b Ile199Ala MAO B 8.8 ± 0.1 8.6 ± 0.3 WT MAO A 15a 19b Ile199Ala-Tyr326Ala MAO B 8.7 ± 0.2 8.2 ± 0.3 MAO B Ile199Ala 12 ± 2 11 ± 3 MAO B Ile199Ala-Tyr326Ala 360 ± 40 17 ± 3

a Taken from work cited in ref. 5. b Taken from work cited in ref. 17. shown this residue is not required for catalytic activity. Therefore, the location of the residue whose ionization is responsible is likely to be near the surface of the A(Ki =15lM) [16]; however, it inhibits the protein in or near the loop (residues 99–112) that Ile199Ala-Tyr326Ala double mutant (Ki =  360 lM) shields the entrance cavity. Possible candidates include with an  100-fold weaker affinity than to WT MAO Lys95 or Lys93. There are His residues (residues 90, B(Ki =3lM) (Table 4). Tranylcypromine binding is 91 and 115) flanking this shielding loop that are possi- not dramatically influenced by mutations to the MAO ble but less likely candidates. B cavity structure and this observation is consistent

Plots of kcat versus pH are expected to follow ioniza- with its known non-specificity for MAO A or MAO B tions of the enzyme–substrate complexes that would [17]. Weak binding of aminoindane or methylaminoin- have catalytic functional importance in MAO B and its dane (R or S isomers) to the MAO B double mutant is mutant forms. As pointed out by Jones et al. [12] the also observed; these were shown previously to also fact that [O2] at air saturation in the catalytic assays is bind solely to the substrate cavity of WT MAO B [18] not saturating with MAO B brings an additional level (data not shown). Therefore, removal of one ‘wall’ (i.e. of complexity in the estimation of pKa values from the Ile199 and Tyr326 side chains, Fig. 2) of the sub- such data. The influence of these mutations on O2 reac- strate cavity of MAO B results in an enzyme with a tivity and KMO2 behavior is not known and will be considerable loss of affinity for compounds that bind investigated in future studies since the monopartite within the MAO B substrate cavity.

MAO A exhibits a low KMO2 and its reaction with O2 Previous work has shown that specific MAO B inhibi- is dependent on the presence of substrate ⁄ products in tors bind by traversing both the entrance and the the active site of the reduced enzyme [15]. substrate cavities, which forces Ile199 into an open con- It is of interest that phenylbutylamine (Table 2) formation allowing fusion of the two cavities [5,6]. These exhibits saturation behavior at pH 7.5 with the double inhibitors exhibit no observable binding to WT MAO A mutant in contrast to what was observed with benzyl- thus demonstrating the role of the bipartite cavity struc- amine. This behavior is not a reflection of their respec- ture of MAO B in specificity. Removal of the gating resi- tive differences in amine pKa values which are quite due in MAO B (the Ile199Ala mutant form) results in similar. The longer alkyl side chain may permit inter- either increased (7- to 24-fold higher) or no difference in actions of the phenyl ring with the residues in the nas- affinities for the MAO B specific inhibitors safinamide cent entrance cavity site of the double mutant leading (Ki =21nM), farnesol (Ki =2.7 lM), 1,4-diphenyl-2- to an environment facilitating amine deprotonation of butene (DPB) (Ki =  9 lM) and 1,4-diphenyl- the free enzyme for optimal catalysis. 1,3-butadiene (DPBD) (Ki = 1.0 lM) (Table 5). The double mutant exhibits decreased affinities for these dual cavity spanning specific inhibitors, binding safinamide Inhibitor binding properties of the mutant forms (K =4lM) with a  10-fold weaker affinity than WT of MAO B i MAO B. A weaker binding of trans,trans-farnesol and To determine the influence of the gating residues on no detectable binding of DPB or DPBD is found with inhibitor binding, several classes of MAO inhibitors the double mutant. In agreement with other functional (MAOIs) were tested (for structures, see Fig. S1). data described above, loss of the dipartite cavity results Isatin is a non-specific reversible inhibitor that binds in an enzyme that functionally resembles human MAO to WT MAO B in the substrate cavity [7]. It competi- A in inhibitor binding properties. tively binds to the Ile199Ala single mutant Known MAO A specific inhibitors are characterized

(Ki =12 lM) with an affinity similar to that of MAO by bulky fused ring systems which exhibit higher

FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS 4865 Gating residue function in human MAO B E. M. Milczek et al.

Table 5. Inhibition constants for MAO B specific reversible inhibitors with WT and mutant enzymes. Data were collected at pH 7.5 for WT and the single mutant. Double mutant data were collected at pH 9.3.

Ki (lM)

Enzyme Farnesol DPB Safinamide CSC DPBD

WT MAO B 2.3 ± 0.4a 34.5 ± 1.4a 0.50 ± 0.10b 0.27 ± 0.08a 7.0 ± 0.2a Ile199Ala MAO B mutant 2.7 ± 0.4 8.1 ± 1.6 0.021 ± 0.002 No inhibition 1.00 ± 0.03 Ile199Ala-Tyr326Ala MAO B mutant 18 ± 3 No inhibition 4.0 ± 0.6 1.7 ± 0.3 No inhibition a Taken from ref. 5. b Taken from ref. 11.

inhibitors show a ‘gain of function’ which results from Table 6. Ki values for MAO A specific reversible inhibitors. Data collected at pH 7.5 for WT and the single mutant. Double mutant formation of a large, monopartite cavity. This struc- data were collected at pH 9.3. tural change also results in a ‘loss of function’ as the

MAO B specific inhibitor zonisamide (Ki = 3.1 lM) Methylene blue [20] exhibits no detectable inhibition of the double Enzyme K (lM) K (lM) K (lM) i i i mutant. WT MAO A 0.92 ± 0.04a 0.58 ± 0.02a 0.025 ± 0.001b WT MAO B No inhibition 140 ± 47 1.01 ± 0.05 MAO B Ile199Ala No inhibition 550 ± 50 0.214 ± 0.032 Discussion MAO B 4.1 ± 0.2 165 ± 17 0.143 ± 0.014 The structural and functional results of this study pres- Ile199Ala-Tyr326Ala ent insights into the role of the dipartite cavities in a Taken from ref. 19. b Taken from ref. 10. human MAO B and on the function of the gating resi- due (Ile199) whose conformation is relevant for inhibi- affinities for its larger monopartite active site. The tor and substrate function. Binding small inhibitors reversible inhibitors pirlindole, harmane and methylene like isatin to WT MAO B results in the Ile199 gate blue inhibit MAO A with high affinities [10,19] and rotating to a closed conformation [5]. Permanently demonstrate weak inhibitory behavior with WT MAO B opening the ‘gate’ through mutation of Ile199 to an (Table 6). The Ile199Ala mutant also exhibits weak Ala results in three- to four-fold reduced binding affin- (micromolar) or no affinities for these inhibitors indi- ity of smaller inhibitors that bind only in the substrate cating that this gating residue between the bipartite cavity. MAO B specific inhibitors that traverse both cavities does not play an important role in this speci- the entrance cavity and the substrate cavity force the ficity difference. Removal of the dipartite cavity struc- Ile199 ‘gate’ to rotate into its open conformation ture results in an MAO B form that exhibits higher resulting in the fusion of the two cavities. The affinities for these MAO A specific reversible inhibitors Ile199Ala mutation has a positive effect on binding than either WT or the single mutant (Table 6). In the of this class of inhibitors with an increase of up to case of pirlindole, neither MAO B nor the Ile199Ala 24-fold higher affinity than to WT. The results of single mutant are inhibited, while the Ile199Ala- inhibitor binding to the MAO B double mutant is con- Tyr326Ala double mutant is competitively inhibited in sistent with the structural data in Fig. 1 showing that the low micromolar range (Ki = 4.1 lM). Methylene the cavity structure of this form is monopartite rather blue is a tight binding inhibitor of MAO A than bipartite. MAO A has a monopartite active site (Ki =25nM) [10], the single mutant (Ki = 250 nM) cavity and therefore analysis of MAO A specific inhib- and the Ile199Ala-Tyr326Ala mutant (Ki = 143 nM). itors provides insights into the properties of the In contrast, it is bound with lower affinity to MAO B Ile199Ala-Tyr326Ala active site. MAO A specific inhib-

(Ki =1lM). Examination of the crystal structure of itors bind to the double mutant with increased affinities the methylene blue complex with the double mutant relative to WT MAO B but with lower affinities than shows that the orientation of the bound ligand observed with MAO A. Smaller inhibitors like isatin (Fig. 1A–C) would clash with the side chain of Tyr326 bind to the double mutant considerably more weakly in either the WT or the Ile199Ala mutant forms. than to WT enzyme. MAO B specific inhibitors which Therefore, we predict the conformation of bound favor a bipartite active site bind to the double mutant methylene blue in these enzymes would be altered to with  10-fold weaker affinity or not at all. accommodate the observed binding. The inhibitory Structural data on the double mutant enzyme show properties of the double mutant with MAO A specific interesting structural effects that deserve some

4866 FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS E. M. Milczek et al. Gating residue function in human MAO B comment. Phe103 is situated on the protein loop that sources or were synthesized using procedures previously guards access to the entrance cavity. When Ile199 is in described [21]. MAO A and MAO B inhibitors that are com- an ‘open’ position, the side chain of Phe103 is forced mercially available were purchased from Tocris Bioscience, into a conformation that results in the protein loop Ellisville, MO, USA, or from Sigma-Aldrich, St Louis, MO, closing off access to the entrance cavity. When Ile199 USA. Safinamide was a gift from Newron Pharma (Milan, is in its ‘closed’ conformation, the Phe103 side chain Italy). trans,trans-Farnesol and CSC were generously does not experience this steric clash and is now in a supplied by N. Castagnoli, Virginia Polytechnic Institute and conformation resulting in an ‘open’ conformation of State University. The structures of the various MAO this ‘guarding’ protein loop. Presumably, in the ligand inhibitors used in this study are given in the supplementary information. free state, these conformations are rapidly inter-con- verting, thus providing access to the active site. Con- version of Ile199 to an Ala residue results in this Enzymes Phe103 side chain being in a conformation that favors Recombinant WT human liver MAO B [22] and MAO A opening the ‘guarding’ loop in front of the entrance [23] were expressed in Pichia pastoris and purified by pub- cavity. Visualization of this process can be observed in lished protocols. The Ile199Ala MAO B and Ile199Ala- Fig. 1B,C. Comparison of the cavity structure in the Tyr326Ala mutant enzymes were prepared by gene muta- double mutant with those determined previously with tions using the Stratagene Quik-Change XL Site-Directed bound inhibitors where both ‘open’ and ‘closed’ con- Mutagenesis kit and confirmed by gene sequence analysis. formations were present show that these mutations The mutant enzymes were expressed and purified using the ˚3 have now resulted in a larger volume ( 65 A )ofa protocol for WT enzyme. monopartite cavity as well as increased its volume rela- tive to that found when the Ile199 gate is open. The functional consequences of this structure in the Catalytic assays double mutant are evident with altered ligand binding Standard MAO A and MAO B activity assays were per- properties and the catalytic properties shown in Fig. 2. formed spectrophotometrically using p-CF3-benzylamine )1 )1 The alteration in KM value for benzylamine in the dou- (Dæ = 11 800 M Æcm , k = 243 nm) and benzylamine ) ) ble mutant (Fig. 2A) is best explained by the failure of (Dæ = 12 800 M 1Æcm 1, k = 250 nm), respectively, at this mutant enzyme to facilitate deprotonation of the 25 Cin50mM phosphate buffer (pH 7.5) containing 0.5% enzyme–substrate complex. Since neither mutated resi- (w ⁄ v) reduced Triton X-100. Assays with other benzylamine due is expected to exhibit pKa values in the range analogues were performed as described previously [21,24]. observed for WT enzyme and the pKa value of the res- MAO B Ile199Ala-Tyr326Ala assays were performed at idue required for optimal catalysis in the free enzyme 25 Cin50mM 2-(cyclohexylamino)-ethane sulfonic acid is not changed, the difference probably originates from (CHES) (pH 9.3) and 0.5% (w ⁄ v) reduced Triton X-100. alterations in catalytic site hydrophobicity and ⁄ or Phenethylamine and other phenylalkylamine oxidations water content. were monitored spectrally using the horseradish peroxidase æ )1 )1 Alterations in structure of the guarding ‘entrance coupled Amplex red assay (D = 54 000 M Æcm , k = 560 nm). Kynuramine oxidation was monitored spec- loop’ by mutagenesis or by membrane interactions are ) ) æ M 1 1 thought to have important influences on the properties trally (D = 12 000 Æcm , k = 316 nm). The interaction of various inhibitors with the WT and of the active site. In this respect, it would be of interest mutant enzymes was determined by measuring the initial to measure the pK for deprotonation of recombinant a rates of substrate oxidation (six different concentrations) in MAO B in its membrane associated form to compare the presence of varying concentrations of inhibitor (a mini- with purified preparations to address the question of mum of four different concentrations). K values were deter- K i membrane influence of p a values. A more detailed mined using global fit analysis of the hyperbolic fits of experimental and theoretical description of the dynam- enzyme activity with inhibitor concentrations using GRAPHPAD ics of this process will be the subject of further work. PRISM 5.0 software (GraphPad Software, Inc. La Jolla, CA, USA). Materials and methods pH studies were performed spectrophotometrically using p-CF3-benzylamine or benzylamine as substrates at 25 C M M Reagents in a buffer containing 50 m potassium phosphate, 50 m sodium pyrophosphate phosphate and 50 mM CHES with The reagents used in this study were obtained from com- 0.5% (w ⁄ v) reduced Triton X-100. The pH was adjusted to mercially available sources unless otherwise stated. All ben- 7.5, 8.0, 8.5, 9.0 or 9.5 depending on the assay. pKa estima- zylamine analogues used were purchased from commercial tions from steady state kinetic data used approaches and

FEBS Journal 278 (2011) 4860–4869 ª 2011 The Authors Journal compilation ª 2011 FEBS 4867 Gating residue function in human MAO B E. M. Milczek et al. equations described by Dunn et al. [25] and Fersht [13]. 5 Huba´lek F, Binda C, Khalil A, Li M, Mattevi A, Fits of experimental data to these equations were per- Castagnoli N & Edmondson DE (2005) Demonstration formed using GRAPHPAD PRISM 5.0 software. of isoleucine 199 as a structural determinant for the selective inhibition of human monoamine oxidase B by specific reversible inhibitors. J Biol Chem 280, 15761– Crystallographic methods 15766. Crystallographic studies were performed as previously 6 Edmondson DE, Binda C, Wang J, Upadhyay AK & described [7]. Briefly, mutant enzyme solutions in the pres- Mattevi A (2009) Molecular and mechanistic properties ence of methylene blue in 25 mM potassium phosphate pH of the membrane-bound mitochondrial monoamine 7.5 and 8.5 mM Zwittergent 3-12 (Anatrace, Affymetix, oxidases. Biochemistry 48, 4220–4230. Maumee, OH, USA) were crystallized by mixing equal vol- 7 Binda C, Li M, Huba´lek F, Restelli N, Edmondson DE umes of protein sample and reservoir solution [12% (w ⁄ v) & Mattevi A (2003) Insights into the mode of inhibition polyethylene glycol 4000, 100 mM N-(2-acetamido)-2-imin- of human mitochondrial monoamine oxidase B from odiacetic acid buffer, pH 6.5, and 70 mM sulfate]. high-resolution crystal structures. Proc Natl Acad Sci Diffraction data were collected at 100 K at the European USA 100, 9750–9755. Synchrotron Radiation Facility in Grenoble, France. Data 8 Binda C, Huba´lek F, Li M, Herzig Y, Sterling J, processing and scaling were carried out using MOSFLM [26] Edmondson DE & Mattevi A (2004) Crystal structures and programs of the CCP4 package [27]. Crystallographic of monoamine oxidase B in complex with four inhibi- refinements were performed with the programs REFMAC5 tors of the N-propargylaminoindan class. J Med Chem [28] and COOT [29]. Structural illustrations were produced 47, 1767–1774. using PYMOL (http://www.pymol.org). 9 Bonivento D, Milczek EM, McDonald GR, Binda C, Holt A, Edmondson DE & Mattevi A (2010) Potentiation of ligand binding through cooperative Acknowledgements effects in monoamine oxidase B. J Biol Chem 285, This work was supported by National Institutes of 36849–36856. Health grant GM-29433 (to DEE), a Ruth Kirschstein 10 Ramsay RR, Dunford C & Gillman PK (2007) Methy- predoctoral fellowship from the National Institute of lene blue and serotonin toxicity: inhibition of mono- Neurological Disorders and Stroke award number amine oxidase A (MAO A) confirms a theoretical prediction. Br J Pharmacol 152, 946–951. F31NS063648 (to EMM), and MIUR-PRIN09 (to CB) 11 Binda C, Wang J, Pisani L, Caccia C, Carotti A, Salvati and Fondazione Cariplo (to AM). The authors thank P, Edmondson DE & Mattevi A (2007) Structures of Mrs Milagros Aldeco for her valuable technical assis- human monoamine oxidase B complexes with selective tance. noncovalent inhibitors: safinamide and coumarin analogs. J Med Chem 50, 5848–5852. References 12 Jones TZE, Balsa D, Unzeta M & Ramsay RR (2007) Variations in activity and inhibition with pH: the pro- 1 Weyler W, Hsu YP & Breakefield XO (1990) Biochem- tonated amine is the substrate for monoamine oxidase, istry and genetics of monoamine oxidase. Pharmacol but uncharged inhibitors bind better. J Neural Transm Ther 47, 391–417. 114, 707–712. 2 Bach AW, Lan NC, Johnson DL, Abell CW, Bembenek 13 Fersht A (1999). In Structure and Mechanism in Protein ME, Kwan SW, Seeburg PH & Shih JC (1988) cDNA Science, pp. 181–182. WH Freeman & Co., New York. cloning of human liver monoamine oxidase A and B: 14 Wu H-F, Chen K & Shi JC (1993) Site-directed molecular basis of differences in enzymatic properties. mutagenesis of monoamine oxidase A and B: role of Proc Natl Acad Sci USA 85, 4934–4938. cysteines. Mol Pharm 43, 888–893. 3 Mallajosyula JK, Kauer D, Chinta SJ, Rajagopalan S, 15 Tan AK & Ramsay RR (1993) Substrate-specific Rane A, Nicholls DG, Di Monte DA, Macarthur H & enhancement of the oxidative half-reaction of mono- Andersen JK (2008) MAO-B elevation in mouse brain amine oxidase. Biochemistry 32, 2137–2143. astrocytes results in Parkinson’s pathology. PLoS ONE 16 Van der Walt EM, Milczek EM, Malan SF, Edmond- 3, e1616. son DE, Castagnoli N Jr, Bergh JJ & Petzer JP 4 Chen JF, Steyn S, Staal R, Petzer JP, Xu K, Van Der (2009) Inhibition of monoamine oxidase by (E)-styryli- Schyf CJ, Castagnoli K, Sonsalla PK, Castagnoli N Jr satin analogues. Bioorg Med Chem Lett 19, & Schwarzschild MA (2002) 8-(3-Chlorostyryl)caffeine 2509–2513. may attenuate MPTP neurotoxicity through dual 17 Binda C, Valente S, Romanenghi M, Pilotto S, Cirilli actions of monoamine oxidase inhibition and A2A R, Karytinos A, Ciossani G, Botrugno OA, Forneris F, receptor antagonism. J Biol Chem 277, 36040–36044. Tardugno M et al. (2010) Biochemical, structural, and

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biological evaluation of tranylcypromine derivatives as monoamine oxidase A indicates stabilization of the neu- inhibitors of histone demethylases LSD1 and LSD2. tral amine in the enzyme-substrate complex. FEBS J J Am Chem Soc 132, 6827–6833. 275, 3850–3858. 18 Binda C, Huba´lek F, Li M, Herzig Y, Sterling J, 26 Leslie AGW (1999) Integration of macromolecular Edmondson DE & Mattevi A (2005) Binding of diffraction data. Acta Crystallographica Section D-Biol -related inhibitors to human monoamine Crystallog 55, 1696–1702. oxidases: a kinetic and crystallographic analysis. J Med 27 Bailey S (1994) The Ccp4 suite – programs for protein Chem 48, 8148–8154. crystallography. Acta Crystallographica Section D-Biol 19 Wang J, Harris J, Mousseau DD & Edmondson DE Crystallog 50, 760–763. (2006) Mutagenic probes of the role of serine 209 on 28 Murshudov GN, Vagin AA & Dodson EJ (1997) the cavity shaping loop of human monoamine oxidase Refinement of macromolecular structures by the A. FEBS J 276, 4569–4581. maximum-likelihood method. Acta Crystallographica 20 Binda C, Aldeco M, Mattevi A & Edmondson DE Section D-Biol Crystallog 53, 240–255. (2010) Interactions of monoamine oxidases with the 29 Emsley P & Cowtan K (2004) Coot: model-building antiepileptic drug zonisamide: specificity of inhibition tools for molecular graphics. Acta Crystallographica and structure of the human monoamine oxidase B Section D-Biol Crystallog 60, 2126–2132. complex. J Med Chem 54, 909–912. 21 Walker MC & Edmondson DE (1994) Structure-activity relationships in the oxidation of benzylamine analogues Supporting information by bovine liver mitochondrial monoamine oxidase B. The following supplementary material is available: Biochemistry 33, 7088–7098. Fig. S1. Structures of reversible MAO inhibitors used 22 Newton-Vinson P, Huba´lek F & Edmondson DE (2000) in this study. High-level expression of human liver monoamine Fig. S2. Graphical representations of the effect of oxidase B in Pichia pastoris. Protein Expr Purif 20, pH on V ⁄ K values for WT MAO B, the Ile199Ala 334–345. MAO B mutant and the Ileu199Ala-Tyr326Ala MAO B 23 Li M, Huba´lek F, Newton-Vinson P & Edmondson DE mutant. (2002) High-level expression of human liver monoamine This supplementary material can be found in the oxidase A in Pichia pastoris: comparison with the online version of this article. enzyme expressed in Saccharomyces cerevisiae. Protein Please note: As a service to our authors and readers, Expr Purif 24, 152–162. this journal provides supporting information supplied 24 Miller JR & Edmondson DE (1999) Structure-activity relationships in the oxidation of para-substituted by the authors. Such materials are peer-reviewed and benzylamine analogues by recombinant human may be re-organized for online delivery, but are not liver monoamine oxidase A. Biochemistry 38, copy-edited or typeset. Technical support issues arising 13670–13683. from supporting information (other than missing files) 25 Dunn RV, Marshall KR, Munro AW & Scrutton NS should be addressed to the authors. (2008) The pH dependence of kinetic isotope effects in

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