title: A pivotal role for tryptophan 447 in enzymatic coupling of human endothelial nitric oxide synthase (eNOS): effects on tetrahydrobiopterin-dependent catalysis and eNOS dimerization.
abstract: Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by NOS. Bioavailability of BH4 is a critical factor in regulating the balance between NO and superoxide production by endothelial NOS (eNOS coupling). Crystal structures of the mouse inducible NOS oxygenase domain reveal a homologous BH4-binding site located in the dimer interface and a conserved tryptophan residue that engages in hydrogen bonding or aromatic stacking interactions with the BH4 ring. The role of this residue in eNOS coupling remains unexplored. We overexpressed human eNOS W447A and W447F mutants in novel cell lines with tetracycline-regulated expression of human GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, to determine the importance of BH4 and Trp-447 in eNOS uncoupling. NO production was abolished in eNOS-W447A cells and diminished in cells expressing W447F, despite high BH4 levels. eNOS-derived superoxide production was significantly elevated in W447A and W447F versus wild-type eNOS, and this was sufficient to oxidize BH4 to 7,8-dihydrobiopterin. In uncoupled, BH4-deficient cells, the deleterious effects of W447A mutation were greatly exacerbated, resulting in further attenuation of NO and greatly increased superoxide production. eNOS dimerization was attenuated in W447A eNOS cells and further reduced in BH4-deficient cells, as demonstrated using a novel split Renilla luciferase biosensor. Reduction of cellular BH4 levels resulted in a switch from an eNOS dimer to an eNOS monomer. These data reveal a key role for Trp-447 in determining NO versus superoxide production by eNOS, by effects on BH4-dependent catalysis, and by modulating eNOS dimer formation.
authors: From the British Heart Foundation Centre of Research Excellence, Division of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom.
authors: Matthew A
authors: MA
authors: Benson
authors: Helen
authors: H
authors: Batchelor
authors: Surawee
authors: S
authors: Chuaiphichai
authors: Jade
authors: J
authors: Bailey
authors: Hanneng
authors: H
authors: Zhu
authors: Dennis J
authors: DJ
authors: Stuehr
authors: Shoumo
authors: S
authors: Bhattacharya
authors: Keith M
authors: KM
authors: Channon
authors: Mark J
authors: MJ
authors: Crabtree
mesh_terms: D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D020134:Catalytic Domain; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D013481:Superoxides / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
chemical_list: D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; D014364:Tryptophan; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin
keywords: Endothelial Dysfunction; Nitric Oxide Synthase; Superoxide; Superoxide Ion; Tetrahydrobiopterin; Tryptophan; Uncoupling
references: Am J Physiol Heart Circ Physiol. 2008 Apr;294(4):H1530-40
references: 18192221
references: Adv Exp Med Biol. 1996;387:163-9
references: 8794208
references: J Biol Chem. 1992 Oct 15;267(29):20547-50
references: 1383204
references: Pharmacogenetics. 2004 Dec;14(12):831-9
references: 15608562
references: J Clin Invest. 2002 Mar;109(6):817-26
references: 11901190
references: Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11771-5
references: 8524846
references: Circ Res. 2000 Feb 4;86(2):E36-41
references: 10666424
references: J Clin Invest. 1997 Jan 1;99(1):41-6
references: 9011574
references: J Cardiovasc Pharmacol. 2000 Feb;35(2):173-8
references: 10672847
references: Am J Physiol Heart Circ Physiol. 2001 Sep;281(3):H981-6
references: 11514262
references: Biochemistry. 2001 Oct 30;40(43):12826-32
references: 11669619
references: Arterioscler Thromb Vasc Biol. 2004 Mar;24(3):445-50
references: 14707037
references: Kidney Int. 1999 Jan;55(1):252-60
references: 9893134
references: FEBS Lett. 2000 Sep 22;481(3):305-6
references: 11041680
references: Circulation. 2003 Sep 9;108(10):1238-45
references: 12925450
references: Circulation. 1996 Apr 1;93(7):1346-53
references: 8641023
references: J Biol Chem. 2009 Jan 9;284(2):1136-44
references: 19011239
references: J Biol Chem. 2000 Aug 25;275(34):26150-7
references: 10846172
references: Biochemistry. 2001 Oct 30;40(43):12819-25
references: 11669618
references: Diabetologia. 2000 Nov;43(11):1435-8
references: 11126415
references: J Clin Invest. 2003 Sep;112(5):725-35
references: 12952921
references: J Biol Chem. 2001 May 4;276(18):14533-6
references: 11279231
references: Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5727-32
references: 15824309
references: J Chromatogr. 1989 Sep 29;494:77-85
references: 2584347
references: FASEB J. 1996 Apr;10(5):552-8
references: 8621055
references: Diabetologia. 2005 Sep;48(9):1933-40
references: 16034613
references: Am J Physiol Cell Physiol. 2004 Oct;287(4):C895-902
references: 15306539
references: J Leukoc Biol. 1994 Nov;56(5):576-82
references: 7525816
references: Lancet. 1995 Aug 19;346(8973):467-71
references: 7637480
references: Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9220-5
references: 9689061
references: Arterioscler Thromb Vasc Biol. 2004 Mar;24(3):413-20
references: 14656731
references: Am J Hypertens. 2002 Apr;15(4 Pt 1):326-32
references: 11991218
references: Eur J Pharmacol. 2011 Jan 10;650(1):371-7
references: 20950600
references: Eur Heart J. 2012 Apr;33(7):829-37, 837a-837d
references: 21890489
references: Biochem Biophys Res Commun. 1995 Oct 24;215(3):1119-29
references: 7488039
references: J Biol Chem. 1992 Jun 5;267(16):10976-81
references: 1375933
references: Circulation. 2002 Apr 9;105(14):1656-62
references: 11940543
references: Trends Cardiovasc Med. 2004 Nov;14(8):323-7
references: 15596110
references: Circulation. 2012 Mar 20;125(11):1356-66
references: 22315282
references: Circulation. 1997 Jul 1;96(1):25-8
references: 9236411
references: J Clin Invest. 2002 Aug;110(3):331-40
references: 12163452
references: J Clin Invest. 2003 Apr;111(8):1201-9
references: 12697739
references: Nitric Oxide. 2007 Feb;16(1):1-9
references: 16647284
references: EMBO J. 1995 Aug 1;14(15):3687-95
references: 7543842
references: Cardiovasc Res. 2002 Sep;55(4):838-49
references: 12176133
references: Heart. 2002 Mar;87(3):264-9
references: 11847169