The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site
2000; Springer Nature; Volume: 19; Issue: 16 Linguagem: Inglês
10.1093/emboj/19.16.4204
ISSN1460-2075
Autores Tópico(s)Hemoglobin structure and function
ResumoArticle15 August 2000free access The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site Peter D. Pawelek Peter D. Pawelek Biochemistry Department and Montréal Joint Center for Structural Biology, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Jaime Cheah Jaime Cheah Biochemistry Department and Montréal Joint Center for Structural Biology, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Rene Coulombe Rene Coulombe Biochemistry Department and Montréal Joint Center for Structural Biology, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Peter Macheroux Peter Macheroux Eidgenössische Technische Hochschule, Institut für Pflanzenwissenschaften, Universitätstrasse 2, CH-8092 Zürich, Switzerland Search for more papers by this author Sandro Ghisla Sandro Ghisla Fachbereich Biologe, University of Konstanz, Konstanz, Germany Search for more papers by this author Alice Vrielink Corresponding Author Alice Vrielink Present address: Biology Department, 225 Sinsheimer Laboratory, University of California, 1156 High St, Santa Cruz, CA, 95064 USA Search for more papers by this author Peter D. Pawelek Peter D. Pawelek Biochemistry Department and Montréal Joint Center for Structural Biology, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Jaime Cheah Jaime Cheah Biochemistry Department and Montréal Joint Center for Structural Biology, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Rene Coulombe Rene Coulombe Biochemistry Department and Montréal Joint Center for Structural Biology, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6 Canada Search for more papers by this author Peter Macheroux Peter Macheroux Eidgenössische Technische Hochschule, Institut für Pflanzenwissenschaften, Universitätstrasse 2, CH-8092 Zürich, Switzerland Search for more papers by this author Sandro Ghisla Sandro Ghisla Fachbereich Biologe, University of Konstanz, Konstanz, Germany Search for more papers by this author Alice Vrielink Corresponding Author Alice Vrielink Present address: Biology Department, 225 Sinsheimer Laboratory, University of California, 1156 High St, Santa Cruz, CA, 95064 USA Search for more papers by this author Author Information Peter D. Pawelek1, Jaime Cheah1, Rene Coulombe1, Peter Macheroux2, Sandro Ghisla3 and Alice Vrielink 4 1Biochemistry Department and Montréal Joint Center for Structural Biology, McIntyre Medical Sciences Building, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6 Canada 2Eidgenössische Technische Hochschule, Institut für Pflanzenwissenschaften, Universitätstrasse 2, CH-8092 Zürich, Switzerland 3Fachbereich Biologe, University of Konstanz, Konstanz, Germany 4Present address: Biology Department, 225 Sinsheimer Laboratory, University of California, 1156 High St, Santa Cruz, CA, 95064 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4204-4215https://doi.org/10.1093/emboj/19.16.4204 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 Å resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 Å long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO–AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals significant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis. Introduction L-amino acid oxidase (LAAO) is a dimeric flavoprotein first described by Zeller in 1944 (Zeller and Maritz, 1944). It contains non-covalently bound FAD as cofactor, and is present in various species (Curti et al., 1992). LAAO catalyzes the stereospecific oxidative deamination of an L-amino acid substrate to an α-keto acid along with the production of ammonia and hydrogen peroxide via an imino acid intermediate (Scheme 1). LAAO enzymes purified from the venoms of a variety of snake species are the best studied members of this family of enzyme. The enzyme is present at significantly high concentrations in venom and is postulated to be a toxin (Li et al., 1994; Torii et al., 1997). These enzymes exhibit a marked preference for hydrophobic amino acids including phenylalanine, tryptophan, tyrosine and leucine. Although the mode of toxicity of snake venom LAAO is not known, it has been shown that the enzymes from Crotalus adamanteus and Crotalus atrox can associate specifically with mammalian endothelial cells (Suhr and Kim, 1996). Furthermore, snake venom LAAO can induce apoptosis in mammalian endothelial cells, possibly through the production of highly localized concentrations of hydrogen peroxide (Suhr and Kim, 1996, 1999). We recently have determined the amino acid sequence of LAAO from Calloselasma rhodostoma (P.Macheroux, personal communication); this protein shares a high degree of sequence identity with the two other known ophidian LAAOs from C.adamanteus (83%) and C.atrox (83%). Also, these proteins share significant similarity (>30% identity) with the murine interleukin 4-inducible Fig 1 protein (Raibekas and Massey, 1998). This protein is predicted to be a flavoprotein homologous to monoamine oxidase (MAO) and may be involved in the allergic inflammatory response (Chu and Paul, 1997). Figure 1.Stereo ribbon representation of the secondary structure elements for L-amino acid oxidase (produced with the program Molscript; Kraulis, 1991) (A) The functional dimer. The individual protomers are colored red and green. (B) A single protomer of L-amino acid oxidase. (C) Topology diagram. The β-strands are depicted as arrows and the α-helices as cylinders. The strands and helices are numbered in the order in which they appear in the primary sequence. The FAD-binding domain is shown in red, the substrate-binding domain in green and the helical domain in blue. The FAD and citrate molecules as well as the glycosylation sites are shown as a ball-and-stick representation. Download figure Download PowerPoint A peculiarity of LAAO from C.adamanteus is the reversible inactivation observed upon freezing (−20°C) or raising the pH to above neutrality (Curti et al., 1968; Coles et al., 1977). Reactivation of freeze- or pH-inactivated enzyme is achieved by heat treatment (37°C) at pH 5. This reversible process is associated with small changes in the absorbance spectrum of the FAD cofactor with no change in the CD spectrum of the protein (Coles et al., 1977). The structural basis for the inactivation/reactivation of C.adamanteus LAAO remains elusive. The structures of three enzymes functionally similar to LAAO have been determined recently: D-amino acid oxidase (DAAO) from pig kidney (Mattevi et al., 1996; Mizutani et al., 1996; Miura et al., 1997), DAAO from the yeast Rhodotorula gracilis (Umhau et al., 1999) and polyamine oxidase (PAO) from Zea mays (Binda et al., 1999). These structural studies reveal that the overall folds of DAAOs and PAO are similar; however, the mode of substrate entry differs between them. Here we present the first high-resolution structure of an LAAO. The enzyme was purified from C.rhodostoma (Malayan pit viper) and solved to 2.0 Å resolution. The enzyme has been crystallized in the presence of citrate and o-aminobenzoate (AB), two ligands that are inhibitory to LAAO activity. Analysis of the LAAO structure in the presence of these ligands, and in conjunction with the structures of DAAO and PAO, provides us with valuable insights into: (i) how these enzymes are related in terms of modes of substrate entry; (ii) how the structural factors dictate enantiomeric specificity; and (iii) the relative arrangement of atoms involved in the chemical catalysis within their respective active sites. Results and discussion Overall structure The structure of the LAAO–citrate (CIT) complex from Malayan pit viper has been determined at 2.0 Å resolution by the method of multiple isomorphous replacement (MIR) combined with solvent flipping and 4-fold averaging. This model was then used to determine the structure of the LAAO–AB complex to 2.0 Å resolution by the method of molecular replacement. Table I gives the final refinement statistics for each of the structures. The Ramachandran plot for both complexes shows that 99.3% of the residues fall within favorable regions. Table 1. Refinement statistics Model refinement statistics LAAO–citrate complex LAAO–AB complex Resolution range (Å) 50.0–2.0 50.0–2.0 Total reflections used in refinement 139 206 283 071 R-factora 0.185 0.205 Rfreeb 0.210 0.225 R.m.s.d. bond lengths (Å) 0.007 0.007 R.m.s.d. bond angles (°) 1.26 1.25 No. of non-hydrogen atoms protein 15 376 30 792 FAD 212 424 citrate 52 – carbohydrate 208 112 o-aminobenzoate – 240 water 1791 2378 Average B-factors (Å2) overall 24.2 19.0 protein atoms 23.1 18.9 FAD atoms 14.0 10.4 citrate atoms 66.3 – carbohydrate atoms 53.6 37.7 o-aminobenzoate atoms – 43.5 water molecules 32.2 21.9 a R-factor = ΣΣh‖Fobs (h)| − k|Fc(h)‖/Σh|Fobs(h)| b Rfree was calculated using a10% randomly selected subset of the total number of reflections. Each protomer includes the FAD cofactor, the N-linked oligosachharide chains at Asn172 and Asn361, and the ligand (CIT or AB). The composition of the sugars making up the oligosaccharide has been determined and the sequence of glycosylation is being confirmed by NMR analysis (O.Geyer, P.Pawelek, P.Fitzpatrick, K.Kitzing, A.Vrielink, S.Ghisla and P.Macheroux, in preparation). Only the proximal sugar residues within the N-linked oligosaccharides are clearly evident in the electron density and have been included in the model. LAAO from Malayan pit viper is a dimeric enzyme of 55 kDa per protomer. Each protomer consists of 15 α-helices and 22 β-strands that fold into three well-defined domains (Figure 1). The FAD-binding domain consists of three discontinuous regions of the structure: residues 35–64, 242–318 and 446–486. The main structural feature of this domain is a six-stranded β-pleated sheet sandwiched between three α-helices and a four-stranded β-pleated sheet. The motif makes up the classical nucleotide-binding fold seen in many FAD- and NAD(P)-binding enzymes. The substrate-binding domain is made up of residues 5–25, 73–129, 233–236 and 323–420. Finally, an entirely helical domain is made up of residues 130–230. This domain comprises one side of a funnel-shaped entrance to the active site of the enzyme. The topology of LAAO most closely resembles that of PAO (Binda et al., 1999) with further similarities to p-hydroxybenzoate hydroxylase (Wierenga et al., 1979), cholesterol oxidase (Vrielink et al., 1991), glucose oxidase (Hecht et al., 1993) and DAAO (Mattevi et al., 1996; Mizutani et al., 1996; Umhau et al., 1999). A structure superposition with PAO reveals an r.m.s. deviation of 2.9 Å with 414 Cα atoms included in the alignment. Given the high degree of topological similarity, analysis of the sequence identity between LAAO and PAO is rather low at only 26% (19% for structurally equivalent residues), with the major structural difference localized in the helical domain. In LAAO, this domain comprises six α-helices (α4–α9). Four of these helices correspond to ones found in the PAO structure (Sα1, Sα2, Sα3 and Sα4); however, in PAO, these helices have been identified as part of the substrate-binding domain and do not comprise a separate domain. Furthermore, the relative orientations of these helices are different between the two enzymes. The average temperature factors for the helical domain in both complexes of LAAO are higher than for other domains in the structure, suggesting that the helical domain may be more flexible than other regions of the protein. Biochemical studies have shown that LAAO is active as a dimer. In the crystal structure of LAAO–CIT we observe four molecules in the asymmetric unit arranged as a dimer of dimers, and in the structure of LAAO–AB the asymmetric unit contains two dimers of dimers. Figure 1A shows the secondary structure representation for a single closely associated dimer in the asymmetric unit. The 2-fold non-crystallographic rotation axis relating the two protomers with a single dimer results in positioning of the isoalloxazine rings of the two FAD molecules along a single plane, with the remaining portions of the prosthetic group extending in opposite directions. Furthermore, the entrance to the active site for each protomer is located on opposite surfaces of the dimer. Studies of LAAO have shown that the enzyme is inactivated reversibly upon freezing or increasing the pH to above neutrality, and reactivation can be achieved by heat treatment at pH 5.0 (Wellner, 1966). The LAAO enzyme that was crystallized had been frozen and thawed prior to crystallization and thus was in the inactive form. However, crystals of the enzyme were obtained at pH 4.5, suggesting that the protein may have been reactivated during the course of the crystallization experiment. In order to verify whether the crystallized enzyme was active, crystals dissolved in 50 mM Tris buffer (pH 7.4) were assayed and the solubilized enzyme was shown to be active (data not shown). In addition, crystals were transferred to a mother liquor solution (at pH 7.4) containing 10 mM phenylalanine. Visual observation of these crystals revealed cracking and a color change from yellow to colorless and back to yellow again within 40 min, indicating a change in the redox state of LAAO-bound FAD within the crystal. A similar experiment, conducted in acetate-buffered mother liquor (pH 4.5), yielded similar observations, however at a substantially slower rate. The enzyme crystallized at pH 4.5 is therefore in the active form. Furthermore, the bleaching experiments support our data suggesting that the catalytic efficiency of the enzyme is significantly lower at pH 4.5 and that a conformational change upon substrate binding may occur, resulting in crystal cracking. Glycosylation sites The protein used for crystallization and subsequent structure solution was that isolated from the native source (snake venom) and is glycosylated. Indeed, studies by Macheroux and co-workers have shown that the enzyme contains up to 3.7 kDa of glycosylation per protomer (Macheroux et al., 1999). LAAO from C.adamanteus has also been shown to be highly glycosylated (de Kok and Rawitch, 1969). From the electron density map of the LAAO–CIT complex, two N-glycosylation sites have been identified in agreement with biochemical analysis: Asn172, located in the loop region between helices α5 and α6 of the helical domain, and Asn361, located in the loop region between β18 and β19 of the substrate-binding domain (Figure 1B). Inspection of the glycosylation sites within the dimer reveals that the carbohydrates connected to Asn172 lie along a single molecular surface (the distance between the sugar moieties is ∼35 Å) whereas those connected to Asn361 lie on opposite surfaces of the dimer (separated by ∼75 Å). In the case of Asn172, three carbohydrate residues are visible in the electron density and have been included in the model: C1 of N-acetylglucosamine (Nag523) is linked to the amide nitrogen of Asn172. This Nag has a further two sugar molecules connected to it: a second Nag (Nag524) in a βO4–C1 linkage and a fucose molecule (Fuc525) in an αO6–C1 linkage. Chemical studies suggest that the composition and branching of the sugar chains at Asn172 and Asn361 are similar (O.Geyer, P.Pawelek, P.Fitzpatrick, K.Kitzing, A.Vrielink, S.Ghisla and P.Macheroux, in preparation). The electron density is of sufficient definition at Asn361 to reveal only a single Nag molecule (Nag522), but small amounts of positive difference electron density are seen near to O4 and O6 of Nag522, indicating branching similar to that observed at Asn172. The structure of PAO also shows glycosylation; however, in contrast to LAAO, only a single glycosylation site (Asn77) is present. Superposition of the structures indicates that the glycosylation sites are located at different positions in the overall structures of LAAO and PAO. However, the sequence of the first three carbohydrates at Asn172 in LAAO and Asn77 in PAO are identical, but with differing linkages. FAD interactions The FAD prosthetic group is buried deeply within the enzyme and makes extensive interactions with protein atoms and with conserved water molecules. The FAD adopts a conformation similar to that seen in a number of other FAD enzymes. The interactions made by the FAD are shown for a single protomer in Figure 2A. All protomers for both complexes show identical interactions between the protein and the prosthetic group. In addition, the water molecules involved in hydrogen bond interactions are structurally conserved for the protomers in the asymmetric unit. Of the residues involved in side chain interactions with FAD, three are identical in PAO (these residues are Glu457–Glu430, Arg71–Arg43 and Glu63–Glu35 coinciding to LAAO–PAO). Figure 2.Stereoview of the LAAO–citrate complex in the region of (A) the FAD prosthetic group and (B) the citrate ligand. The protein main chain is shown as a green coil, and specific amino acid residues and the FAD molecule are depicted as ball-and-stick models with yellow and gray bonds, respectively. Water molecules are represented as red spheres. The hydrogen bond contacts are shown as black dashed lines. Download figure Download PowerPoint The isoalloxazine ring is positioned at the interface between the FAD-binding domain and the substrate-binding domain in a similar fashion to that observed for a number of FAD-binding enzymes. The dimethylbenzene ring is surrounded by a number of hydrophobic residues (Ile374, Trp420 and Ile430) (Figure 2A). An identical environment is observed in the structure of PAO with amino acid conservation seen for Ile374 (Thr350 in PAO), Trp420 (Trp393 in PAO) and Ile430 (Phe403 in PAO). In the structure of PAO, Mattevi and co-workers have identified a water molecule interacting with N5 of the isoalloxazine ring and with the side chain amino group of a lysine residue (Lys300). They have suggested that this water molecule may be important in the hydrolytic attack on the imino intermediate. In the structure of LAAO, we also see a water molecule (Wat605) hydrogen bonded to N5 as well as to the side chain amino group of Lys326 in the β16 strand of the substrate-binding domain. A comparison of the sequences of LAAO and PAO around this conserved lysine residue reveals significant identity (TKIFL) along the β16-strand. Furthermore, the environment surrounding the side chain of Lys326 reveals a large number of hydrophobic residues (see Figure 2A) (Phe328, Ile370, Tyr372, Tyr356, Met89 and Leu86) similar to that observed around Lys300 in PAO. This suggests that the Lys326 may have a decreased pKa and may act as a base to increase the nucleophilicty of Wat605 for attack on the imino intermediate. Neither a water molecule nor a lysine residue is observed at the identical positions in the structures of DAAO. Thus, if hydrolysis of the imino acid was to be mediated by the protein, this might suggest that in LAAO and DAAO it occurs through different mechanisms. Indeed, with DAAO, hydrolysis is thought to occur non-enzymatically. LAAO–CIT complex The active site of the enzyme is located at the base of a long funnel extending 25 Å from the surface into the interior of the protein. The entrance to the funnel is bounded by the side chains of residues within four α-helices in the helical domain: α4, α5, α8 and α9, as well as residues in the β6 strand. The wide entrance to the funnel is filled with water molecules and gradually narrows to a constricted region located ∼15 Å into the interior of the molecule and formed by the side chains of His223 and Glu209. This constriction is not complete since a continuous solvent-accessible channel is observed from the surface to the active site. However, the opening is not large enough to allow the passage of either substrate or α-keto acid product. Interior to the constricted region of the funnel, near to the N5 of the isoalloxazine ring of FAD, a widened internal cavity constitutes the substrate-binding site. Inspection of the residual electron density in the active site region as well as the crystallization conditions for the original LAAO structure indicate that a citrate molecule is positioned ∼17 Å from the external surface of the protein, blocking the entrance to the active site. Extensive hydrogen bond interactions are made between the citrate and protein side chains and with water molecules located in the active site cleft as shown in Figure 2B; however, there are no interactions between citrate and the FAD cofactor. In all four protomers, the temperature factors for the citrate molecules are high (average 70 Å2) and extensive residual difference electron density indicates that the citrate molecules are not tightly bound to the enzyme and are highly mobile. Steady-state kinetic studies performed on LAAO indicate that citrate is an inhibitor at pH 4.6 but does not inhibit the enzyme at pH 7.4 (data not shown). At pH 4.5, its balance of charge is less negative than at physiological pH which may facilitate more favorable contacts with LAAO. Increasing the pH will result in deprotonation of the ligand and will adversely affect its binding in the active site of the enzyme. Extending from the main cavity where the citrate molecule is bound is a pocket filled with water molecules and bounded by helix α9 and the loop between β22 and α15. An alternative entrance to this pocket, from the external surface of the molecule, is restricted by the N-terminal region of the structure, specifically the main chain of residues 10–14. In PAO, this region of the structure forms one of the entrances to the U-shaped tunnel. The N-terminal region of LAAO is not present in the structure of PAO and thus the tunnel is only open at one end. In LAAO, this N-terminal region is held to the helical domain by hydrogen bond interactions between the side chain of Asn5 and Asp225 as well as van der Waals interactions between the side chain of Phe11 and those of Leu221, and the alkyl portion of Lys222 along the α9 helix. LAAO–AB complex AB is an inhibitor of C.adamanteus LAAO (de Kok and Veeger, 1968). Through preliminary steady-state kinetic assays, we have confirmed that AB is also a mixed inhibitor of C.rhodostoma LAAO at pH 4.6 (data not shown). We were able to obtain crystals of the complex of LAAO with AB, which grew in a crystal form different from those of the original LAAO–CIT crystals. The change in cell dimensions results in a doubling of the asymmetric unit such that four dimers are present instead of two as in the case of the LAAO–CIT crystals. The structure of the LAAO–AB complex was determined using molecular replacement techniques and refined to 2.0 Å resolution (Table I). The LAAO–AB structure shows the ligand bound in three discrete positions within each of the eight protomers in the asymmetric unit (Figure 3A) (AB1, AB2 and AB3). A comparison of the temperature factors of AB1, AB2 and AB3 averaged across the eight protomers in the asymmetric unit indicates that AB1 has the most well ordered position since its Bavg is 35.7 Å2 compared with 43.3 Å2 for AB2 and 41.1 Å2 for AB3. This is consistent with both the well-defined electron density and the increased number of contacts observed for AB1. Although the density is weaker for AB2 and AB3, one can still clearly model the ligand into the density and orient both the carboxylate moiety and the amino group with confidence in all eight protomers within the asymmetric unit. Interestingly, the recently solved structure of yeast DAAO (yDAAO) indicates the presence of two AB molecules bound within its active site funnel (Umhau et al., 1999). The yDAAO funnel is substantially shallower than that seen for LAAO, and could not accommodate a third AB molecule. Figure 3.(A) Electron density in the region of the active site of the LAAO–AB complex. Some protein side chains, the FAD and the three AB positions are shown. The electron density is from a 2Fo − Fc map, averaged over eight protomers in the asymmetric unit, and contoured at 1.0σ. (B) Molecular surface representation of the LAAO–AB complex with the electrostatic potential mapped between −15.0 kT (red) and +15.0 kT (blue). The protein main chain is shown as a colored tube with blue as the helical domain, green as the substrate-binding domain and red as the FAD-binding domain. The AB and FAD molecules are represented as capped sticks. A magnified view of the funnel and the active site containing the AB ligands is shown in the inset. (C) Stereo representation of the interactions between the protein and the three AB ligands in the LAAO–AB complex. The protein main chain regions are represented as a green coil. Amino acid residues, the FAD and the AB molecules are shown in yellow, gray and cyan bonds, respectively. The alternative conformation of His223 is colored magenta. Hydrogen bonds are depicted as black dashed lines. Download figure Download PowerPoint In the LAAO–AB structure, the positions of the three bound AB molecules suggest the trajectory taken by the substrate from the surface of the molecule to the active site (Figure 3B). The orientation of the AB molecules appears to be determined by the electrostatics of the funnel. The surface closest to the carboxylates of AB1, AB2 and AB3 is uniformly electropositive, whereas the surface most proximal to the amino groups of the ligands is predominantly electronegative in character. The three AB molecules make contacts with a number of residues within the funnel and the active site (Figure 3C). The outermost ligand, AB3, lies ∼10 Å within the funnel between helices α5, α8 and α9 and makes van der Waals contacts with side chain residues within these three helices. Further stabilization at this position is due primarily to an ionic interaction between the amino group of AB3 and the carboxylate of Asp224. AB2 lies 5.5 Å closer to the active site than AB3. The AB2 carboxyl group is within 3.7 Å of the imidazole portion of His223, and the amino group makes contact with the carboxyl group of Glu209; the side chains of both of these residues protrude from the lining of the active site funnel. The most well defined of the three inhibitors, AB1, lies within the active site nearest to the isoalloxazine ring with C1 positioned 3.9 Å from N5 of FAD. A salt bridge exists between Arg90 and the carboxylate moiety of AB1. The AB1 carboxylate is also stabilized by a hydrogen bond with the side chain hydroxyl group of Tyr372. Finally, the AB1 amino group is within hydrogen bonding distance of the backbone carbonyl oxygen atom of Gly464. In addition, His223 in the LAAO–AB complex can adopt an alternative conformation to that seen in the LAAO–CIT complex (Figure 3C). This indicates that the active site of the enzyme has space to accommodate alternative positions for the imidazole side chain of His223, which may be required for catalysis. Comparison of the orientations of the three AB molecules reveals that the carboxylate group for each ligand points in the same direction, upward towards the funnel entrance to the active site, and the aromatic ring is directed downwards to the ribityl moiety of FAD. In AB1, the aromatic ring is sandwiched between the side chains of Ile430 and Ile374. The orientations of AB2 and AB3 are similar; however, that of AB1 is different, with the benzyl ring rotating by 108° relative to that of AB2 such that the amino group is positioned over the pyrimidine portion of the isoalloxazine ring. This orientation places the amino group within hydrogen bonding distance of the carbonyl oxygen of Gly464, at the N-terminus of the C-terminal helix in the structure. Interestingly, comparisons of the protein–ligand contacts seen in LAAO, DAAO and PAO all reveal a similar interaction of the ligand amino nitrogen atom with the carbonyl oxygen atom of a conserved glycine residue at the N-terminus of the final α-helix in the structures. Apart from the above-mentioned hydrogen bond interaction, the amino group makes no further contacts with the protein. Structural comparisons with D-amino acid oxidase LAAO speci
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