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The nitric oxide synthase-like protein from Bacillus cereus (bcNOS) has been cloned, expressed, and characterized. This small hemeprotein (356 amino acids in length) has a mass of 43 kDa and forms a dimer. The recombinant protein showed similar spectral shifts to the mammalian NOS proteins and could bind the substrates L-arginine and NG-hydroxy-L-arginine as well as the ligand imidazole. Low levels of activity were recorded for the hydrogen peroxide-dependent oxidation of NG-hydroxy-L-arginine and L-arginine by bcNOS, while a reconstituted system with the rat neuronal NOS reductase domain showed no activity. The recombinant bcNOS protein adds to the complement of bacterial NOS-like proteins that are used for the investigation of the mechanism and function of NO in microorganisms.
Nitric oxide has many diverse functions in the mammalian body and is produced in mammals by a family of nitric oxide synthase enzymes (NOS: EC 184.108.40.206) . These enzymes are composed of an N-terminus oxygenase domain containing a heme, tetrahydrobiopterin (H4B), and the substrate L-arginine as well as a C-terminus reductase domain. There are over thirty NOS-like proteins in prokaryotes including eight different types reported in bacilli . Bacterial NOS-like enzymes lack the associated NOS reductase domain found in mammalian enzymes . The bacterial NOSs have many properties in common with the mammalian oxygenase domain including dimer structure, L-Arginine as a substrate, and typical heme spectroscopy . Very little is known about the function of these proteins in prokaryotes, and they are not required for nitrification and denitrification pathways .
The opportunistic pathogen B. cereus causes food poisoning and is closely related to the animal and human pathogen B. anthracis used as a biological weapon as well as the insect pathogen B. thuringiensis that is used as a pesticide. We report the cloning, expression, purification, and characterization of a NOS-like protein from Bacillus cereus (bcNOS).
All reagents were purchased from Sigma-Adrich Canada Ltd. (Oakville, ON, Canada) and Fisher-Scientific Ltd. (Ottawa, ON, Canada) and were of high quality chemical grade.
The bcNOS gene from B. cereus (ATCC strain number 10987) was amplified by PCR from genomic DNA. The following PCR primers generated an Nde I site (bold) before the 5′ start codon in BCNOSF1 and an EcoR I site (underlined) after the 3′ stop codon in BCNOSR1: BCNOSF1, 5′-GAA GAT CTC ATA TGA GTA AAA CGA AGC AAT TAA TAG AGG AAG CG-3′; BCNOSREV, 5′-GGG AAT TCC TAT TTA TGA AAA AAA TTC GGC TTC AAA ATT TC-3′. The amplified fragment was cloned into the pET28a expression vector (Novagen, Madison, WI), which contains a His6 coding region upstream from the Nde I site.
E. coli strain BL21(DE3) pLysS transfected with the bcNOS plasmid and grown in Luria-Bertani broth containing 100μg/mL ampicillin, induced at an OD (600nm) of 0.6 with 1mM IPTG and grown for 4 hours and then harvested. The recombinant bcNOS was purified from the bacterial cells using metal chelating chromatography  (see Supplementary Material available online at doi: 10.1155/2010/489892). Recombinant rat nNOS reductase protein was overexpressed in Escherichia coli strain BL21(DE3) and purified as previously described .
Mass spectrometry of the purified bcNOS was performed at the WATSPEC Mass Spectroscopy Facility at the University of Waterloo . Gel exclusion chromatography was used to estimate the molecular weight of bcNOS dimer .
Spectroscopy performed on a Varian-Cary 1. The bcNOS exhibited a typical heme peak around 400nm. Enzyme (10μM) was incubated in the presence of 1mM imidazole, 1mM L-arginine, 100μM H4B, or CO gas. Difference spectroscopy was used to measure the binding affinities of imidazole, NOHA, and L-arginine as previously described .
The H2O2-dependent bcNOS oxidation of NOHA and L-arginine to nitrite was monitored at 25°C on a 96-well plate reader as presiously reported [8, 9]. The hemoglobin capture assay  was used to monitor nitric oxide production from bcNOS in the presence of L-arginine and the rat neuronal NOS reductase domain.
The sequence alignment of the bcNOS protein with the NOS-like proteins from B. subtilis (bsNOS), Staphylococcus aureus (saNOS), and D. radiodurans (deiNOS) revealed a 42 to 52% identity and 61 to 68% conservation of the sequence and full conservation of the residues involved in binding to the heme (see Figure 1 in supplementary materials). A comparison of the bcNOS protein with the human inducible NOS (iNOS) revealed a 40% identity and 57% conservation of the sequence. Recombinant bcNOS was purified to greater than 90% homogeneity based upon SDS-PAGE and mass spectrometry analysis (see Figure 2 in supplementary materials). A yield of 8mg of pure bcNOS was obtained per liter of culture. Nondenaturing native-PAGE and gel exclusion chromatography both showed that bcNOS forms a dimer.
The bcNOS UV-visible spectrum showed the presence of a heme chromophore in a high spin state (Figure 1). The observed spectral changes of bcNOS due to the binding of different heme ligands are summarized in Table 1. The characteristic Soret absorbance peak of 399nm was observed for ferric bcNOS (Figure 1), likely corresponding to a mixture of 5-coordinated high-spin species and a water-bound 6-coordinated low-spin species. The binding of DDT shifts the equilibrium towards a fully 5-coordinated high-spin species. When H4B was added the Soret peak shifted to 397nm. The slight blue shift in the spectrum indicates that the H4B displaced the DTT ligand. Adding L-arginine produced the same result as H4B, resulting in a Soret absorbance peak shift to 397nm. The addition of imidazole resulted in a low-spin heme state with a peak absorbance of 426nm that is typical of imidazole serving as a distal sixth ligand in the active site of NOS enzymes. Dithionite-reduced, carbon monoxide (CO) bound bcNOS gave absorbance peaks at 443nm and 545nm, comparable to the mammalian iNOS and neuronal NOS (nNOS) oxygenase domains and other bacterial NOS-like proteins (Table 1) indicating a similar heme iron coordination structure.
Difference spectrometry, used to study heme-substrate interactions, revealed that a substrate-binding site exists in bcNOS close to the heme group and that this site is capable of binding imidazole, NOHA, or L-arginine. The displacement of heme-bound imidazole due to the binding of NOHA or L-arginine was monitored to determine the binding affinity of NOHA and L-arginine to bcNOS. Double-reciprocal analysis of the binding of imidazole to bcNOS gave a K d value of 181 ± 13μM. The binding affinities of NOHA and L-arginine were much greater with K s values of 1.12 ± 0.01 and 11 ± 3, respectively. The apparent binding affinity of L-arginine to bcNOS was found to be similar to those determined for other bacterial NOS proteins and the mammalian iNOSoxy protein (Table 2). This is consistent with bcNOS containing a conserved glutamate residue found to be essential for high affinity L-arginine binding in mammalian NOS enzymes .
Tetrahydrobiopterin-free nNOS can oxidize NOHA or L-arginine to nitrite in the presence of H2O2 . Despite low binding constants for both NOHA and L-arginine, low levels of activity were found for the bcNOS protein in the presence of H2O2 and either NOHA (0.100 ± 0.014min−1) or L-arginine (0.048 ± 0.001minute). When compared to mammalian NOS enzymes, slow turnover numbers for H2O2-supported NOHA oxidation have also been reported for other bacterial NOS-like proteins [11, 15, 16]. The addition of H4B did not significantly increase the activity of bcNOS, but in the presence of NOHA and THF the activity doubled (0.231 ± 0.023). This suggests that a biological ligand, such as THF or a related pterin, may be required by bcNOS for catalytic activity. A similar catalytic rate was determined for saNOS in the absence of a cofactor (0.15 ± 0.01nmol nitrite min−1nmol saNOS) with no increase in activity observed in the presence of THF  and a recent report indicates that THF may replace H4B as a redox-active cofactor in deiNOS .
The bcNOS protein was not catalytically active when coupled with the rat nNOS reductase domain protein in the presence of L-arginine and NADPH despite the fact that the nNOS reductase domain protein readily transfers electrons to reduce cytochrome c . The relevance of such a reconstituted system comes into question, as a reductase protein similar to the mammalian NOS reductase domain could not be found in the B. cereus genome. Bacterial flavodoxins have been reported to support nitric oxide production by B. subtilis nitric oxide synthase . We were unable to identify the electron donor(s) of bcNOS which is consistent with a recent report showing that bacterial NOS proteins do not appear to accept electrons from a specific reductase but more likely accept electrons from several different sources .
The NO produced by bacterial NOS enzymes has been associated with a number of novel functions. In Streptomyces NOS mediates the nitration of the tryptophan moiety of the phytotoxin dipeptide L-tryptophan-L-phenylalanine . Disruption of the NOS gene in B. subtilis renders the strain more susceptible to oxidative damage . Our B. cereus nosgene knock out strain was also found to be more vulnerable to hydrogen peroxide exposure (results not shown). Notably, B. anthracis derived-NO is correlated with pathogen virulence and survival in macrophages . Clearly, there are several unanswered questions regarding bacterial NOS enzymes including cofactor requirements, evolutionary traits, catalytic mechanism, their biological reductase partners, and their in vivo function(s).
The supplementary material includes sequence alignments of bacterial NOS-like proteins and three mammalian NOS oxygenase domains. The results of our complete characterization of the the Bacillus cereus NOS-like protein are also included in the supplementary section.
This research was funded by the Natural Sciences and Engineering Research Council of Canada Grant no. 183521.