Characterization of the cdhD and cdhE genes encoding subunits of the corrinoid/iron-sulfur enzyme of the CO dehydrogenase complex from Methanosarcina thermophila.
The CO dehydrogenase enzyme complex from Methanosarcina thermophila contains a corrinoid/iron-sulfur enzyme composed of two subunits (delta and gamma). The cdhD and cdhE genes, which encode the delta and gamma subunits, respectively, were cloned and sequenced. The cdhD gene is upstream of and separated by 3 bp from cdhE. Both genes are preceded by apparent ribosome-binding sites. Northern (RNA) blot and primer extension analyses indicated that cdhD and cdhE are cotranscribed from a promoter located several kilobases upstream of cdhD. The putative CdhD and CdhE sequences are 37% identical to the sequences deduced from the genes encoding the beta and alpha subunits of the corrinoid/iron-sulfur enzyme from Clostridium thermoaceticum. The CdhE sequence had a four-cysteine motif with the potential to bind a 4Fe-4S cluster previously identified in the corrinoid/iron-sulfur enzyme by electron paramagnetic resonance spectroscopy. A T7 RNA polymerase/promoter system was used to produce CdhD and CdhE independently in Escherichia coli. The purified CdhD protein was reconstituted with hydroxocobalamin in the base-on configuration. The purified CdhE protein exhibited an Fe-S center and base-off cobalamin binding in which the benzimidazole base nitrogen atom was no longer a lower axial ligand to the cobalt atom.
Maupin-Furlow
J
J
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802-4500, USA.
Ferry
J G
JG
D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D045728:Corrinoids; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006879:Hydroxocobalamin / Q000378:metabolism; D002479:Inclusion Bodies; D017020:Methanosarcina / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D011166:Porphyrins / Q000378:metabolism; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D045728:Corrinoids; D009097:Multienzyme Complexes; D011166:Porphyrins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C097641:SlpM protein, Thermus thermophilus; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D006879:Hydroxocobalamin