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J. Biol. Chem., Vol. 282, Issue 29, 21301-21307, July 20, 2007
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From the Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Sendai 980-8577, Japan
Received for publication, March 5, 2007 , and in revised form, May 9, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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180 amino acids long and has a conserved amino acid sequence, GG(D/E)(D/E)F. The EAL subdomain has a phosphodiesterase activity that hydrolytically cleaves cyclic diGMP into l-diGMP and/or GMP, and is 260 residues in length, including the conserved amino acid sequence EAL. Metabolism of c-diGMP may be physiologically important because the genome of Escherichia coli K-12, for example, encodes 19 proteins with GGDEF subdomains and 17 with EAL subdomains.
Heme-regulated phosphodiesterase from E. coli (Ec DOS) contains a heme-bound PAS domain in the N-terminal region and a phosphodiesterase domain with GGDEF and EAL subdomains in the C-terminal region (11, 12). Although Ec DOS also contains a GGDEF subdomain, it does not appear to have guanylate cyclase activity. Thus, the precise roles of the GGDEF domain in the catalysis and structure of Ec DOS remain unclear. In contrast, the EAL domain in Ec DOS appears to contain the catalytic domain, so that this domain retains high phosphodiesterase activity that rapidly converts c-diGMP into linear dinucleotide GMP (l-diGMP) (3, 4). Furthermore, E. coli with a knock-out of the Ec DOS gene display a morphology and growth rate different from those of the wild-type strain, suggesting that the enzyme is important for cell development and/or proliferation (13).
In well known heme-bound gas-sensor proteins, binding of specific gases to the Fe(II) heme (for example, CO for CooA, NO for soluble guanylate cyclase, and O2 for FixL) causes a change in the heme environment that is transduced intramolecularly to the other domain to regulate the function (14). Each of these heme-bound gas-sensor enzymes can recognize only a single physiologically relevant gas. We recently reported, however, that the phosphodiesterase activity of full-length, dithionite-reduced Ec DOS is up-regulated upon binding of either O2 or CO binding to the Fe(II) heme (15). Although the amino acid sequence and overall structure of the heme-containing PAS domains of Ec DOS and FixL are similar, Ec DOS is activated by both O2 and CO, whereas FixL is down-regulated only by O2. Thus, Ec DOS appears to be a novel heme-bound gas-sensor enzyme with the unprecedented ability to recognize more than one type of gas molecule.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Construction of Expression Plasmid and Site-directed Mutagenesis—The cloning of Ec DOS and construction of the expression plasmid pET28a(+) were previously described (19). Mutants were generated as described in the supplementary materials. All constructs were confirmed by DNA sequencing.
Protein Overexpression and Purification—The wild-type and mutant Ec DOS proteins were overexpressed in E. coli BL21(DE3) using the pET28a(+) expression plasmid, which contains an N-terminal His6 tag and a thrombin cleavage site. A single colony was put in 50 ml of Luria-Bertani (LB) medium containing 20 µg/ml kanamycin and 0.5% glucose and then shaken overnight at 200 rpm and 37 °C. The cultured medium was added to 1 liter of terrific broth (TB) medium containing 20 µg/ml kanamycin. Then, the medium was shaken for 4 h at 120 rpm and 37 °C. After the medium was cooled down to 20 °C, 
-aminolevulinic acid was added to 0.45 mM and incubated with shaking for another 20 h. The E. coli cells were harvested by centrifugation for 10 min at 5000 x g and 4 °C, and finally stored at -80 °C.
The following protein purification procedures were performed on ice or at 4 °C. E. coli cells frozen at -80 °C were suspended in 50 mM potassium phosphate buffer (pH 7.5) containing 20 mM imidazole, 150 mM NaCl, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride (buffer A), and 0.1 mg/ml lysozyme. The solution was sonicated and then centrifuged at 100,000 x g for 30 min. The supernatants were applied to a nickel-nitrilotriacetic acid-agarose column. The Ec DOS fractions were eluted with a linear gradient of 20–300 mM imidazole in buffer A. The solution containing Ec DOS was dialyzed against 20 mM Tris-HCl (pH 8.0) with 5% glycerol (buffer B), and then thrombin protease (Wako Chemicals, Osaka, Japan) (1 unit/mg of protein) was added to cleave the His tag. Subsequently, the sample solution was applied to a nickel-nitrilotriacetic acid-agarose column, and the His tag-free Ec DOS was eluted in the flow-through fractions using buffer B. The Ec DOS fractions were then applied to DEAE column, and Ec DOS was eluted with a linear gradient of 0–1 M NaCl in buffer B. After the proteins were dialyzed against buffer B, the purified Ec DOS was concentrated with Centriprep (Millipore, Billerica, MA) and Centrisalt I (Sartorius AG, Goettigen, Germany). The purified proteins were >95% homogenous as confirmed by SDS-PAGE. The sample was stored at -20 °C in the presence of 40% glycerol.
Optical Absorption Spectroscopy—All spectral data were obtained using a UV-1600PC (Shimadzu, Kyoto, Japan) spectrophotometer in a glove box that can maintain the oxygen concentration under 100 ppm (Hirasawa, Tokyo, Japan). The heme of Ec DOS proteins was reduced using either 10 mM DTT or small quantities of sodium dithionite in PDE buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 5 mM MgCl2) under anaerobic conditions. The Fe(II) heme, Fe(II) heme-O2, and Fe(II) heme-CO complexes were prepared in anoxic (10% H2 and 90% N2 gas), O2-saturated, or CO-saturated buffer, respectively. The gas-saturated buffer solutions were obtained by bubbling the buffer with the appropriate gas for at least 1 h. The Fe(II) heme-NO complexes were prepared by adding of 50 or 100 µM NOC9 to the solution of Fe(II) heme complex.
Phosphodiesterase Assay—Ec DOS hydrolyzes c-diGMP to l-diGMP as demonstrated by reversed-phase liquid chromatography (supplementary materials Fig. 1S). On the other hand, in our phosphodiesterase assay, we used a colorimetric method, namely, l-diGMP that is converted to a guanylyl (3',5')guanosine by Ec DOS. CIAP reacts with only l-diGMP because CIAP lacks phosphodiesterase activity and thus reacts only with terminal phosphate groups. CIAP therefore converts l-diGMP to GpG (guanylyl (3',5')guanosine) and phosphate. One product, phosphate, was quantified by colorimetrically using BIOMOL GREEN reagent (Biomol). The change in absorbance at 630 nm (supplementary materials Fig. 2S, A) was measured with a Benchmark Microplate ReaderTM (Bio-Rad) and used to determine phosphate concentration by comparison with a standard curve (supplementary materials Fig. 2S, B).
All steps were performed in the globe box. Wild-type and mutant Ec DOS proteins were diluted in PDE buffer. The wild-type and Arg97 mutant enzymes were fully reduced to the Fe(II) form by adding 10 mM DTT. For the Met95 mutants, a trace amount of sodium dithionite was used to completely reduce the heme iron to the Fe(II) complex because DTT cannot reduce the heme iron in the Met95 mutant proteins. Subsequently, excess dithionite was removed by gel filtration on Superdex G-25. After complete reduction the Fe(II) form was confirmed by optical absorption spectroscopy, Fe(II)-CO and Fe(II)-O2 complexes were prepared by diluting the sample in gas-saturated PDE buffer containing 10 mM DTT. The Fe(II)-NO sample was prepared by adding 50 µM or 100 µM NOC9 to the sample of Fe(II) enzyme.
The phosphodiesterase reaction was performed at 25 °C in a mixture with 71.4 mM Tris-HCl (pH 8.0), 71.4 mM NaCl, 7.14 mM MgCl2, 2.14 units/µl of CIAP, 14.3 mM DTT, and 0.29 µM Ec DOS and initiated by the addition of 0.3 volumes of 0.33 mM c-diGMP. After the desired time, the reaction was terminated by the addition of an equal volume of 1 M HCl. The solution was then centrifuged for 5 min at 15,000 x g to remove denatured proteins as precipitates. The supernatant (100 µl) was mixed with 200 µl of BIOMOL GREEN, and the mixture incubated at 25 °C for 30 min. The change in absorbance at 630 nm was measured as described above. The initial rates of the reactions are averages of at least three time course experiments. The experimental errors are shown in the l-diGMP hydrolysis time course data of Fig. 3, and also in Figs. 5S and 6S (supplementary materials). Note that the stick diagrams in Figs. 3B, 4, and supplemental materials Fig. 7S do not have error bars because values were calculated from averaged data points.
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RESULTS |
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In the remaining experiments, we reduced the wild-type and Arg97 mutant enzymes to the Fe(II) heme state using DTT, whereas we reduced the Met95 mutant proteins with sodium dithionite. It was, therefore, examined whether there were differences in the optical absorption spectra of the Fe(II) heme complexes when the enzymes were reduced with DTT and sodium dithionite. We found that the spectra of the Fe(II) heme complexes were essentially the same, even if the heme iron was reduced by DTT or sodium dithionite, suggesting that the heme environment of the full-length enzyme is not influenced by the reducing agent.
Phosphodiesterase Activity of the Wild-type Enzyme—Activities of other heme-bound oxygen-sensor enzymes, such as SmFixL, in the presence of DTT are much more sensitive to oxygen and appear to reflect the activity of the native enzyme under hypoxic conditions (18). In the present study, therefore, we used 10 mM DTT to reduce the wild-type enzyme, and we measured the catalytic activity in the glove box to provide anoxic conditions. High pressure liquid chromatography (as shown in supplementary materials Fig. 1S) is used in general to measure phosphodiesterase cleavage of c-diGMP. However, this method is time consuming. In this report, we use colorimetric detection of products in the development of a rapid c-diGMP cleavage assay ("Experimental Procedures" and supplementary materials Fig. 2S).
Fig. 3 shows the time course of l-diGMP generation by Fe(II), Fe(II)-O2, Fe(II)-CO, and Fe(II)-NO complexes of wild-type Ec DOS in the presence of 10 mM DTT. The initial velocities are compared in Fig. 3B and are summarized in Table 2. The binding of O2, CO, or NO to the heme enhanced the activity 6–7-fold. The finding that even NO binding markedly enhanced the catalytic activity was surprising because other heme gas-sensor proteins such as FixL, CooA, and soluble guanylate cyclase strictly distinguish O2, CO, and NO, respectively (14). This emphasizes that Ec DOS is a novel gas-sensor enzyme that has the unprecedented ability to be activated by all three of these gases.
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Interestingly, the gas-free M95A and M95L mutants showed high activities without gas binding; specifically, the activities of these two mutants were 7–8-fold higher than that of the gas-free wild-type enzyme (Table 1 and supplementary materials Fig. 2S). The activities of the gas-bound bound forms of the mutants were almost as high as the gas-free forms (Table 2). In other words, the catalytic activities of the mutant enzymes were not enhanced by gas binding. In contrast, the activity of the gas-free M95H mutant was 2-fold higher than that of the wild-type enzyme, and the gas molecules enhanced its activity 2–3-fold.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-helices flanking a five-stranded antiparallel 
-sheet scaffold (17, 29–33). PAS domains are found in many signaling proteins, where they are used either as signal sensor domains or for protein dimerization (29–32). Several PAS domain proteins are known to detect signals through the use of prosthetic groups such as heme or flavin (30, 31). Heme-based PAS sensor proteins detect oxygen, CO, and possibly the cellular redox state (14, 31). Comparison of the crystal structures of the oxygen-free and oxygen-bound PAS domains of Ec DOS (16, 17) shows that Arg97 undergoes a large change in position when oxygen binding occurs. It might thus be expected that Arg97 should play a prominent role in DOS function. Notably, however, our study clearly shows that this is not the case. The R97A and R97I mutants have activity profiles similar to that of the wild-type enzyme. On the other hand, the M95A and M95L mutants are clearly constitutively "on." In the absence of Met95, the PAS domain presumably assumes a conformation similar to that of the gas-bound protein. Consistent with this idea, the M95H mutant, with a histidine bound to the heme, exhibits properties similar to those of the wild-type enzyme. These data strongly suggest that changes in Met95 coordination structure caused by gas binding (mimicked by mutations at Met95) result in larger conformational changes at the distal side, which subsequently communicate ligand binding information to the catalytic domain.
Effects of Sodium Dithionite and DTT on the Phosphodiesterase Activity of Ec DOS—Although the optical absorption spectra of the Fe(II) complexes for the wild-type and Arg97 mutant proteins were essentially the same when reduced by DTT or sodium dithionite (Fig. 2 and Table 1), the phosphodiesterase activities of the DTT-reduced enzymes were at least 5-fold higher than those of the dithionite-reduced enzymes (Fig. 3 and supplementary materials Fig. 6S). Similar results have been reported for other heme-bound oxygen-sensor enzymes, including the histidine kinase SmFixL (18). The low activity of the dithionite-reduced forms of SmFixL were thought to be due to the formation of an aberrant disulfide bond at Cys301 between Fe(III) homodimers during the preparation of samples under aerobic conditions in the absence of reducing agents. In SmFixL treated with DTT, the disulfide bond was cleaved, leading to more enhancing the enzyme activity.
The wild-type and some mutants of Ec DOS are sometimes purified as the Fe(II)-O2 form (data not shown), suggesting that Ec DOS is present as the Fe(II) form in cells. In general, however, the Fe(III) form along with minor components of Fe(II) and Fe(II)-O2 forms are obtained when Ec DOS is purified under aerobic conditions. Ec DOS contains eight cysteine residues in each monomer. It is possible, therefore, that some cysteine residues in Ec DOS are already oxidized and form disulfide bond(s), leading to lower activity. The following results support the idea that dispensable oxidation caused partial inactivation of Ec DOS. When DTT was removed by G-25 column chromatography, DTT-reduced Ec DOS showed low phosphodiesterase activities for Fe(II), Fe(II)-O2, and Fe(II)-NO complexes, but showed high activity only for the Fe(II)-CO complex (supplementary materials Fig. 5S). O2 and NO molecules are oxidants, and thus, may oxidize cysteine residue(s) or cleave disulfide bonds in Ec DOS, partially inactivating the phosphodiesterase activity.
Role of Arg97 in the Catalysis—The Arg97 mutants autoxidized rapidly (>10 s-1) and/or had low affinities for oxygen. Therefore, Arg97 must be important for stabilizing the O2 molecule bound to the Fe(II) heme by directly binding to the O2 molecule on the heme distal side (16). In contrast, optical absorption spectra suggest that Arg97 is not important for stabilizing the CO and NO molecules bound to the Fe(II) heme. The enhancement of catalytic activity by CO and NO was similar for the Arg97 mutants and the wild-type enzyme. Therefore, Arg97 must not be critical for the enhancement of catalysis by CO and NO.
The structure of the heme-bound sensor PAS domains of SmFixL and Ec DOS are similar. Arg214 in SmFixL, which corresponds to Arg97 in Ec DOS, in the heme distal side directly interacts with the O2 molecule bound to the Fe(II) heme (23). The Fe(II)-O2 complex of SmFixL is inactive, whereas the gas-free Fe(II) complex is active, and Arg214 forms salt bridges with the heme propionate in the gas-free Fe(II) complex (23). Therefore, the O2 molecule has opposite roles in the catalysis of SmFixL and Ec DOS. For SmFixL, the distal Arg214 is thought to be essential for the regulation of kinase activity by ligand binding, O2 binding, and, therefore, reduced autoxidation of the Fe(II)-O2 complex (24, 25). The optical absorption spectra suggest that Arg97 in Ec DOS stabilizes the Fe(II)-O2 form and that the interaction of Arg97 with the gas ligand may not be important for the activation of catalysis by ligand binding (Table 2 and Fig. 4).
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Role of Met95 in the Catalysis—Met95 is the sixth axial ligand in the gas-free Fe(II) complex. Met95 must move away from the heme iron upon binding of exogenous ligand to Fe(II) heme (Fig. 1). Previous studies indicate that mutations at Met95 in the isolated heme-bound PAS domain of Ec DOS substantially increase the affinities of the heme iron for O2, CO, and CN- and that the heme redox potential is also markedly changed by Met95 mutations (20, 21, 26). The role of Met95 in catalytic regulation, however, has been unclear.
In the present study, we found that the activities of the gas-free M95A and M95L mutant enzymes were already similar to those of the gas-bound wild-type enzymes (Table 2). Therefore, it appears that Met95 is critical for regulating the catalytic activity of Ec DOS. In the gas-free form, the M95A and M95L mutants are 5-coordinate high-spin complexes, in contrast to the wild-type and Arg97 mutant enzymes, which are 6-coordinate low-spin complexes. In the wild-type and Arg97 mutant enzymes, cleavage of the Met95 coordination to the Fe(II) heme occurs upon gas binding. On the other hand, in the M95A and M95L mutants, as in the gas-bound wild-type and Arg97 mutant proteins, there is no bond between the residue at position 95 and the Fe(II) heme iron. Certain structural change(s) near the main or side chain of Met95 on the heme distal side must occur upon binding of the gas ligand. There may be a similar structure(s) in the Fe(II) complex of the M95A and M95L mutants even in the absence of the gas molecules, which lead to the high activities of the gas-free Fe(II) heme enzymes (Table 2 and Fig. 4).
It is interesting that the M95H mutant is the 6-coordinate low-spin complex, probably as a result of His95 coordination to the Fe(II) heme iron. The catalytic activity of the gas-free form of this mutant was lower than those of other Met95 mutants that are 5-coordiante high-spin complexes. On the other hand, gas binding by this mutant enhanced its activity 2–3-fold, which is higher than that observed for other Met95 mutant proteins. Perhaps a certain conformational change(s) caused by the M95H mutation influences the activity of the gas-free M95H mutant enzyme, leading to activities intermediate between the wild-type/Arg97 and M95A/M95L mutants. Collectively, the results suggest that Met95 coordination to the Fe(II) heme is a key factor in the down-regulation of catalysis so that its removal by gas binding or the M95A or M95L mutations enhances the catalytic activity.
Comparison with Catalysis of Ec DOS toward cAMP—Recent studies of the cAMP phosphodiesterase activity of Ec DOS indicate that cAMP is hydrolyzed by the Fe(II) heme form but not the Fe(III) form of Ec DOS (19). Furthermore, binding of CO or NO to the Fe(II) heme complex eliminates the ability to hydrolyze cAMP. X-ray crystal structural studies of the isolated heme-bound PAS domain of Ec DOS indicate that profound structural changes of the heme-bound PAS domain occur upon changes in the redox state (17). Such redox-dependent structural changes appear to reflect the redox-dependent regulation in the cAMP phosphodiesterase activity of Ec DOS. Furthermore, knocking down the Ec DOS gene in E. coli causes changes in cell growth rate, cell morphology, and intracellular cAMP concentration, suggesting that Ec DOS plays an important role in modulating the cellular effects of cAMP (13).
In the present study, we showed that, in addition to cAMP, Ec DOS hydrolyzes c-diGMP. The mechanism by which c-diGMP hydrolysis is regulated differs from that regulating cAMP hydrolysis. Namely, the c-diGMP phosphodiesterase activity of Ec DOS is substantially up-regulated by the binding of gas molecules. Furthermore, it is important to note that, unlike other heme-bound gas-sensor enzymes and proteins (14), Ec DOS does not discriminate between O2, CO, and NO. Further studies are needed to understand the mechanisms of gas sensing and intramolecular signal transduction by this novel enzyme. It will also be interesting to investigate the intracellular cross-talk between the two substrates of Ec DOS, cAMP and c-diGMP (27).
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental data and Figs. S1–S7.
1 To whom correspondence should be addressed: 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Tel.: 81-22-217-5604/5605/5606; Fax: 81-22-217-5604/5605/5390; E-mail: shimizu{at}tagen.tohoku.ac.jp.
2 The abbreviations used are: c-diGMP, cyclic dinucleotide-GMP; GGDEF subdomain, a subdomain that characteristically expresses diguanylate cyclase activity, used to synthesize cyclic diGMP; EAL subdomain, a subdomain that characteristically expresses phosphodiesterase activity and hydrolytically cleaves cyclic diGMP; l-diGMP, linear dinucleotide GMP; Ec DOS, Escherichia coli direct oxygen sensor or heme-regulated phosphodiesterase from E. coli; PAS, Per (Drosophila period clock protein)-Arnt (vertebrate aryl hydrocarbon receptor nuclear translocator)-Sim (Drosophila single-minded protein); SmFixL, an oxygen sensor heme protein from Sinorhizobium meliloti; CooA, CO-sensing heme-bound transcriptional regulator from Rhodospirillum rubrum; DTT, dithiothreitol; CIAP, calf intestine alkaline phosphatase; NOC9, 6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl-1-hexanamine; PDE, phosphodiesterase.
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), Fe(II)-O2 (
), Fe(II)-CO (•), and Fe(II)-NO (
) complexes of wild-type Ec DOS. PDE activities were measured in the presence of 10 mM DTT and are given as mean ± S.D. of at least three determinants. B, product generation of the PDE reaction in the absence or presence of gas molecules.