Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene.
Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-3H]S-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr congruent to 27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr congruent to 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 +/- 0.2 microM for precorrin-6x and 23.5 +/- 3.5 microM for NADPH and a Vmax value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand.
Blanche
F
F
Département Analyse, Centre de Recherche de Vitry-Alfortville, Vitry-sur-Seine, France.
Thibaut
D
D
Famechon
A
A
Debussche
L
L
Cameron
B
B
Crouzet
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D005798:Genes, Bacterial / Q000235:genetics*; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D014578:Uroporphyrins / Q000737:chemistry* / Q000378:metabolism; D014805:Vitamin B 12 / Q000096:biosynthesis
D014578:Uroporphyrins; C066509:precorrin 6x; C072473:precorrin 6y; D009249:NADP; D010088:Oxidoreductases; C072472:precorrin 6x oxidoreductase; D014805:Vitamin B 12