Cloning, sequencing and immunological characterization of the corrinoid-containing subunit of the N5-methyltetrahydromethanopterin: coenzyme-M methyltransferase from Methanobacterium thermoautotrophicum.
A 3.5-kb EcoRI fragment of the Methanobacterium thermoautotrophicum chromosome contains five open reading frames, mtrA to mtrE. The deduced N-terminal amino acid sequence of mtrA is identical with 26 N-terminal amino acids of a corrinoid-containing membrane protein from Methanobacterium. Computer-aided analyses of mtrA predicts 237 amino acids with a molecular mass of 25,603 Da for its gene product. A hydropathy plot of this amino acid sequence indicates one hydrophobic helical conformation near the N-terminus of the peptide which represents a tentative membrane-spanning region. The main part of the protein, however, shows hydrophilic domains, suggesting a location outside the cytoplasmic membrane. These domains are probably accessible by monospecific polyclonal antibodies raised previously against the corrinoid-containing membrane protein. The immunogold-labeling technique revealed that the corrinoid-dependent membrane protein was detectable at the cytoplasmic face of the membranes and of vesicle preparations. No significant identity of the deduced amino acid sequence was found with sequences of several corrinoid-containing enzymes. In contrast to the hydrophilic gene product of mtrA, four other gene products from the gene cluster encode extremely hydrophobic proteins. The N-terminal sequences of mtrC and mtrD are identical with two peptides of the N5-methyltetrahydromethanopterin:coenzyme-M methyltransferase complex from Methanobacterium, indicating that the mtr genes encode this membrane protein.
Stupperich
E
E
Institut für Mikrobiologie, Göttingen, Germany.
Juza
A
A
Hoppert
M
M
Mayer
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D045728:Corrinoids; D003545:Cysteine / Q000032:analysis; D015246:Deoxyribonuclease EcoRI; D005798:Genes, Bacterial; D017014:Methanobacterium / Q000201:enzymology* / Q000235:genetics; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017433:Protein Structure, Secondary; D014805:Vitamin B 12 / Q000032:analysis*
D045728:Corrinoids; D008780:Methyltransferases; C048755:tetrahydromethanopterin S-methyltransferase; D015246:Deoxyribonuclease EcoRI; D003545:Cysteine; D014805:Vitamin B 12