Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii.
An S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) activity has been identified in Methanobacterium ivanovii and was purified 4,500-fold to homogeneity with a 38% yield. The enzyme had an apparent molecular weight of 58,200 by gel filtration and consisted of two identical subunits of Mr 29,000, as estimated by gel electrophoresis under denaturing conditions. The Km value for uroporphyrinogen III was 52 nM. The enzyme catalyzed the two C-2 and C-7 methylation reactions converting uroporphyrinogen III into precorrin-2. Unlike Pseudomonas denitrificans SUMT, the only SUMT characterized to date (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet and B. Cameron, J. Bacteriol. 171:4222-4231, 1989), M. ivanovii SUMT did not show substrate inhibition at uroporphyrinogen III concentrations of up to 20 microM. Oligonucleotide probes from limited peptide sequence information were used to clone the corresponding gene. The encoded polypeptide showed more than 40% strict homology with P. denitrificans SUMT. The M. ivanovii SUMT structural gene is likely to be, as is P. denitrificans cobA, involved in corrinoid synthesis.
Blanche
F
F
Département de Chimie Analytique, Rhône Poulenc Rorer S.A., Vitry sur Seine, France.
Robin
C
C
Couder
M
M
Faucher
D
D
Cauchois
L
L
Cameron
B
B
Crouzet
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D019605:Euryarchaeota / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry
D001426:Bacterial Proteins; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase