Bacterial nitric-oxide synthases operate without a dedicated redox partner. VOLUME 283 (2008) PAGES 13140-13147 (original) (raw)
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Scientific Reports
The enzyme nitric oxide synthase (NOS) oxidizes L-arginine to NO and citrulline. In this work, we characterise the NOS from the cyanobacteria Synechococcus PCC 7335 (SyNOS). SyNOS possesses a canonical mammalian NOS architecture consisting of oxygenase and reductase domains. In addition, SyNOS possesses an unusual globin domain at the N-terminus. Recombinant SyNOS expressed in bacteria is active, and its activity is suppressed by the NOS inhibitor L-NAME. SyNOS allows E. coli to grow in minimum media containing L-arginine as the sole N source, and has a higher growth rate during N deficiency. SyNOS is expressed in Synechococcus PCC 7335 where NO generation is dependent on L-arginine concentration. The growth of Synechococcus is dramatically inhibited by L-NAME, suggesting that SyNOS is essential for this cyanobacterium. Addition of arginine in Synechococcus increases the phycoerythrin content, an N reservoir. The role of the novel globin domain in SyNOS is discussed as an evolutionary advantage, conferring new functional capabilities for N metabolism. Nitric oxide (NO) is a free radical and a signal molecule with functional activities in all living organisms. The biological functions of NO are well documented in animals, and range from vasorelaxation, smooth muscle relaxation, platelet inhibition, neurotransmission and cytotoxicity to immunoregulation of pathophysiological processes 1-3. Nitric oxide synthases (NOSs) are enzymes that catalyse the oxidation of the substrate L-arginine to L-citrulline and NO. Three different enzymes have been described in animals: inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS). All of them present two main domains: the oxygenase containing a heme group and a tetrahydrobiopterin (BH 4) cofactor, and the reductase domain, which provides the electrons for L-arginine oxidation. NOSs are homodimers joined by a Zn binding motif at the N-terminus 4. Most Gram-positive bacteria contain NOSs, though their structure differs from their animal counterparts, since they only contain the oxygenase domain. Therefore, the electron required for the catalytic activity of NOS is provided by non-specific cellular reductases 5. An exception is a NOS from the Gram-negative bacterium Sorangium cellulosum, which contains a reductase module at its N-terminal end 6. The functions of NOS in bacteria are less understood, and the actions that affect cell physiology are diverse and described less frequently. In Bacillus, NOS-derived NO contributes to oxidative stress resistance by activating catalases or reducing Fenton chemistry 5,7. In Deinococcus radiodurans, NO regulates the recovery from UV-B radiation damage 8. Recent results show that NOS modulates aerobic respiration and the switch to nitrate-based respiration during low-oxygen growth in pathogenic bacteria 9. NOS also participates in the response to oxidative stress, in the biosynthesis of nitrated compounds during defence responses and in transcriptional regulation, among others 10. The first NOS from a photosynthetic microorganism was characterised in the microalgae Ostreococcus tauri (OtNOS) 11. OtNOS is a canonical NOS with similar biochemical and spectral properties to animal NOSs. It has a high activity and seems to be independent of Ca 2+-calmodulin, similar to animal iNOS 11,12. Recently, new sequence similarity searches have revealed several NOS proteins in diatoms 13 and in the green lineage, except for land plants 14,15. Phylogenetic analysis showed that a NOS sequence from the cyanobacteria Synechococcus PCC 7335 (SyNOS) presents with high similarity to OtNOS 11. Cyanobacteria are one of the most important primary producers on the Earth, and are responsible for the spread of the eukaryotic green lineage that originated
Update on Mechanism and Catalytic Regulation in the NO Synthases
Journal of Biological Chemistry, 2004
Nitric-oxide synthases (NOSs, EC 1.14.13.39) 1 oxidize L-arginine to nitric oxide (NO) and are interesting for several reasons. They are present in many life forms (1, 2), their gene regulation is complex (3), they are the only flavoheme enzymes that utilize tetrahydrobiopterin (H 4 B) as a redox cofactor, and their electron transfer reactions are regulated by a Ca 2ϩ-binding protein (calmodulin). In the past 5 years, crystal structures of NOS heme (oxygenase) domains and bacterial NOS-like proteins have shown how Arg, heme, and H 4 B bind in the active site (4, 5). Reviews are available on NOS biochemistry (6), regulation (7, 8), protein-protein interactions (9), and posttranslational modifications (10). This minireview updates the NO biosynthetic mechanism and describes a global catalytic model that highlights the role of NO as an intrinsic regulator.
In search of the prototype of nitric oxide synthase
FEBS Letters, 2003
Recent identi¢cation of the prokaryotic genes related to the catalytic oxygenase domain of mammalian nitric oxide synthase (NOS) has led to speculations on the origins of the NO signaling network. NOS activity in eukaryotes relies on the concerted action of the oxygenase domain with an electron-donating reductase domain that is fused to it. A fused reductase domain is, however, absent in prokaryotes. Consequently, we searched bacterial genomes for homologs of the reductase domain and identi¢ed candidate genes. On the basis of genomic sequence and protein structural analysis, we here propose that sul¢te reductase £avoprotein is a prototype of the mammalian NOS reductase domain and a complementing interaction partner of the bacterial NOS oxygenase protein. Abbreviations: NOS, nitric oxide synthase; bNOSoxy, bacterial NOS oxygenase domain precursor; NOSoxy, oxygenase domain of NOS; NOSred, reductase domain of NOS; BH 4 , tetrahydrobiopterin FEBS 27716 FEBS Letters 554 (2003) 1^5
Journal of Biological Chemistry, 2002
Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound L-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from L-Arg when incubated with NADPH and a mammalian NOS reductase domain.
From no-confidence to nitric oxide acknowledgement: A story of bacterial nitric-oxide reductase
Folia Microbiologica, 2000
The review briefl> summarizes current knowledge of the bacterial nitric-oxide reductase (NOR). This membrane enzyme consists of two subunits, the smaller one contains hmm C and the larger one two haems B and nonhaem iron. The protein sequence and structure of metal centres demonstrate the relationship of NOR to the family of terminal oxidases. The binuelear Fe-Fe reaction centre, consisting of antiferro-magneticall~ coupled h~em B and nonhmm iron, is analogous to Fe-Cu centre of terminal oxidases. The data on the structure and function of NOR and terminal oxidases suggest that all these enzymes are closely evolutionally related The catalytic properties are determined most of all by the relatively high toxicity of nitric oxide as a substrate and the resulting strong need to maintain its concentration at nanomolar levels. A kinetic model of the action of the enzyme comprises substrate inhibition. NOR does not conserve the free energy of nitric oxide reduction because it does not work as a proton pump and, moreover, the protons coming into the reaction are taken from periplasm, i.e. they do not cross the membrane. A bbreviat~ons CCCP 3-chlorophenylhydrazono-malononitrile ('carbonyl cyanide 3-chlorophenylhydrazone') COX aa3-cytochrome-c oxidase NIR nitrite reductase NOR PMS TMPD nitric-oxide reductase N-methylphenazonium methanesulfonate Cphenazine methosulfate')
Nitrogen oxide cycle regulates nitric oxide levels and bacterial cell signaling
Scientific Reports, 2016
Nitric oxide (NO) signaling controls various metabolic pathways in bacteria and higher eukaryotes. Cellular enzymes synthesize and detoxify NO; however, a mechanism that controls its cellular homeostasis has not been identified. Here, we found a nitrogen oxide cycle involving nitrate reductase (Nar) and the NO dioxygenase flavohemoglobin (Fhb), that facilitate inter-conversion of nitrate, nitrite, and NO in the actinobacterium Streptomyces coelicolor. This cycle regulates cellular NO levels, bacterial antibiotic production, and morphological differentiation. NO down-regulates Nar and up-regulates Fhb gene expression via the NO-dependent transcriptional factors DevSR and NsrR, respectively, which are involved in the auto-regulation mechanism of intracellular NO levels. Nitrite generated by the NO cycles induces gene expression in neighboring cells, indicating an additional role of the cycle as a producer of a transmittable inter-cellular communication molecule. Nitric oxide (NO) is a freely diffusible neutral gas that acts as an important signaling molecule to control metabolic pathways in bacteria and higher eukaryotes. Since NO is highly reactive and toxic for living cells, cells must have strict control over intracellular NO levels. The genus Streptomyces includes bacteria that produce many commercially useful secondary metabolites that are extremely important to humans. They follow an elaborate life cycle that includes vegetative (or substrate) mycelial growth, aerial mycelial growth, and sporulation. Recent studies suggest that actinobacteria require NO to regulate various metabolic pathways 1-5 , however, little is known about the mechanism by which actinobacteria generate NO, except for NO synthase (NOS) distribution in a limited number of actinobacterial species. Recently, we reported a unique nitrogen metabolism in Streptomyces antibioticus, in which organic nitrogen was mineralized to form nitrogen oxide species, nitrite (NO 2 −), nitrate (NO 3 −), and NO during vegetative cell growth under aerobic conditions, and NO 2 − was excreted into the medium 6. Since arginine analogs inhibited the production of NO 2 − and NO, we suggested that NOS is involved in the NO 2 −-formation, and proposed a NO 2 −-forming pathway (pathway 1, below), although presence of a NOS enzyme has not been demonstrated in S. antibioticus.