Importance of valine 567 in substrate recognition and oxidation by neuronal nitric oxide synthase
Introduction
Nitric oxide (NO) is a recently discovered messenger molecule in mammals that plays key roles in a variety of physiological processes such as neurotransmission, vasorelaxation, platelet aggregation, and immune responses [1], [2], [3]. NO is synthesized by heme proteins called nitric oxide synthases (NOSs) that catalyse the two-step oxidation of l-Arginine (l-Arg) to citrulline and NO with intermediate formation of Nω-hydroxy-l-Arg (NOHA). NOSs are active as homodimeric enzymes consisting of an NH2-terminal oxygenase domain that contains binding sites for the heme prosthetic group, substrate l-Arg and cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4). The CO2H-terminal reductase domain contains binding sites for flavins FMN and FAD as well as the substrate NADPH. These two domains are fused by a Ca++-dependent calmodulin (CaM)-binding sequence. The CaM binding triggers the electron flow necessary for catalysis from the reductase domain to the heme domain [4], [5], [6]. The reductase domain has a close homology to NADPH-cytochrome P450 reductase [7] and is able to catalyse the reduction of exogenous electron acceptors such as ferric cytochrome c (cyt c). Under- as well as over-productions of NO are related to various pathological processes and immunological diseases [1], [8], [9]. Therefore, NOSs constitute interesting targets for drug designs and many studies are undertaken to get a better understanding of the mechanism of NO-synthesis catalyzed by NOSs.
Crystal structures of the oxygenase domains of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) have been solved recently and show strong conservation among isoforms [10], [11], [12], [13], [14]. Based on these structures, key amino acid residues involved in the l-Arg and NOHA recognition at the heme-active site have been identified. In rat nNOS, Glu 592 (Glu 363 in bovine eNOS, Glu 371 in murine iNOS) establishes crucial hydrogen-bonds with the guanidino- or hydroxyguanidino-group of l-Arg and NOHA, thus governing their binding close to the heme (Fig. 1). Mutation of this Glu residue to a non-polar residue totally abolished the binding of these substrates and the formation of NO [15], [16], [17], [18]. Hydrogen-bonds between the α-carboxylate group of l-Arg and the hydroxyl group of Tyr588 in rat nNOS are also of importance for recognition of α-amino acids at the active site, and mutation of Tyr588 strongly alters the substrate specificity [19]. Recently, NOSs have been shown to catalyse NO-formation by oxidation of some non-amino acid alkylguanidines and N-alkyl- and N-aryl-N′-hydroxyguanidines following reactions similar to the oxidations of natural substrates, l-Arg and NOHA [20], [21], [22]. The latest crystal structures reveal a new binding mode for two alternative substrates that accounts for their ability to be oxidised in nNOS active site [13]. The critical hydrogen-bonds with Glu592 are conserved. Therefore, compared to l-Arg, the alkyl chains have moved towards a hydrophobic pocket consisting of residues Phe584, Pro565 and Val567 (Fig. 1) [13]. Indeed, the distances between one CH3 group of Val567 and the α-CH of l-Arg or the terminal CH3 group of N-(n-butyl)-N′-hydroxyguanidine are 5.11 and 3.75 Å, respectively, suggesting stronger hydrophobic interaction of Val567 with N-(n-butyl)-N′-hydroxyguanidine than with l-Arg. Hydrophobic interactions with the corresponding Val338 residue of bovine eNOS have also been observed with an N-aryl-N′-hydroxyguanidine [23] and inhibitors such as alkyl- and aryl-iso thioureas [12], [24], [25].
The Val residue is highly conserved among the different NOS isoforms (Fig. 2). To investigate its importance in substrate recognition and NO-forming activity, we constructed four Val567 mutants of nNOS. Here we report the spectral characterisation of these proteins and compare their ability to bind and to metabolize l-Arg, NOHA, and N-hydroxyguanidines 1–3 (Fig. 3). Our results demonstrate, for the first time, the key role of Val567 in substrate recognition and oxidation to NO.
Section snippets
Materials
BH4 was purchased from Schircks Laboratories (Jona, Switzerland). Calmodulin, 2′,5′-ADP-Sepharose, CaM-Sepharose and Sephadex G25 were products of Amersham-Pharmacia Biotech Inc. l-Arg, NADPH, superoxide dismutase (SOD), catalase, hemoglobin, and other reagents were obtained from Sigma or Wako Pure Chemicals, and were of the highest purity commercially available. [Guanido-14C]l-Arginine (2.2 GBq/mmol) was purchased from NEN Life Science Products.
The synthesis and physico-chemical
Results
To investigate the active site of nNOS and to identify the key residues involved in the binding and transformation of substrates, we mutated Val567 of nNOS for hydrophobic (Leu, Phe) or polar (Glu, Arg) residues. Val to Leu mutation should give information on the importance of the size of this side-chain in the active site. Val to Phe mutation would introduce different steric hindrances but also possible π–π interactions with N-aryl-N′-hydroxyguanidines. Lastly, the effects of the introduction
Discussion
Comparisons of the eNOS, iNOS and nNOS X-ray crystallographic structures have revealed a striking degree of active-site conservation among NOS isoforms [10], [11], [12], [13], [14]. This high structural resemblance in the oxygenase domains of NOSs hinders the design of highly selective inhibitors or efficient alternative substrates. A better understanding of the role(s) of amino acid residues present at the active site of NOS isoforms is thus of importance for the discovery of more potent and
Abbreviations
- l-Arg
l-arginine
- BH4
(6R)-5,6,7,8-tetrahydro-l-biopterin
- CaM
calmodulin
- cyt c
cytochrome c
- DTT
dithiothreitol
- Hepes
N-(2-hydroxyethyl)piperazine-N′-2-ethane sulfonic acid
- HS
high spin
- ImH
imidazole
- NOS
nitric oxide synthase
- nNOS
iNOS and eNOS, neuronal, inducible and endothelial NOS, respectively
- NOHA
Nω-hydroxy-l-Arg
- iPr-NOHG
N-(iso-propyl)-N′-hydroxyguanidine
- LS
low spin
- nBu-NOHG
N-(n-butyl)-N′-hydroxyguanidine
- NO2Arg
Nω-nitro-l-Arg
- OH-Ph-NOHG
N-(4-hydroxyphenyl)-N′-hydroxyguanidine
- RP-HPLC
reverse phase high performance
Acknowledgements
This study was partly supported by Human Frontier Science Program and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan. M.M. was supported by a fellowship from the French Ministry of Education, Research and Technology. The authors thank M. Jaouen for her assistance in preparing NOSs proteins and electrophoresis, L. Casse for her help in experiments and M. Hayes for his critical reading of the manuscript.
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