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Crystallographic Snapshots of the Complete Reaction Cycle of Nicotine Degradation by an Amine Oxidase of the Monoamine Oxidase (MAO) Family

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Crystallographic Snapshots of the Complete Reaction Cycle of Nicotine Degradation by an Amine Oxidase of the Monoamine Oxidase (MAO) Family Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family Galina Kachalovaa, Karl Deckerb, Andrew Holtc, and Hans D. Bartunika,1 aMax Planck Unit for Structural Molecular Biology, Notkestrasse 85, 22607 Hamburg, Germany; bInstitut für Biochemie und Molekularbiologie, Albert-Ludwig University, Stefan-Meier-Strasse 17, 79104 Freiburg im Breisgau, Germany; and cDepartment of Pharmacology, School of Molecular and Systems Medicine, Faculty of Medicine and Dentistry, 9-58 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7 Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved February 4, 2011 (received for review November 9, 2010) FAD-linked oxidases constitute a class of enzymes which catalyze alditol oxidase (17). However, substrate and product were found dehydrogenation as a fundamental biochemical reaction, followed to coexist in the active site and could not be distinguished, despite by reoxidation of reduced flavin. Here, we present high-resolution the high resolution reached in these studies. crystal structures showing the flavoenzyme 6-hydroxy-L-nicotine Here, we describe discrete structural states populated during oxidase in action. This enzyme was trapped during catalytic degra- productive enzymatic turnover in crystalline 6HLNO. The flavo- dation of the native substrate in a sequence of discrete reaction enzyme is a close structural neighbor (15) of human MAO-A states corresponding to the substrate-reduced enzyme, a complex and MAO-B. It is involved in the degradation of nicotine in of the enzyme with the intermediate enamine product and forma- the soil bacterium Arthrobacter nicotinovorans, which starts with tion of the final aminoketone product. The inactive D-stereoisomer 6-hydroxylation of the pyridine ring. In a second step, catalyzed binds in mirror symmetry with respect to the catalytic axis, reveal- by 6HLNO, the pyrrolidine ring of 6-hydroxy-L-nicotine (6HLN) ing absolute stereospecificity of hydrogen transfer to the flavin. is oxidized to 6-hydroxy-N-methylmyosmine and cleaved to yield The structural data suggest deprotonation of the substrate when 6-hydroxy-pseudooxynicotine (18) (Fig. 1A). The enzyme acts bound at the active site, an overall binary complex mechanism with absolute stereospecificity on 6HLN and is competitively in- BIOCHEMISTRY and oxidation by direct hydride transfer. The amine nitrogen has hibited by the antistereomer 6-hydroxy-D-nicotine (6HDN). This a critical role in the dehydrogenation step and may activate carbo- paper describes the functioning of 6HLNO in action, based on cation formation at the α-carbon via delocalization from the lone high-resolution structures trapped during productive turnover of pair to σ*Cα-H. Enzymatically assisted hydrolysis of the intermedi- the L-substrate. The structural data and studies of steady-state ate product occurs at a remote (P site) cavity. Substrate entry and kinetics provide an experimental basis for assessing the structural product exit follow different paths. Structural and kinetic data mechanism of 6HLNO and revisiting the reaction pathway in suggest that substrate can also bind to the reduced enzyme, asso- MAO. ciated with slower reoxidation as compared to the rate of reoxida- tion of free enzyme. The results are of general relevance for the Results mechanisms of flavin amine oxidases. Structures of 6HLNO Trapped During Enzymatic Turnover. Crystal structures of 6HLNO in different reaction states were obtained catalytic mechanism ∣ chiral stereospecificity ∣ cryocrystallography ∣ at high resolution following initiation of the catalytic reaction reaction intermediate ∣ structural kinetics in the crystalline enzyme and cryotrapping (Table S1). The structures 3NGC (E-S-P1) and 3NH3 (E-S-P2) showed 6HLN lavin-dependent amine oxidases take part in a number of bound with full occupancy at the active site, which is located Fcellular processes ranging from degradative metabolism to at the interface between the FAD-binding domain and the sub- chromatin remodeling. A large body of work has examined the strate-binding subdomains S1 and S2 of the 6HLNO subunit, and common kinetic mechanism of flavoprotein oxidases, which a further ligand bound with high occupancy at a second, remote includes a reductive half-reaction yielding the oxidized substrate site (Figs. 1B–3). The additional ligand was structurally identified bound to the reduced enzyme and subsequent reoxidation of the as the intermediate 6-hydroxy-N-methylmyosmine (P1) in E-S-P1 enzyme by oxygen in a binary or ternary complex scheme. The and as the final product 6-hydroxy-pseudooxynicotine (P2) in mechanism of individual steps remains controversial as discussed E-S-P2. The binding sites of P1 and P2 are located in a (“P site”) in a number of reviews (1–5). Hypotheses put forward for the cavity, which is lined by residues from S1 (81–88, 99–100, dehydrogenation step of the catalytic reaction include radical 163–167, 195–199) and S2 (300–302, 309–311). It is further lined mechanisms involving single-electron transfer from the substrate by one of the acyl chains of the diglycerophospholipid (Fig. 2), to the flavin as a first step (6), nucleophilic mechanisms involving which is noncovalently bound to the S1 subdomain of all 6HLNO an intermediate substrate-flavin adduct (7, 8), a carbanion me- structures (15). Binding of P1 or P2 to the P-site cavity is asso- chanism (9), and a direct hydride transfer mechanism, which has ciated with a movement of the main chain segment 81–85 and a been established for D-amino-acid oxidase (DAAO) (4, 10, 11). For a number of flavin amine oxidases including DAAO (11), Author contributions: H.D.B. designed research; A.H. designed kinetic studies; G.K., A.H., L -amino-acid oxidase (LAAO) (12), monoamine oxidase (MAO) and H.D.B. performed research; K.D. contributed new reagents/analytic tools; G.K., K.D., (1, 13, 14), and 6-hydroxy-L-nicotine oxidase (6HLNO) (15), a A.H., and H.D.B. analyzed data; and H.D.B. wrote the paper. carbanion mechanism may be excluded because the active sites The authors declare no conflict of interest. do not contain residues capable of and properly placed for acid- This article is a PNAS Direct Submission. base catalysis. Most of the investigations of structural mechan- Data deposition: The atomic coordinates and structure factors have been deposited in isms involved enzymes in unproductive complexes with substrate the Protein Data Bank, www.pdb.org [PBD ID codes 3NGC (E-S-P1), 3NH3 (E-S-P2), analogues or inhibitors. Crystallographic studies of amine oxi- 3NK0 (E-I-P2), 3NG7 (E-S-I), 3NHO (E-P2), 3NK1 (E-5HT), 3NK2 (E-DA), and 3NN6]. dases under productive oxidizing conditions were reported for 1To whom correspondence should be addressed. E-mail: [email protected]. Rhodosporidium toruloides D-amino-acid oxidase (11), porcine This article contains supporting information online at www.pnas.org/lookup/suppl/ kidney D-amino-acid oxidase (16), and Streptomyces coelicolor doi:10.1073/pnas.1016684108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1016684108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 26, 2021 H H H A O O O A B 6 N N H2O2 O2 N Y59 1 K287 L198 82 3 918 H 2‘ E-FAD E-FADH2 6HDN CH3 CH3 O 1‘N N + H2O H P1 N CH3 P2 660 W163 (S) (P1) (P2) P1 B 470 PHL 85 F85 S2 E300 Y311 E300 D312 A100 D C S1 P2 6HDN S83 FD Fig. 1. Reaction catalyzed by 6HLNO. (A) Dehydrogenation of the pyrroli- dine moiety of 6HLN (S) results in the formation of the intermediate product 6-hydroxy-N-methylmyosmine (P1). Subsequent hydration of the intermedi- Fig. 3. Binding geometry at P site. (A) The structures E-S-P1 (orange), E-S-P2 E ate opens the pyrrole ring to form the final product 6-hydroxy-pseudooxy- (green), E-S-I (magenta), E-S (yellow), and nat (gray) are superimposed. nicotine (P2). (B) Crystal structure of 6HLNO subunit with substrate and Ligand binding to the P site is associated with backbone motions of the – product sites. The substrate (teal) is bound at the active site and the inter- segment 81 85. The orientation in the P-site cavity is shown for (B) the inter- mediate product P1 (orange) at the P site. PHL denotes a noncovalently mediate P1 (orange), (C) the final product P2 (green), and (D) the inhibitor F − F bound phospholipid. The inset shows the solvent-inaccessible active-site cav- 6HDN (magenta). Simulated annealed omit ( o c) maps are contoured at 3 3 σ σ σ ity (yellow; volume 195 Å ), the exit (P site) cavity (brown; 290Å ) opening 2.2 for P1, 1.8 for P2, and 3.0 for 6HDN. 3 toward the protein surface, and the channel (red; 80 Å ) for gated transfer of the intermediate product. Arrows indicate possible paths for entry and exit. its orientation with respect to the isoalloxazine ring and the con- tacts with the protein environment are closely similar as in the reorientation of the Phe85 ring (Fig. 3A), which opens the cavity previously described dithionite-reduced enzyme–substrate com- toward the protein surface, enabling release of the final product plex (E-S; 3K7Q) (15). The pyrrolidine moiety of the substrate to the solvent at a location close to the subunit–subunit interface is present in a slightly twisted envelope conformation (Fig. 4) ′ of the 6HLNO dimer. A further structure, E-I-P2, was derived with the amine nitrogen N1 in the apex position pointing away ′ from the reaction of the enzyme with a racemic mixture of the from the isoalloxazine ring (distance N1 -C4a about 4.1 Å). L-substrate with 6HDN. E-I-P2 contains 6HDN bound at the ac- Flavin and ligand-binding geometry data are summarized in ′ tive site and, in addition, the product P2 resulting from turnover Table S2.
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