Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans

Abstract

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.G enes coding for NO synthases (NOSs, EC 4.14.23) are present throughout the plant and animal kingdom. NOS activities are present in plants and lower eukaryotes (1-5). Their primary structures and activities are strikingly similar to the mammalian NOSs, suggesting that NO has been important throughout evolution. All eukaryotic NOSs catalyze the NADPH-and O 2 -dependent oxidation of L-arginine (Arg) to citrulline and NO, with N-hydroxy-L-arginine (NOHA) formed as an enzyme-bound intermediate (6). Structurally, all animal NOSs are bi-domain proteins containing an N-terminal oxygenase domain (NOSoxy) that binds protoporphyrin IX (heme), 6R-tetrahydrobiopterin (H 4 B), and Arg and is linked to a C-terminal f lavoprotein domain (NOS reductase domain, NOSred) by a central calmodulin (CaM) binding sequence (7,8). NOSred bears strong sequence and functional similarity to NADPH-cytochrome P450 reductase and related electron transfer flavoproteins (9, 10), and function to transfer NADPHderived electrons to the ferric heme for O 2 activation during NO synthesis. In contrast, NOSoxy and cytochromes P450 have completely different primary, secondary, and tertiary structures, even though both enzyme families use a thiolate-ligated heme for O 2 activation (11, 12). Moreover, unlike P450s, NOSoxy must dimerize to become active (13,14). Dimerization produces functional binding sites for Arg and H 4 B and sequesters the heme catalytic center from solvent. These distinguishing features imply that NOSs evolved separately from other heme-thiolate enzymes and can so provide unique perspectives on their structure-function relationships.Although oxidative (nitrification) or reductive (denitrification) pathways for prokaryote NO biosynthesis are well established (15), there have been few reports on NOS-like proteins in bacteria (16,17). To date no prokaryotic NOS proteins have been completely sequenced, purified, or cloned. However, some have been shown to have nitrite-forming activity that depended on Arg, NADPH, and H 4 B. More recent genome sequencing of Bacillus subtilis, Deinococcus radiodurans, and other bacteria (18)(19)(20) confirm that a subset contain ORFs that code for proteins homo...

Endogenous nitric oxide regulates the recovery of the radiation-resistant bacterium Deinococcus radiodurans from exposure to UV light

“…However, unlike bacilli, Dr does not appear to contain the biosynthetic enzymes necessary to produce the mammalian NOS cofactor H 4 B (10, 38). In vitro, DrNOS (as with other bacterial NOSs) can use the alternative reduced pterin tetrahydrofolate (THF), a ubiquitous cofactor that can be generated by Dr (10,29). Consistent with the binding of an alternative cofactor by bacterial NOSs, structural studies indicate that there is substantial variation in the region of the bacterial enzymes that recognize the pterin side chain (10).…”

“…In vitro, DrNOS (as with other bacterial NOSs) can use the alternative reduced pterin tetrahydrofolate (THF), a ubiquitous cofactor that can be generated by Dr (10,29). Consistent with the binding of an alternative cofactor by bacterial NOSs, structural studies indicate that there is substantial variation in the region of the bacterial enzymes that recognize the pterin side chain (10). DrNOS must also produce NO in the absence of a flavodoxin reductase module, as the Dr genome lacks flavodoxin-like proteins.…”

“…Mammalian NOSs (mNOSs) are homodimers that contain two domains: an N-terminal heme oxygenase domain (NOS ox ) that binds the substrate L-arg and cofactors heme and tetrahydrobiopterin (H 4 B), and a C-terminal reductase domain (NOS red ) that binds FAD, FMN, and NADPH (7)(8)(9). Proteins with homology to the mNOS ox domain are found in several mainly Gram-positive bacterial genera such as Streptomyces, Bacillus, Staphylococcus, and Deinococcus (10)(11)(12)(13). These proteins lack NOS red , but retain structural and catalytic properties similar to their mNOS counterparts (10,(12)(13)(14)(15).…”

“…Proteins with homology to the mNOS ox domain are found in several mainly Gram-positive bacterial genera such as Streptomyces, Bacillus, Staphylococcus, and Deinococcus (10)(11)(12)(13). These proteins lack NOS red , but retain structural and catalytic properties similar to their mNOS counterparts (10,(12)(13)(14)(15). Only a few studies have explored the functional role of bacterial NOSs.…”

Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts

“…Note that deleting folX or folM caused complementation to fail only in the P. aeruginosa control. activity with THF in vitro (Fujisawa and Nakata, 1987) and THF can replace H 4 BPt as cofactor for bacterial NO synthase, again in vitro (Adak et al, 2002). However, the use of folates as cofactors in vivo is unprecedented for Phe hydroxylase or any other pterindependent enzyme.…”