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Structural chemistry of assembly and...

Aoyagi, Mika.

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Structural chemistry of assembly and activation for nitric oxide synthase isozymes.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Structural chemistry of assembly and activation for nitric oxide synthase isozymes./
作者:	Aoyagi, Mika.
面頁冊數:	169 p.
附註:	Source: Dissertation Abstracts International, Volume: 65-01, Section: B, page: 0199.
Contained By:	Dissertation Abstracts International65-01B.
標題:	Chemistry, Biochemistry. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3120528

Structural chemistry of assembly and activation for nitric oxide synthase isozymes.
Aoyagi, Mika.

Structural chemistry of assembly and activation for nitric oxide synthase isozymes. - 169 p.

Source: Dissertation Abstracts International, Volume: 65-01, Section: B, page: 0199.

Thesis (Ph.D.)--The Scripps Research Institute, 2004.

Nitric oxide synthases (NOSs) catalyze the biosynthesis of nitric oxide (NO), a signaling molecule involved in diverse physiological and pathophysiological processes. Three homologous isozymes, namely endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS), share a common modular architecture: the N-terminal catalytic oxygenase module (NOSox), a central calmodulin (CaM) recognition site and the C-terminal electron-supplying reductase module (NOSred). Electron transfer in NOS catalysis is triggered by CaM binding and occurs across the subunits in a homodimer. Dimerization and CaM binding serve as molecular switches for regulating activities. Furthermore, the NOS isozymes differ in Ca2+-dependence for CaM binding. To elucidate the molecular basis for (6R)-5,6,7,8-tetrahydrobiopterin (H 4B-dependent dimerization/electron transfer and Ca2+-(in)dependent CaM binding, the following crystal structures were determined: (1) iNOS ox H4B-site mutants (W457F and W457A), (2) Ca 2+/CaM bound to an eNOS peptide, and (3) Ca2+/CaM bound to mutant eNOS peptides. In addition, the CaM-binding affinities of several iNOS (wild-type) and eNOS (wild-type and mutant) peptides were measured by surface plasmon resonance and equilibrium fluorescence. The W457F and W457A iNOSox structures showed that decreased pterin pi-stacking at the mutation sites contributes to higher mobility at the dimer interface and slower electron transfer to heme, compared to the wild-type. Conserved Trp457 thus stabilizes H4B-bound dimers and pterin radical formation. The Ca2+/CaM/eNOS peptide structure indicated that the alpha-helical peptide binds CaM via extensive hydrophobic interactions. Unique interactions of eNOS with the CaM central linker and C-terminus support their specific roles in NOS activation. Based on the eNOS CaM-binding mode, testable models are proposed for eNOS deactivation by Thr495 phosphorylation and for iNOS Ca2+ independence. Comparative analyses of the CaM-binding properties of iNOS and mutant eNOS peptides indicated that the unique CaM-binding region contributes to the seemingly irreversible, Ca2+-independent CaM binding of iNOS. The iNOS CaM-binding region is differentiated from the relatively homologous eNOS region by its dependency on increased hydrophobicity, and additional residues C-terminal to the canonical target site. Collectively, results from this work provide a structural basis for H4B- and CaM-dependent assembly and activation of NOS isozymes. Tightly controlled NOS activity is vital for specific NO signaling, while limiting cytotoxicity.Subjects--Topical Terms:

1017722
Chemistry, Biochemistry.

Structural chemistry of assembly and activation for nitric oxide synthase isozymes.
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502 $a Thesis (Ph.D.)--The Scripps Research Institute, 2004.
520 $a Nitric oxide synthases (NOSs) catalyze the biosynthesis of nitric oxide (NO), a signaling molecule involved in diverse physiological and pathophysiological processes. Three homologous isozymes, namely endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS), share a common modular architecture: the N-terminal catalytic oxygenase module (NOSox), a central calmodulin (CaM) recognition site and the C-terminal electron-supplying reductase module (NOSred). Electron transfer in NOS catalysis is triggered by CaM binding and occurs across the subunits in a homodimer. Dimerization and CaM binding serve as molecular switches for regulating activities. Furthermore, the NOS isozymes differ in Ca2+-dependence for CaM binding. To elucidate the molecular basis for (6R)-5,6,7,8-tetrahydrobiopterin (H 4B-dependent dimerization/electron transfer and Ca2+-(in)dependent CaM binding, the following crystal structures were determined: (1) iNOS ox H4B-site mutants (W457F and W457A), (2) Ca 2+/CaM bound to an eNOS peptide, and (3) Ca2+/CaM bound to mutant eNOS peptides. In addition, the CaM-binding affinities of several iNOS (wild-type) and eNOS (wild-type and mutant) peptides were measured by surface plasmon resonance and equilibrium fluorescence. The W457F and W457A iNOSox structures showed that decreased pterin pi-stacking at the mutation sites contributes to higher mobility at the dimer interface and slower electron transfer to heme, compared to the wild-type. Conserved Trp457 thus stabilizes H4B-bound dimers and pterin radical formation. The Ca2+/CaM/eNOS peptide structure indicated that the alpha-helical peptide binds CaM via extensive hydrophobic interactions. Unique interactions of eNOS with the CaM central linker and C-terminus support their specific roles in NOS activation. Based on the eNOS CaM-binding mode, testable models are proposed for eNOS deactivation by Thr495 phosphorylation and for iNOS Ca2+ independence. Comparative analyses of the CaM-binding properties of iNOS and mutant eNOS peptides indicated that the unique CaM-binding region contributes to the seemingly irreversible, Ca2+-independent CaM binding of iNOS. The iNOS CaM-binding region is differentiated from the relatively homologous eNOS region by its dependency on increased hydrophobicity, and additional residues C-terminal to the canonical target site. Collectively, results from this work provide a structural basis for H4B- and CaM-dependent assembly and activation of NOS isozymes. Tightly controlled NOS activity is vital for specific NO signaling, while limiting cytotoxicity.
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