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Paul W Ludden - One of the best experts on this subject based on the ideXlab platform.

  • new insights into the mechanism of nickel insertion into Carbon Monoxide Dehydrogenase analysis of rhodospirillum rubrum Carbon Monoxide Dehydrogenase variants with substituted ligands to the fe3s4 portion of the active site c cluster
    Journal of Biological Inorganic Chemistry, 2005
    Co-Authors: Won Bae Jeon, Paul W Ludden, Steven W Singer, Luis M Rubio
    Abstract:

    Carbon Monoxide Dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO2. A unique [NiFe4S4] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH•Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe3S4] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step.

  • converting the nifes Carbon Monoxide Dehydrogenase to a hydrogenase and a hydroxylamine reductase
    Journal of Bacteriology, 2002
    Co-Authors: Jongyun Heo, Christopher R Staples, Marcus T Wolfe, Paul W Ludden
    Abstract:

    Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing Carbon Monoxide Dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys531 exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His265 exhibits hydroxylamine reductase activity. Both Cys531 and His265 are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.

  • Carbon Monoxide Dehydrogenase from rhodospirillum rubrum produces formate
    Journal of Biological Inorganic Chemistry, 2002
    Co-Authors: Jongyun Heo, Lars Skjeldal, Christopher R Staples, Paul W Ludden
    Abstract:

    Carbon Monoxide Dehydrogenase (CODH) from Rhodospirillumrubrum reversibly catalyzes the oxidation of CO to CO2 at the active site C-cluster. In this article, the reduction of CO2 to formate is reported as a slow side reaction catalyzed by both Ni-containing CODH and Ni-deficient CODH. Recently, the structures of R. rubrum CODH and its active site NiFeS cluster (the C-cluster) have been solved. The data in this manuscript describe the formate-producing capability of CODH with or without Ni in the active site.

  • spectroscopic studies of nickel deficient Carbon Monoxide Dehydrogenase from rhodospirillum rubrum nature of the iron sulfur clusters
    Biochemistry, 2002
    Co-Authors: Jennifer L Craft, Paul W Ludden, Thomas C Brunold
    Abstract:

    Carbon Monoxide Dehydrogenase (CODH) from Rhodospirillum rubrum utilizes three types of Fe-S clusters to catalyze the reversible oxidation of CO to CO2:  a novel [Ni4Fe5S] active site (C cluster) and two distinct [4Fe4S] electron-transfer sites (B and D clusters). While recent X-ray data show the geometric arrangement of the five metal centers at the C cluster, electronic structures of the various [Ni4Fe5S] oxidation states remain ambiguous. These studies report magnetic circular dichroism (MCD), variable temperature, variable field MCD (VTVH MCD), and resonance Raman (rR) spectroscopic properties of the Fe-S clusters contained in Ni-deficient CODH. Essentially homogeneous sample preparations aided in the resolution of the reduced [4Fe4S]1+ (S = 1/2) B cluster and the reduced Ni-deficient C cluster (denoted C*, S > 1/2) by MCD. The three Fe atoms derived from the [Ni3Fe4S] cubane component appear to dominate the reduced C* cluster MCD spectrum, while the presence of a fourth Fe center can be inferred from...

  • purification and characterization of membrane associated cooc protein and its functional role in the insertion of nickel into Carbon Monoxide Dehydrogenase from rhodospirillum rubrum
    Journal of Biological Chemistry, 2001
    Co-Authors: Won Bae Jeon, Jiujun Cheng, Paul W Ludden
    Abstract:

    Abstract The accessory protein CooC, which contains a nucleotide-binding domain (P-loop) near the N terminus, participates in the maturation of the nickel center of Carbon Monoxide Dehydrogenase (CODH). In this study, CooC was purified from the chromatophore membranes of Rhodospirillum rubrum with a 3,464-fold purification and a 0.8% recovery, and its biochemical properties were characterized. CooC is a homodimer with a molecular mass of 61–63 kDa, contains less than 0.1 atom of Ni2+ or Fe2+ per dimer, and has a λmax at 277.5 nm (e277.5 32.1 mm −1cm−1) with no absorption peaks at the visible region. CooC catalyzes the hydrolysis of ATP and GTP with K mvalues of 24.4 and 26.0 μm andV max values of 58.7 and 3.7 nmol/min/mg protein for ATP and GTP hydrolysis, respectively. The P-loop mutated form of K13Q CooC was generated by site-specific replacement of lysine by glutamine and was purified according to the protocol for wild-type CooC purification. The K13Q CooC was inactive both in ATP hydrolysis andin vivo nickel insertion. In vitro nickel activation of apoCODH in the cell extracts from UR2 (wild type) and UR871 (K13Q CooC) showed that activation of nickel-deficient CODH was enhanced by CooC and dependent upon ATP hydrolysis. The overall results suggest that CooC couples ATP hydrolysis with nickel insertion into apoCODH. On the basis of our results and models for analogous systems, the functional roles of CooC in nickel processing into the active site of CODH are presented.

Stephen W Ragsdale - One of the best experts on this subject based on the ideXlab platform.

  • crystallographic characterization of the Carbonylated a cluster in Carbon Monoxide Dehydrogenase acetyl coa synthase
    ACS Catalysis, 2020
    Co-Authors: Steven E Cohen, Mehmet Can, Elizabeth C Wittenborn, Rachel A Hendrickson, Stephen W Ragsdale, Catherine L Drennan
    Abstract:

    The Wood–Ljungdahl pathway allows for autotrophic bacterial growth on Carbon dioxide, with the last step in acetyl-CoA synthesis catalyzed by the bifunctional enzyme Carbon Monoxide Dehydrogenase/a...

  • dramatic conformational flexibility of Carbon Monoxide Dehydrogenase acetyl coa synthase revealed by electron microscopy
    The FASEB Journal, 2015
    Co-Authors: Edward J Brignole, Elizabeth C Wittenborn, Stephen W Ragsdale, Samuel Thompson, Catherine L Drennan
    Abstract:

    Carbon Monoxide Dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a 310-kDa bifunctional enzyme involved in anaerobic fixation of atmospheric Carbon (CO2) by acetogenic bacteria. The CODH subunit red...

  • efficient and clean photoreduction of co2 to co by enzyme modified tio2 nanoparticles using visible light
    Journal of the American Chemical Society, 2010
    Co-Authors: Thomas W Woolerton, Stephen W Ragsdale, Sally Sheard, Erwin Reisner, Elizabeth Pierce, Fraser A Armstrong
    Abstract:

    A hybrid enzyme−nanoparticle system is described for achieving clean reduction of CO2 to CO using visible light as the energy source. An aqueous dispersion of TiO2 nanoparticles modified by attachment of Carbon Monoxide Dehydrogenase (CODH) and a Ru photosensitizer produces CO at a rate of 250 μmol of CO (g of TiO2)−1 h−1 when illuminated with visible light at pH 6 and 20 °C.

  • 13c nmr characterization of an exchange reaction between co and co2 catalyzed by Carbon Monoxide Dehydrogenase
    Biochemistry, 2008
    Co-Authors: Javier Seravalli, Stephen W Ragsdale
    Abstract:

    Carbon Monoxide Dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel−iron−sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s−1 at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidatio...

  • pulse chase studies of the synthesis of acetyl coa by Carbon Monoxide Dehydrogenase acetyl coa synthase evidence for a random mechanism of methyl and Carbonyl addition
    Journal of Biological Chemistry, 2008
    Co-Authors: Javier Seravalli, Stephen W Ragsdale
    Abstract:

    Carbon Monoxide Dehydrogenase/acetyl-CoA synthase catalyzes acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic Carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the acetyl-CoA synthase active site. Furthermore, Carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulse-chase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60–70% of the CO employed in the pulse phase can be trapped in the product acetyl-CoA.

Paul A Lindahl - One of the best experts on this subject based on the ideXlab platform.

  • tunnel mutagenesis and ni dependent reduction and methylation of the α subunit of acetyl coenzyme a synthase Carbon Monoxide Dehydrogenase
    Journal of Biological Inorganic Chemistry, 2008
    Co-Authors: Xiangshi Tan, Paul A Lindahl
    Abstract:

    Two isolated alpha subunit mutants (A110C and A222L) of the alpha(2)beta(2) acetyl coenzyme A synthase (ACS)/Carbon Monoxide Dehydrogenase (CODH) from Moorella thermoacetica were designed to block the CO-migrating tunnel in the alpha subunit, allowing comparison with equivalent mutants in ACS/CODH. After Ni activation, both mutants exhibited electron paramagnetic resonance spectra indicating that the A-cluster was properly assembled. ACS activities were similar to those of the wild-type recombinant Ni-activated alpha subunit, suggesting that CO diffuses directly to the A-cluster from solvent rather than through the tunnel as is observed for the "majority" activity of ACS/CODH. Thus, CO appears to migrate to the A-cluster through two pathways, one involving and one not involving the tunnel. The kinetics and extent of reduction of the Fe(4)S(4) cubane in the apo-alpha subunit and the Ni-activated alpha subunit upon exposure to titanium(III) citrate were examined using the stopped-flow method. The extent of reduction was independent of Ni, whereas the kinetics of reduction was Ni-dependent. Apo-alpha subunit reduction was monophasic while Ni-activated alpha subunit reduction was biphasic, with the more rapid phase coincident with that of apo-alpha subunit reduction. Thus, binding of Ni to the A-cluster slows the reduction kinetics of the [Fe(4)S(4)](2+) cubane. An upper limit of two electrons per alpha subunit are transferred from titanium(III) citrate to the Ni subcomponent of the A-cluster during reductive activation. These electrons are accepted quickly relative to the reduction of the [Fe(4)S(4)](2+) cubane. This reduction is probably a prerequisite for methyl group transfer. CO appears to bind to reduced nonfunctional subunits, thereby inhibiting reduction (or promoting reoxidation) of the cubane subcomponent of the A-cluster.

  • nickel dependent oligomerization of the alpha subunit of acetyl coenzyme a synthase Carbon Monoxide Dehydrogenase
    Biochemistry, 2007
    Co-Authors: Ioannis Kagiampakis, Xiangshi Tan, Ivan V Surovtsev, Borries Demeler, Paul A Lindahl
    Abstract:

    After activation with NiCl2, the recombinant α subunit of the Ni-containing α2β2 acetyl-CoA synthase/Carbon Monoxide Dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The α subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-α contain only an Fe4S4 cluster. Ni-activated α subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of α subunits contained in ACS/CODH. Evidence presented here indicates that apo-α is a monomer whereas Ni-treated α oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-α was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-α was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/α and exhibited, upon tr...

  • function of the tunnel in acetylcoenzyme a synthase Carbon Monoxide Dehydrogenase
    Journal of Biological Inorganic Chemistry, 2006
    Co-Authors: Xiangshi Tan, Anne Volbeda, Juan C Fontecillacamps, Paul A Lindahl
    Abstract:

    Acetylcoenzyme A synthase/Carbon Monoxide Dehydrogenase (ACS/CODH) contains two Ni-Fe-S active-site clusters (called A and C) connected by a tunnel through which CO and CO2 migrate. Site-directed mutants A578C, L215F, and A219F were designed to block the tunnel at different points along the region between the two C-clusters. Two other mutant proteins F70W and N101Q were designed to block the region that connects the tunnel at the betabeta interface with a water channel also located at that interface. Purified mutant proteins were assayed for Ni/Fe content and examined by electron paramagnetic resonance spectroscopy. Analyses indicate that same metal clusters found in wild-type (WT) ACS/CODH (i.e., the A-, B-, C-, and probably D-clusters) are properly assembled in the mutant enzymes. Stopped-flow kinetics revealed that these centers in the mutants are rapidly reducible by dithionite but are only slowly reducible by CO, suggesting an impaired ability of CO to migrate through the tunnel to the C-cluster. Relative to the WT enzyme, mutant proteins exhibited little CODH or ACS activity (using CO2 as a substrate). Some ACS activity was observed when CO was a substrate, but not the cooperative CO inhibition effect characteristic of WT ACS/CODH. These results suggest that CO and CO2 enter and exit the enzyme at the water channel along the betabeta subunit interface. They also suggest two pathways for CO during synthesis of acetylcoenzyme A, including one in which CO enters the enzyme and migrates through the tunnel before binding at the A-cluster, and another in which CO binds the A-cluster directly from the solvent.

  • the tunnel of acetyl coenzyme a synthase Carbon Monoxide Dehydrogenase regulates delivery of co to the active site
    Journal of the American Chemical Society, 2005
    Co-Authors: Xiangshi Tan, Huaykeng Loke, Shawn B Fitch, Paul A Lindahl
    Abstract:

    The effect of [CO] on acetyl-CoA synthesis activity of the isolated α subunit of acetyl-coenzyme A synthase/Carbon Monoxide Dehydrogenase from Moorella thermoacetica was determined. In contrast to the complete α2β2 enzyme where multiple CO molecules exhibit strong cooperative inhibition, α was weakly inhibited, apparently by a single CO with KI = 1.5 ± 0.5 mM; other parameters include kcat = 11 ± 1 min-1 and KM = 30 ± 10 μM. The α subunit lacked the previously described “majority” activity of the complete enzyme but possessed its “residual” activity. The site affording cooperative inhibition may be absent or inoperative in isolated α subunits. Ni-activated α rapidly and reversibly accepted a methyl group from CH3−Co3+FeSP affording the equilibrium constant KMT = 10 ± 4, demonstrating the superior nucleophilicity of αred relative to Co1+FeSP. CO inhibited this reaction weakly (KI = 540 ± 190 μM). NiFeC EPR intensity of α developed in accordance with an apparent Kd = 30 μM, suggesting that the state exhibit...

  • inactivation of acetyl coa synthase Carbon Monoxide Dehydrogenase by copper
    Journal of the American Chemical Society, 2003
    Co-Authors: Matthew R Bramlett, Xiangshi Tan, Paul A Lindahl
    Abstract:

    Two recent crystal structures of acetyl-CoA synthase (ACS) from Moorella thermoacetica exhibited different metal contents and geometries at their active site, called the A-cluster. This led to the proposal of two catalytic mechanisms, one Ni-based, the other Cu-based. ACS was studied with respect to synthase activity, methyl group transfer activity, metal content, and EPR spectroscopy. Our results indicate that Cu is not required for catalysis and that it inactivates ACS by binding to the proximal site of the A-cluster. With Cu in this site, the A-cluster cannot accept a methyl group from the corrinoid-iron-sulfur protein, nor can it exhibit the NiFeC EPR signal after treatment with CO.

Young Min Kim - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of the genes encoding Carbon Monoxide Dehydrogenase in terrabacter carboxydivorans
    Research in Microbiology, 2017
    Co-Authors: Jae Ho Lee, Sae Woong Park, Young Min Kim
    Abstract:

    Terrabacter carboxydivorans is able to grow aerobically at low concentrations of Carbon Monoxide (CO) as a sole source of Carbon and energy. The genes for Carbon Monoxide Dehydrogenase (CO-DH) were cloned from T. carboxydivorans and analyzed. The operon encoding T. carboxydivorans CO-DH was composed of three structural genes with the transcriptional order of cutB, cutC and cutA, as well as an additional accessory gene (orf4). Phylogenetic analysis of CutA revealed that T. carboxydivorans CO-DH was classified into a group distinct from previously characterized CO-DHs. Expression of antisense RNA for the cutB or cutA gene in T. carboxydivorans led to a decrease in CO-DH activity, confirming that cutBCA genes are the functional genes encoding CO-DH. The CO-DH operon was expressed even in the absence of CO and further inducible by CO. In addition, CO-DH synthesis was increased in the stationary phase compared to the exponential phase during heterotrophic growth on glucose and glycerol. Point mutations of a partially inverted repeat sequence (TCGGA-N6-GCCCA) in the upstream region of the cutB gene almost abolished expression of the CO-DH operon, indicating that the inverted-repeat sequence might be a cis-acting regulatory site for the positive regulation of the CO-DH operon.

  • functional characterization of the cuti gene for the transcription of Carbon Monoxide Dehydrogenase genes in mycobacterium sp strain jc1 dsm 3803
    Journal of Microbiology, 2017
    Co-Authors: Jae Ho Lee, Sae Woong Park, Young Min Kim
    Abstract:

    Carbon Monoxide Dehydrogenase (CO-DH) in Mycobacterium sp. strain JC1 is a key enzyme for the carboxydotrophic growth, when Carbon Monoxide (CO) is supplied as a sole source of Carbon and energy. This enzyme is also known to act as nitric oxide Dehydrogenase (NO-DH) for the detoxification of NO. Several accessory genes such as cutD, cutE, cutF, cutG, cutH, and cutI, are clustered together with two copies of the CO-DH structural genes (cutB1C1A1 and cutB2C2A2) in Mycobacterium sp. strain JC1 and are well conserved in carboxydotrophic mycobacteria. Transcription of the CO-DH structural and accessory genes was demonstrated to be increased significantly by acidified sodium nitrate as a source of NO. A cutI deletion (ΔcutI) mutant of Mycobacterium sp. strain JC1 was generated to identity the function of CutI. Lithoautotrophic growth of the ΔcutI mutant was severely affected in mineral medium supplemented with CO, while the mutant grew normally with glucose. Western blotting, CO-DH activity staining, and CO-DH-specific enzyme assay revealed a significant decrease in the cellular level of CO-DH in the ΔcutI mutant. Northern blot analysis and promoter assay showed that expression of the cutB1 and cutB2 genes was significantly reduced at the transcriptional level in the ΔcutI mutant, compared to that of the wildtype strain. The ΔcutI mutant was much more susceptible to NO than was the wild type.

  • Carbon Monoxide Dehydrogenase in mycobacteria possesses a nitric oxide Dehydrogenase activity
    Biochemical and Biophysical Research Communications, 2007
    Co-Authors: Sae Woong Park, Taeksun Song, Seo Young Kim, Eungbin Kim, Chi Yong Eom, Young Min Kim
    Abstract:

    Abstract CO Dehydrogenase (CO-DH) catalyzes the oxidation of CO to CO 2 in carboxydobacteria. Cell-free extracts prepared from several mycobacteria, including Mycobacterium tuberculosis H37Ra, showed NO Dehydrogenase (NO-DH) activity in a reaction mixture containing sodium nitroprusside (SNP) as the source of NO. The association of the NO-DH activity with CO-DH was revealed by activity staining and confirmed by enzyme assay with purified CO-DH from Mycobacterium sp. strain JC1, a carboxydotrophic mycobacterium. SNP stimulated the production of CO-DH with a coincidental increase in NO-DH activity in the bacterium, further supporting this association and implying the existence of a possible SNP-induced CO-DH gene expression. The addition of purified CO-DH to cultures of Escherichia coli revealed that the enzyme protected E. coli from SNP-induced killing in a dose-dependant way. The present results indicate that mycobacterial CO-DH also acts as a NO-DH, which may function in the protection of mycobacterial pathogens from nitrosative stress during infection.

Sae Woong Park - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of the genes encoding Carbon Monoxide Dehydrogenase in terrabacter carboxydivorans
    Research in Microbiology, 2017
    Co-Authors: Jae Ho Lee, Sae Woong Park, Young Min Kim
    Abstract:

    Terrabacter carboxydivorans is able to grow aerobically at low concentrations of Carbon Monoxide (CO) as a sole source of Carbon and energy. The genes for Carbon Monoxide Dehydrogenase (CO-DH) were cloned from T. carboxydivorans and analyzed. The operon encoding T. carboxydivorans CO-DH was composed of three structural genes with the transcriptional order of cutB, cutC and cutA, as well as an additional accessory gene (orf4). Phylogenetic analysis of CutA revealed that T. carboxydivorans CO-DH was classified into a group distinct from previously characterized CO-DHs. Expression of antisense RNA for the cutB or cutA gene in T. carboxydivorans led to a decrease in CO-DH activity, confirming that cutBCA genes are the functional genes encoding CO-DH. The CO-DH operon was expressed even in the absence of CO and further inducible by CO. In addition, CO-DH synthesis was increased in the stationary phase compared to the exponential phase during heterotrophic growth on glucose and glycerol. Point mutations of a partially inverted repeat sequence (TCGGA-N6-GCCCA) in the upstream region of the cutB gene almost abolished expression of the CO-DH operon, indicating that the inverted-repeat sequence might be a cis-acting regulatory site for the positive regulation of the CO-DH operon.

  • functional characterization of the cuti gene for the transcription of Carbon Monoxide Dehydrogenase genes in mycobacterium sp strain jc1 dsm 3803
    Journal of Microbiology, 2017
    Co-Authors: Jae Ho Lee, Sae Woong Park, Young Min Kim
    Abstract:

    Carbon Monoxide Dehydrogenase (CO-DH) in Mycobacterium sp. strain JC1 is a key enzyme for the carboxydotrophic growth, when Carbon Monoxide (CO) is supplied as a sole source of Carbon and energy. This enzyme is also known to act as nitric oxide Dehydrogenase (NO-DH) for the detoxification of NO. Several accessory genes such as cutD, cutE, cutF, cutG, cutH, and cutI, are clustered together with two copies of the CO-DH structural genes (cutB1C1A1 and cutB2C2A2) in Mycobacterium sp. strain JC1 and are well conserved in carboxydotrophic mycobacteria. Transcription of the CO-DH structural and accessory genes was demonstrated to be increased significantly by acidified sodium nitrate as a source of NO. A cutI deletion (ΔcutI) mutant of Mycobacterium sp. strain JC1 was generated to identity the function of CutI. Lithoautotrophic growth of the ΔcutI mutant was severely affected in mineral medium supplemented with CO, while the mutant grew normally with glucose. Western blotting, CO-DH activity staining, and CO-DH-specific enzyme assay revealed a significant decrease in the cellular level of CO-DH in the ΔcutI mutant. Northern blot analysis and promoter assay showed that expression of the cutB1 and cutB2 genes was significantly reduced at the transcriptional level in the ΔcutI mutant, compared to that of the wildtype strain. The ΔcutI mutant was much more susceptible to NO than was the wild type.

  • cloning and expression analysis of the duplicated genes for Carbon Monoxide Dehydrogenase of mycobacterium sp strain jc1 dsm 3803
    Microbiology, 2010
    Co-Authors: Taeksun Song, Sae Woong Park, Sujeong Park, Ji Hyang Kim, Young Man Kim
    Abstract:

    Carbon Monoxide Dehydrogenase (CO-DH) is an enzyme catalysing the oxidation of CO to Carbon dioxide in Mycobacterium sp. strain JC1 DSM 3803. Cloning of the genes encoding CO-DH from the bacterium and sequencing of overlapping clones revealed the presence of duplicated sets of genes for three subunits of the enzyme, cutB1C1A1 and cutB2C2A2, in operons, and a cluster of genes encoding proteins that may be involved in CO metabolism, including a possible transcriptional regulator. Phylogenetic analysis based on the amino acid sequences of large subunits of CO-DH suggested that the CO-DHs of Mycobacterium sp. JC1 and other mycobacteria are distinct from those of other types of bacteria. The growth phenotype of mutant strains lacking cutA genes and of a corresponding complemented strain showed that both of the duplicated sets of CO-DH genes were functional in this bacterium. Transcriptional fusions of the cutB genes with lacZ revealed that the cutBCA operons were expressed regardless of the presence of CO and were further inducible by CO. Primer extension analysis indicated two promoters, one expressed in the absence of CO and the other induced in the presence of CO. This is believed to be the first report to show the presence of multiple copies of CO-DH genes with identical sequences and in close proximity in carboxydobacteria, and to present the genetic evidence for the function of the genes in mycobacteria.

  • Carbon Monoxide Dehydrogenase in mycobacteria possesses a nitric oxide Dehydrogenase activity
    Biochemical and Biophysical Research Communications, 2007
    Co-Authors: Sae Woong Park, Taeksun Song, Seo Young Kim, Eungbin Kim, Chi Yong Eom, Young Min Kim
    Abstract:

    Abstract CO Dehydrogenase (CO-DH) catalyzes the oxidation of CO to CO 2 in carboxydobacteria. Cell-free extracts prepared from several mycobacteria, including Mycobacterium tuberculosis H37Ra, showed NO Dehydrogenase (NO-DH) activity in a reaction mixture containing sodium nitroprusside (SNP) as the source of NO. The association of the NO-DH activity with CO-DH was revealed by activity staining and confirmed by enzyme assay with purified CO-DH from Mycobacterium sp. strain JC1, a carboxydotrophic mycobacterium. SNP stimulated the production of CO-DH with a coincidental increase in NO-DH activity in the bacterium, further supporting this association and implying the existence of a possible SNP-induced CO-DH gene expression. The addition of purified CO-DH to cultures of Escherichia coli revealed that the enzyme protected E. coli from SNP-induced killing in a dose-dependant way. The present results indicate that mycobacterial CO-DH also acts as a NO-DH, which may function in the protection of mycobacterial pathogens from nitrosative stress during infection.