Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans.
We cloned, expressed, and characterized a hemeprotein from Deinococcus radiodurans (D. radiodurans NO synthase, deiNOS) whose sequence is 34% identical to the oxygenase domain of mammalian NO synthases (NOSoxys). deiNOS was dimeric, bound substrate Arg and cofactor tetrahydrobiopterin, and had a normal heme environment, despite its missing N-terminal structures that in NOSoxy bind Zn(2+) and tetrahydrobiopterin and help form an active dimer. The deiNOS heme accepted electrons from a mammalian NOS reductase and generated NO at rates that met or exceeded NOSoxy. Activity required bound tetrahydrobiopterin or tetrahydrofolate and was linked to formation and disappearance of a typical heme-dioxy catalytic intermediate. Thus, bacterial NOS-like proteins are surprisingly similar to mammalian NOSs and broaden our perspective of NO biochemistry and function.
Adak
Subrata
S
Department of Immunology, Cleveland Clinic, Cleveland, OH 44195, USA. adaks@ccf.org
Bilwes
Alexandrine M
AM
Panda
Koustubh
K
Hosfield
David
D
Aulak
Kulwant S
KS
McDonald
John F
JF
Tainer
John A
JA
Getzoff
Elizabeth D
ED
Crane
Brian R
BR
Stuehr
Dennis J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D002384:Catalysis; D002956:Citrulline / Q000737:chemistry; D003001:Cloning, Molecular; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D006418:Heme / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000201:enzymology*; D013997:Time Factors; D015032:Zinc / Q000378:metabolism
D008024:Ligands; D001708:Biopterins; D002956:Citrulline; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D001120:Arginine; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; C003402:sapropterin; D015032:Zinc; D004229:Dithiothreitol