Purification and properties of the formate dehydrogenase and characterization of the fdhA gene of Sulfurospirillum multivorans.
The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene ( fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe(4)/S(4) cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.
Schmitz
Roland P H
RP
Institut für Mikrobiologie, FSU Jena, Philosophenweg 12, Lehrstuhl für Angewandte und Okologische Mikrobiologie, 07743 Jena, Germany. roland.schmitz@uni-jena.de
Diekert
Gabriele
G
D000595:Amino Acid Sequence; D002845:Chromatography; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004795:Enzyme Stability; D020565:Epsilonproteobacteria / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D006150:Guanine Nucleotides / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010269:Paraquat / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011622:Pterins / Q000032:analysis; D017279:Selenocysteine / Q000032:analysis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005561:Formates; D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011622:Pterins; C408585:twin-arginine translocase complex, E coli; D017279:Selenocysteine; D009243:NAD; C030544:formic acid; C063761:molybdopterin guanine dinucleotide; D009249:NADP; D005560:Formate Dehydrogenases; D010269:Paraquat