Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin.
A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH4) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds N omega-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH4. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH4. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH4 binding site by analogy to its homology with DHFR, but that tight binding of BH4 requires amino acids 220-577.
Nishimura
J S
JS
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Martasek
P
P
McMillan
K
K
Salerno
J
J
Liu
Q
Q
Gross
S S
SS
Masters
B S
BS
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D018076:DNA, Complementary; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D011622:Pterins / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D018076:DNA, Complementary; D011622:Pterins; D011993:Recombinant Fusion Proteins; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D013762:Tetrahydrofolate Dehydrogenase; D005982:Glutathione Transferase