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Comparative Study
. 1999 Sep;181(18):5581-90.
doi: 10.1128/JB.181.18.5581-5590.1999.

Cloning and molecular characterization of the genes for carbon monoxide dehydrogenase and localization of molybdopterin, flavin adenine dinucleotide, and iron-sulfur centers in the enzyme of Hydrogenophaga pseudoflava

B S Kang  1 , Y M Kim
Affiliations
Comparative Study

Cloning and molecular characterization of the genes for carbon monoxide dehydrogenase and localization of molybdopterin, flavin adenine dinucleotide, and iron-sulfur centers in the enzyme of Hydrogenophaga pseudoflava

B S Kang et al. J Bacteriol. 1999 Sep.

Abstract

Carbon monoxide dehydrogenases (CO-DH) are the enzymes responsible for the oxidation of CO to carbon dioxide in carboxydobacteria and consist of three nonidentical subunits containing molybdopterin flavin adenine dinucleotide (FAD), and two different iron-sulfur clusters (O. Meyer, K. Frunzke, D. Gadkari, S. Jacobitz, I. Hugendieck, and M. Kraut, FEMS Microbiol. Rev. 87:253-260, 1990). The three structural genes of CO-DH in Hydrogenophaga pseudoflava were cloned and characterized. The genes were clustered on the chromosome in the transcriptional order cutM-cutS-cutL. The cloned cutM, cutS, and cutL genes had open reading frames of 864, 492, and 2,412 nucleotides, coding for proteins with calculated molecular weights of 30,694, 17,752, and 87,224, respectively. The overall identities in the nucleotide sequence of the genes and the amino acid sequence of the subunits with those of other carboxydobacteria were 64.5 to 74.3% and 62.8 to 72.3%, respectively. Primer extension analysis revealed that the transcriptional start site of the genes was the nucleotide G located 47 bp upstream of the cutM start codon. The deduced amino acid sequences of the three subunits of CO-DH implied the presence of molybdenum cofactor, FAD, and iron-sulfur centers in CutL, CutM, and CutS, respectively. Fluorometric analysis coupled with denaturing polyacrylamide gel electrophoresis of fractions from hydroxyapatite column chromatography in the presence of 8 M urea of active CO-DH and from gel filtration of spontaneously inactivated enzyme revealed that the large and medium subunits of CO-DH in H. pseudoflava bind molybdopterin and FAD cofactors, respectively. Iron-sulfur centers of the enzyme were identified to be present in the small subunit on the basis of the iron content in each subunit eluted from the denaturing polyacrylamide gels.

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Figures

FIG. 1
FIG. 1
Southern hybridization of H. pseudoflava DNA. Southern hybridization was performed with random primed probes synthesized by using a 0.5-kb PCR product covering the CO-DH small subunit as a template. Shown are gels subjected to agarose gel electrophoresis (A) and Southern hybridization with random primed probes (B). H. pseudoflava chromosomal and plasmid DNAs were digested with HindIII (H), EcoRI (E), BamHI (B), SalI (S), and SmaI (M). λ DNA digested with HindIII (λ) was used as a size marker.
FIG. 2
FIG. 2
Restriction map of a 17-kb insert DNA in lambda clone 311 containing H. pseudoflava CO-DH genes. EcoRI (E), SmaI (M), and SalI (S) were used for restriction mapping. The probe binding site, a 1.5-kb SalI fragment in pBKS2, and a 3.5-kb SmaI fragment in pBKX1 are indicated under the map. ORFs for large (cutL), medium (cutM), and small (cutS) subunits of CO-DH are shown by arrows.
FIG. 3
FIG. 3
Identification of transcriptional start site of cut genes. The transcriptional start of cut genes was identified by primer extension mapping using a 32P-labeled 30-mer oligonucleotide primer which is complementary to codons 7 to 16 of the HpsCut medium subunit. Extension products (lane P) were analyzed in parallel with the sequencing ladder (lanes G, A, T, and C) primed with the same primer. The asterisk shows the transcriptional start site.
FIG. 4
FIG. 4
Comparison of amino acid sequence of the HpsCut large subunit with those of other molybdopterin-containing subunits and domains. The sequences compared are those of large subunits of HpsCut (HpsCutL), PthCut (PthCutA) (34), OcaCox (OcaCoxL) (40), AniNdh (AniNdhC) (9), PpuQor (PpuQorL) (3), and RpaHba (RpaHbaC) (8) and molybdopterin-containing domains of DmeXdh (DmeXdhL) (17) and DgiMop (DgiMopM) (41). Conserved residues are indicated by a capital letter (identical) or an asterisk (similar). Marked amino acids (▾) are those changed in the DmeXdh large domain from rosy mutants of D. melanogaster (14). Five molybdopterin contacting regions in the DgiMop molybdopterin-containing domain are boxed. Sequences were aligned by using the PCGENE program.
FIG. 5
FIG. 5
Comparison of amino acid sequence of the HpsCut medium subunit with those of other medium subunits of FAD-containing proteins. The sequences compared are those of medium subunits of HpsCut (HpsCutM), PthCut (PthCutB) (34), OcaCox (OcaCoxM) (40), AniNdh (AniNdhA) (9), and PpuQor (PpuQorM) (3). Conserved residues are indicated by a capital letter (identical) or an asterisk (similar). Marked amino acids (▾) are those changed in DmeXdh medium domain from rosy mutants of D. melanogaster (DmeXdh) (14). Sequences were aligned by using the PCGENE program.
FIG. 6
FIG. 6
Comparison of amino acid sequence of the HpsCut small subunit with other [Fe-S]-containing subunits and domains. The sequences compared are those of small subunits of HpsCut (HpsCutS), PthCut (PthCutC) (34), OcaCox (OcaCoxS) (40), AniNdh (AniNdhB) (9), PpuQor (PpuQorS) (3), and RpaHba (RpaHbaB) (8) and iron-sulfur center-containing domains of DmeXdh (DmeXdhS) (17) and DgiMop (DgiMopF) (41). Conserved residues are indicated by a capital letter (identical) or an asterisk (similar). Cysteines bound to Fe-S clusters are shown as white letters on a black background. Marked amino acids (▾) are those changed in the DmeXdh small domain from rosy mutants of D. melanogaster (14). Sequences were aligned by using the PCGENE program.
FIG. 7
FIG. 7
(A) Hydroxyapatite column chromatography of active CO-DH. Active purified CO-DH was applied to a hydroxyapatite column (1 by 3 cm) and eluted successively with standard buffer, standard buffer containing 8 M urea, 8 M urea-containing 50 mM phosphate buffer, and 8 M urea-containing 500 mM phosphate buffer under 15 cm of hydrostatic pressure as described in Materials and Methods. Protein was monitored by absorbance at 280 nm (–––). Cofactors were analyzed by fluorescence of emission at 460 nm after excitation at 360 nm and emission at 525 nm after excitation at 370 nm for molybdopterin (○) and FAD (●), respectively. (B) CO-DH subunits in fractions from hydroxyapatite chromatography. Denaturing PAGE of CO-DH subunits present in fractions obtained from hydroxyapatite chromatography was carried out on a 12.5% polyacrylamide gel in the presence of 0.1% SDS following the method of Laemmli (24). Twenty microliters each of several fractions eluted with standard buffer (fractions 3 and 4), standard buffer containing 8 M urea (fractions 9, 10, and 11), 50 mM phosphate buffer containing 8 M urea (fractions 18, 19, and 20), and 500 mM phosphate buffer containing 8 M urea (fractions 25 and 26) were analyzed together with active purified enzyme. Arrows indicate large (L), medium (M), and small (S) subunits of CO-DH.
FIG. 8
FIG. 8
(A) Elution pattern of inactive CO-DH on a Sephacryl S-300 column. Spontaneously inactivated purified CO-DH was subjected to gel filtration on a Sephacryl S-300 column (1.6 by 100 cm) with standard buffer as described in Materials and Methods. Protein and FAD were monitored by absorbance at 280 nm (–––) and fluorescence of emission at 525 nm after excitaion at 370 nm (●), respectively. (B) Denaturing PAGE of fractions from Sephacryl S-300 column chromatography. CO-DH subunits in 20 μl each of fractions 18, 20, 22, 24, 26, 28, 30, and 32 from Sephacryl S-300 chromatography of the inactivated CO-DH were subjected to denaturing PAGE together with inactive purified enzyme (7 μg) (P) on a 12.5% polyacrylamide gel in the presence of 0.1% SDS by the method of Laemmli (24). The large (L), medium (M), and small (S) subunits of CO-DH are indicated by arrows.
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