The 1.25Acrystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters
1997; Springer Nature; Volume: 16; Issue: 24 Linguagem: Inglês
10.1093/emboj/16.24.7219
ISSN1460-2075
AutoresGünter Auerbach, Anja Herrmann, Markus Gütlich, Markus Fischer, Uwe Jacob, Adelbert Bacher, Robert Huber,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoArticle15 December 1997free access The 1.25 Å crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters Günter Auerbach Corresponding Author Günter Auerbach Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany Search for more papers by this author Anja Herrmann Anja Herrmann Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Markus Gütlich Markus Gütlich Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Markus Fischer Markus Fischer Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Uwe Jacob Uwe Jacob Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany Search for more papers by this author Adelbert Bacher Adelbert Bacher Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Robert Huber Robert Huber Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany Search for more papers by this author Günter Auerbach Corresponding Author Günter Auerbach Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany Search for more papers by this author Anja Herrmann Anja Herrmann Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Markus Gütlich Markus Gütlich Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Markus Fischer Markus Fischer Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Uwe Jacob Uwe Jacob Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany Search for more papers by this author Adelbert Bacher Adelbert Bacher Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany Search for more papers by this author Robert Huber Robert Huber Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany Search for more papers by this author Author Information Günter Auerbach 1, Anja Herrmann2, Markus Gütlich2, Markus Fischer2, Uwe Jacob1, Adelbert Bacher2 and Robert Huber1 1Max-Planck-Institut für Biochemie, Abt. Strukturforschung, Am Klopferspitz 18a, D-82152 Martinsried, Germany 2Technische Universität München, Institut für Organische Chemie und Biochemie, Lichtenbergstrasse 4, 85748 Garching, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:7219-7230https://doi.org/10.1093/emboj/16.24.7219 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co-factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 Å in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single-domain α/β-fold with a central four-helix bundle connecting two seven-stranded parallel β-sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin-substrates, which positions the substrate side chain C1′-carbonyl group near Tyr171 OH and NADP C4′N. The catalytic mechanism of SR appears to consist of a NADPH-dependent proton transfer from Tyr171 to the substrate C1′ and C2′ carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N-acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N-acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo. Introduction Tetrahydrobiopterin (BH4) is a multifunctional cofactor for phenylalanine, tyrosine and tryptophan hydroxylases, which catalyse the initial steps in phenylalanine degradation in the liver, and are the rate-limiting steps in the biosynthesis of the neurotransmitters, catecholamines and indoleamines in the brain. A function to promote release of dopamine, serotonin and noradrenaline from the striatal and cortical nerve terminals has also been proposed for BH4 (Mataga et al., 1991). Serotonin (5-hydroxytryptamine; 5-HT) and its derivatives are neurotransmitters present in brain or pituitary gland, regulating a great number of physiological mechanisms such as sleep, appetite, thermoregulation, control of pituitary secretions and behaviour (McGeer et al., 1987). BH4 has furthermore an essential role in the biosynthesis of nitric oxide (NO) (Lowenstein and Snyder, 1992; Marletta, 1994) as an allosteric activator of nitric oxide synthase (NOS) and seems to be necessary for catalytic turnover involving a redox-function of the co-factor (Hemmens and Mayer, 1996). Recently, it was shown that an increase in BH4 biosynthesis in a pancreatic B-cell line (INS-1) is followed by enhanced NO production and subsequently, inhibition of insulin secretion (Laffranchi et al., 1997). BH4 regulates human melanogenesis by forming a stable complex with the α-melanocyte-stimulating hormone (Schallreuter et al., 1994). Finally, BH4 is known as an essential co-factor for alkylglyceryl monooxygenases (Pfleger et al., 1967). Reduced levels of BH4 in the brain and cerebrospinal fluid are associated with several neuropsychiatric diseases such as Parkinson's disease, Alzheimer's disease, depression and dystonia (Lovenberg et al., 1979; Curtius et al., 1983). In atypical phenylketonuria (PKU), BH4 deficiency results in neurological disorders as a result of decreased biosynthesis of brain catecholamines and serotonin. BH4 is involved in proliferation and growth regulation of erythroid cells. Partial depletion of BH4 in a murine erythroleukaemia cell line caused inhibition of cell growth (Tanaka et al., 1989). The complex organic chemistry involved in de novo formation of BH4 from GTP is catalysed by only three enzymes. The first committed step is catalysed by GTP cyclohydrolase I (EC 3.5.4.16; CYH) conducive to the formation of dihydroneopterin triphosphate. This intermediate is transformed to 6-pyruvoyl tetrahydropterin (6-PPH4) by 6-pyruvoyl tetrahydropterin synthase (EC 4.6.1.10; PTPS, PPH4S). Finally, sepiapterin reductase (EC 1.1.1.153; SR) reduces this diketo compound in an NADPH-dependent step to BH4. Recently, the crystal structures of Escherichia coli CYH (Nar et al., 1995a,b) and rat liver PTPS (Nar et al., 1994; Bürgisser et al., 1995) were solved by isomorphous replacement techniques (Auerbach and Nar, 1997). The crystal structure of mouse sepiapterin reductase (mSR) completes the structural analysis of all three enzymes involved in the BH4 pathway (Figure 1). The biosynthetic pathway of BH4 includes minimally these three enzymes; the participation of a fourth enzyme, aldose reductase, is suggested but still controversial. However, it was shown that aldose reductase is not important for BH4 biosynthesis in liver (Milstien and Kaufman, 1989). Besides the de novo biosynthesis of BH4, SR is also known to be involved in the pterin salvage pathway catalysing the conversion of sepiapterin to dihydrobiopterin (BH2) which is transformed by dihydrofolate reductase to BH4 (Nichol et al., 1983). Furthermore, a regeneration system for the cofactor is known involving pterin-4a-carbinolamine dehydratase (PCD) (Endrizzi et al., 1995; Ficner et al., 1995) and dihydropteridine reductase (DHPR) (Varughese et al., 1992). Figure 1.The complete pathway of the de novo biosynthesis of BH4. The cofactor BH4 is synthesized by only three enzymes, namely CYH, PTPS and SR. The crystal structure of E.coli CYH was recently solved by single isomorphous replacement and averaging techniques. The enzyme complex, a decamer consisting of a pentamer of tightly associated dimers, has perfect D5 symmetry and is doughnut-shaped with dimensions of 65×100 Å. The MIR-solved crystal structure of rat PTPS shows a hexameric enzyme composed of a dimer of trimers with D3 symmetry. Each trimer forms a 12–stranded antiparallel β-barrel, enclosing a basic pore with 6–12 Å diameter. The crystal structure of mSR completes the structural analysis of all three enzymes involved in BH4 biosynthesis, providing the essential information for the interpretation of the complex biochemical regulation of this pathway. Download figure Download PowerPoint The full complement of the three BH4-biosynthesizing enzymes can be found in significant amounts in many tissues of various species (Smith and Nichol, 1984). The richest sources of SR are erythrocytes, liver and brain. Katoh and co-workers (1982) have obtained evidence for a regulation mechanism of the BH4 pathway by feedback inhibition of the activity of SR in rat brain by a catecholamine and an indoleamine. SRs from mouse, rat and human origin have been cloned and sequenced (Citron et al., 1990; Ichinose et al., 1991; Ota et al., 1995) revealing a sequence identity of the mouse SR with rat and human enzymes of 88% and 74% (94% and 88% sequence similarity) (Figure 2A). Figure 2.Structure of mSR. (A) Primary structure of mSR. The sequence alignment of mSR with the rat and human enzyme reveals a weighted sequence similarity of 94% and 88%. Residues involved in cofactor binding are marked with a blue, residues involved in subunit interactions with a black triangle. Central residues for substrate binding, catalysis or inhibition are red inverted. The sequence numbering and secondary structure assignment is according to mSR sequence. (B) Stereo view of the quaternary structure of mSR. The 56 kDa dimer shows two parallel β-strands in anti-parallel orientation enclosing a four-helix bundle forming the subunit interactions. The substrate sepiapterin (SPT) and the cofactor NADP/NADPH (NAP) bind from opposite sides to the enzyme. The secondary structure elements and ligands are labelled, the components of the second monomer are marked with an apostrophe. Download figure Download PowerPoint Here we describe a crystallographic structure analysis of mouse SR. All structures are complexes with the natural cofactor NADP, which was added during purification and crystallization. Surprisingly, recombinantly expressed protein contained oxaloacetate bound to the active site. The ternary complex structure of mouse SR with NADP and oxaloacetate was refined to a resolution of 1.25 Å. In order to investigate the catalytic mechanism of the enzyme, ternary complex structures either with the substrate, sepiapterin, or the product, tetrahydrobiopterin, have been analysed. Inhibitory complex structures were determined as quaternary complex with N-acetyl serotonin (NAS) and oxaloacetate (OAA), and as ternary complex structures with either NAS or noradrenaline. Results and discussion Overall structure of the SR SR is a homodimer with 261 amino acids per monomer, each forming a single domain α/β-structure. A seven-stranded parallel β-sheet in the centre of the molecule is sandwiched by two arrays of three α-helices (αC, αB, αG and αD, αE, αF). Six strands of them constitute a classical dinucleotide-binding motif (Rossmann et al., 1975) composed of βαβ units (Figure 2B). The association of two SR monomers to a dimer leads to the formation of a four-helix bundle by helices αE and αF of each monomer, stabilized by numerous hydrogen bonds and two charged interactions involving Asn116 and Asn141 from each monomer. Exceptionally, the two parallel β-sheets of the dimer are in an anti-parallel orientation enclosing an angle of 90°. The two substrate pockets are opened to the same side of the molecule, each formed by elongation of the three loops between strand βD and helix αE, between βE and αF, and between βF and αG. The latter insertion contains two additional helices αFG1 and αFG2 (Figure 2). From both amino acid sequence and three-dimensional structure, SR can be unequivocally assigned to the family of short-chain dehydrogenases/reductases (SDR). Although sequence comparison of these enzymes show sequence identities between 12% and 35% and their substrates are very different, they share a common tertiary structure. They all use either NAD(H) or NADP(H) as the cofactor and contain a strictly conserved Tyr-X-X-X-Lys sequence motif (Ghosh et al., 1994). Active site for reductase and isomerase activity The presence of the nicotinamide ring of the bound cofactor NADP already indicates the location of the active site of SR, which is situated at the C-terminal end of the β-strands. The acceptor site for the substrate contains a 15 Å-deep pocket that is ideally suited to receive pterin and small carbonyl substrates. The active site cavity is formed by the hydrophobic residues Leu105, Leu159, Tyr165, Trp168, Tyr171, Met206 and Cys160. The crystal structure of SR in ternary complex with NADP and the substrate, sepiapterin, clearly shows the pterin substrate positioned in the active site anchored with its guanidino moiety to Asp258. This positions the pterin moiety with its side chain C1′-carbonyl function in direct proximity to Tyr171 OH and NADP C4′N. The crystal structures clearly show Tyr171 as the central active site residue in an orientation for optimal proton transfer to the carbonyl functional groups of the substrate. Furthermore, the nicotinamide group is co-planar with the side-chain carbon atoms for optimal hydride transfer to the carbonyl group (Figure 3). The electron density at the C2′-position in the ternary complex of SR with sepiapterin and NADP appears planar, thus indicating that sepiapterin is oxidized to the diketo compound in the soaked crystals (Figure 4A). The crystal structure of the ternary complex of the enzyme with NADPH and BH4, the product of the catalysed reaction, confirms this orientation of the side chain also for the bound product (Figure 4B). Figure 3.Active site of mSR in complex with substrate sepiapterin (SPT) and cofactor NADP (NAP). Anchoring of sepiapterin with its purine moiety to Asp258 positions its C1′ carbonyl function in direct proximity to NADP and Tyr171, clearly indicating the latter as the central catalytic site for hydrogen transfer to the carbonyl function of the substrate. Download figure Download PowerPoint Figure 4.Electron densities for the substrate sepiapterin (SPT), the product tetrahydrobiopterin (BH4), the cofactor NADP, and the inhibitors N-acetyl serotonin (NAS) and oxaloacetate (OAA). (A) Stereo diagram showing the substrate sepiapterin in the enzyme-bound conformation placed in its 3σ contoured Fo−Fc density refined to 1.95 Å resolution and (B) the product tetrahydrobiopterin refined to 2.6 Å resolution. (C) Stereo view of the cofactor NADP placed in its 1 σ contoured 2Fo−Fc density refined to 1.25 Å resolution. (D) Stereo view of the quaternary complex structure of SR with the two inhibitors OAA and NAS. NAS, anchored by Asp258, is a specific competitive inhibitor only for pterin substrates, while OAA bound to Tyr171 is probably an inhibitor for pterin- and non-pterin carbonyl compounds. Download figure Download PowerPoint Three residues can be regarded as a central feature in the active site of SR for both reductase and isomerase activity. The two basic residues Lys175 and Arg178 may facilitate the proton transfer from the hydroxyl function of Tyr171 to the substrate's carbonyl oxygen by stabilizing the resulting tyrosinate. The guanidinium group of Arg178 is in hydrogen bond distance to the side-chain amide groups of the three Asn residues, Asn100, Asn128 and Asn155, which are conserved at least within SR, carbonyl reductase, and Drosophila alcohol dehydrogenase. From the crystal structure, a central role can also be proposed for residue Ser158, which is located in hydrogen bond distance to the substrate N5 atom and the C1′-side chain oxygen. Ser158 is presumably involved in stabilizing the position of the substrate, according to results from 7α-hydroxy steroid dehydrogenase (HSDH), mouse lung carbonyl reductase (MLCR) (Tanaka et al., 1996a,b), and 3β/17β-HSDH (Oppermann et al., 1997). Ser158 is close to the residues Cys160 and Cys172, providing possible additional proton sources. The conserved cysteine residues are in the reduced state in all analysed crystals, independent of the presence of dithiothreitol. Conformational changes by substrate-binding as seen in a so-called substrate-binding loop in the structure of 7α-HSDH (Tanaka et al., 1996b), which aligns with the mSR region Asn205 to Asp229 (helices αFG1 and αFG2), do not occur in mSR. This part seems rather rigid with temperature factors below 20 Å2 in mSR. As confirmed by several complex structures, the active site is highly accessible in the crystals of SR by opening the substrate-binding pockets towards honeycomb-like cavities. Major structural changes induced on binding of active site ligands are not observed. Coenzyme-binding mode The MIR-phased electron density already clearly identified the functional cofactor binding site of the enzyme. The adenine moiety of NADP is bound sandwiched between the side chains of Arg43, Leu71 and Leu127, and is anchored via hydrogen bonds to Asp70 and Leu71. On the basis of the first crystal structure at very high resolution of an NAD(P) co-factor in the enzyme-bound state, we are able to determine not only the conformation of the co-factor with high precision, but also the stereochemistry for the optimal hydride transfer reaction. The bound NADP molecule is in an extended conformation with the adenine ring in anti and the nicotinamide ring in syn conformation (Figure 4C), quite similar to the structures of HSDH and DHPR. The distance between C6 of the adenine and C2 of the nicotinamide is 14.2 Å for mSR, which is close to the values of 14.4 Å for 7α-HSDH, 14.6 Å for 3α,20β-HSDH and 15.1 Å for DHPR (Tanaka et al., 1996b). The syn conformation for the nicotinamide ring allows a B-face hydride transfer reaction. Both ribose rings have a C2-endo puckering. The distances between Tyr171 OH and the carbonyl oxygen atom and between the C-4 of the nicotinamide and the carbonyl carbon atom are 2.7 Å and 3.2 Å, respectively. The stacking interaction of Arg43 with the adenine ring stabilizes the position of this residue similar to glutathione reductase reported by Pai et al. (1988). Arg43 is conserved in all known members of the SDR family which prefer NADP(H) (Tanaka et al., 1996a) instead of NAD(H). Catalytic mechanism SR catalyses the NADPH-dependent reduction of various monocarbonyl compounds including ketones and aldehydes. SR is capable of catalysing the NADPH-dependent reduction of both side-chain keto groups of 6-pyruvoyl tetrahydropterin (PH4) to produce BH4 with proper stereochemistry (Katoh and Sueoka, 1984; Smith, 1987). Asp258 seems essential in determining the substrate binding specificity and anchors pterin derivatives for the reduction at their monoketo or diketo side chains. Mouse SR represents the first member of the SDR family in complex with a substrate and a product. Thus, the complex structures of SR set the stage for the detailed scenario of the catalytic reaction. The catalytic mechanism of SR appears to consist of a stereospecific transfer of a hydride ion from the exposed C-4 of the nicotinamide to the carbon of the carbonyl group of the substrate and abstraction of a proton from Tyr171 by the incipient negatively charged carbonyl oxygen. Lys175 stabilizes the position and orientation of the nicotinamide nucleoside moiety of the bound cofactor NADP through a bifurcated hydrogen bond to both the 2′-hydroxyl and 3′-hydroxyl group of the ribose group comparable with carbonyl reductase reported by Tanaka et al. (1996a). After side chain motion, Lys175 is able to stabilize the position of the hydroxyl group of the tyrosine base catalyst by concomitantly lowering its pKa. A common mechanism within the SDR family was suggested to involve a tyrosine and a lysine residue, both conserved within short-chain dehydrogenases/reductases (Jörnvall et al., 1981; Ghosh et al., 1994). The first isolated intermediate in the reduction of PH4 by SR is 1′-hydroxy-2′-oxo tetrahydropterin (Smith, 1987); this is also the compound produced in the reverse reaction from BH4 (Curtius et al., 1985). These data indicate that the enzyme preferentially reduces the 1′ keto group, consistent with the high reactivity of the enzyme toward sepiapterin and lactoyl tetrahydropterin as compared with 1′-hydroxy-2′-oxo tetrahydropterin. There are two possible pathways for complete reduction of the diketo substrate. First, after reduction of the C1′ carbonyl function and side-chain reorientation towards Tyr171 OH and NADPH C4′N, caused by the change in hybridization of the C1′ carbon atom, the second reduction step could take place at the C2′ carbonyl function. The second, stereochemically more attractive, alternative is that the reduction only occurs at the C1′ carbon atom and the second carbonyl function is reduced after isomerization at the C1′ and C2′ via an enediol intermediate which is stereospecifically stabilized by the Tyr171 anion. Evidence for an enediol form of sepiapterin was obtained by NMR studies (Iwanami and Akino, 1975). This isomerization is also catalysed in the absence of NADPH in the conversions of 6-lactoyl H4 pterin and 6-lactoyl 7,8-BH2 (sepiapterin) into the 6-1′-hydroxy-2′-oxopropyl pterins (Katoh and Sueoka, 1987). The tetrahydro isomers were observed as the monoketo intermediates in the de novo BH4 biosynthesis (Smith, 1987). There are other examples of isomerization of a keto-hydroxy group before reduction to the diol: reduction of camphoroquinone by 3α-HSDH (Boutin, 1986) and in the reduction of cortisol to cortol in corticosteroid metabolism (Monder and Bradlow, 1980). Thus, the initial step of the conversion of PH4 to BH4 is the NADPH-dependent reduction at the side-chain C1′-keto function conducive to the formation of 1′-OH-2′-oxopropyl PH4 (Figure 5; reaction 1). Internal rearrangement of the keto group via side chain isomerization forms the 1′-keto compound LPH4 (6-lactoyl tetrahydropterin) (Figure 5; reaction 2). Finally, this intermediate is reduced to BH4 in an NADPH-dependent step (reaction 3). Because of an unfavourable stereochemistry at the C1′-carbonyl atom of PH4, the alternative reduction of PH4 first at the 2′-oxo function is presumably not catalysed by SR (reaction 5). However, reduction at the 1′-position (reaction 1) could reorientate the side-chain C2′-carbonyl towards Tyr171 and NADPH, enabling at least a less favoured reduction at the 2′-oxo function toward BH4 (reaction 4). Figure 5.Catalysed reaction of SR: reductase and isomerase. SR catalyses the sequential diketo reduction of PH4 (6-pyruvoyl tetrahydropterin) conducive to the formation of BH4 via an essential isomerization step of the mono-keto intermediates (reactions 1–3). For C2′-reduction of the 2′–mono keto intermediate (reaction 4), an essential reorientation of the substrate's side chain toward Tyr171 and NADPH can be proposed. The reduction of the 2′-oxo function of PH4 (reaction 5) is presumably not catalysed by SR. Download figure Download PowerPoint The binding pockets for substrate and cofactor are opening to opposite sides of the molecule. Thus, the oxidized cofactor may be exchanged after the first reduction step for a second equivalent of NADPH while the substrate remains bound. The kinetic mechanism of the SR reaction is then proposed as an ordered bi-bi mechanism (Sueoka and Katoh, 1982). Inhibition of SR Competitive inhibition of SR seems feasible for pterin-and non-pterin substrates. Anchoring of an analogue to Asp258 inhibits the reduction and isomerization of pterin derivatives, whereby the catalysis of small non-pterin substrates remains possible. However, binding of an inhibitor, like oxaloacetate, directly to the catalytic centres of the active site, Tyr171 and NADP C4′N, disables the enzyme for both pterin- and non-pterin substrates (Figure 4D). Katoh et al. (1982) reported that N-acetyl serotonin (NAS) is a potent inhibitor of partially purified rat brain and homogeneous rat erythrocyte SR (Ki = 0.2 and 0.17 μM, respectively). These data suggested that NAS binds at the substrate site and Smith et al. (1992) proposed the N-acetyl group and indole hydroxyl function of NAS critical for binding. The binding mode of NAS is shown in Figure 6, derived from a 2.1 Å refined complex structure of mSR with NAS. The high specificity of SR for this inhibitor is achieved by anchoring the indoleamine with its hydroxyl function to Asp258 like the substrate and with its N-acetyl function to Tyr165 via a π-electron interaction. The presumed role of the aromatic ring is also indicated by a reorientation of the Tyr side chain after NAS binding. A sequence comparison shows a corresponding Phe residue in the enzymes from rat and human origin. Furthermore, anchoring via the substrate N-acetyl function explains the strongly reduced Ki value with regard to serotonin. The inhibitor NAS is bound with its indole moiety to the hydrophobic bottom of the pterin binding site formed by Leu159, Leu226 and Tyr260, and with its acetyl group into a hydrophobic side pocket of the active site formed by the side chains of the residues Tyr165, Trp168, Leu219 and Leu223. Thereby, the phenyl ring of NAS binds in the pyrimidine pocket of the enzyme with its 5-hydroxyl aligned at the pyrimidine 3-position. The same binding mode can be proposed for melatonin, however, with a sterically less favourable methoxy group replacing the hydroxyl position of NAS. Figure 6.Different inhibition modes for pterin- and non-pterin carbonyl substrates. The quaternary complex with NADP (nicotinamide moiety; green) shows the potent inhibitor and natural neurotransmitter N-acetyl serotonin NAS (red) bound with its indole moiety to Asp258. Compared with the complex structures with substrate sepiapterin (SPT) or product tetrahydrobiopterin (BH4) (sepiapterin shown in black, hydrogen bond distances for sepiapterin shown in bold), the hydroxyl function of NAS is imitating the N3-nitrogen of the natural substrate. The inhibitor oxaloacetate OAA (blue) is imitating with its α-keto terminal carboxylate group the C1′-carbonyl function of the substrate's side chain. Hydrogen bonds are indicated by broken lines and the corresponding distances (Å) are shown according to the colour coding of the ligands. Download figure Download PowerPoint In summary, SR evaluates two very specific binding motifs for competitive inhibition of the enzyme (versus sepiapterin): the Asp258 anchor, embedded in a hydrophobic pterin binding site, and Tyr165, forming a nitrogen anchor within a hydrophobic side pocket of the active site close to Asp258 and Tyr171. In vivo, only NAS seems to be optimally able to occupy both of these two motifs by forming a strong inhibitory complex with SR (Ki = 0.12 μM). Neither serotonin (Ki = 2.3 mM), an intermediate in the indoleamine pathway just before NAS and lacking the N-acetyl group, nor melatonin (Ki = 30 μM), directly after NAS with an additional methoxy group at the aromatic hydroxyl position of NAS, can achieve this tight binding mode. Because the acetyl group of NAS is not completely filling the hydrophobic pocket around Tyr165, elongation of the inhibitor's side chain should increase the binding ability. This is realized in the case of synthetic inhibitors, like N-chloro- or N-methoxy-acetyl serotonin, which are one of the tightest inhibitors known so far (Ki value = 0.006 μM, respectively 0.008 μM). Based on the crystal structure of the ternary complex with NAS and OAA, it should be possible to design synthetic inhibitors, which bind to three specific sites within the active site of SR, namely Asp258, the hydrophobic pocket near Tyr165, and Tyr171. Oxaloacetate is already bound to native crystals of the recombinant protein expressed in E.coli and was crystallographically identified. Its α-keto terminal carboxylate group bound to Tyr171 imitates the C1′-carbonyl function of the substrate side chain. The inhibitory role of oxaloacetate requires further investigations by kinetic studies. Oxaloacetate can be replaced by soaking the protein with sepiapterin. It is remarkable that malate dehydrogenase (MDH), which catalyses the regeneration of oxaloacetate in the final reaction step of the citric acid cycle, shows significant overall structural homology with SR. In order to confirm the binding mode of NAS to the active site also without OAA, the latter was removed from the protein using sepiapterin and charcoal before crys
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