Abstract
A 5.4-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment carries five different cob genes (cobA to cobE). Four of these genes present the characteristics of translationally coupled genes. cobA has been identified as the structural gene of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) because the encoded protein has the same NH2 terminus and molecular weight as those determined for the purified SUMT. For the same reasons the cobB gene was shown to be the structural gene for cobyrinic acid a,c-diamide synthase. Genetic and biochemical data concerning cobC and cobD mutants suggest that the products of these genes are involved in the conversion of cobyric acid to cobinamide.
Highlights
DNA Fragment Containing Five cob Genes and Identification of Structural Genes Encoding S-Adenosyl-L-Methionine: Uroporphyrinogen III Methyltransferase and Cobyrinic Acid a,c-Diamide Synthase
Four of these genes present the characteristics of translationally coupled genes. cobA has been identified as the structural gene of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) because the encoded protein has the same NH2 terminus and molecular weight as those determined for the purified SUMT
The cobalamin biosynthetic pathway probably involves 20 to 30 different enzymatic steps, consisting of (i) formation of uroporphyrinogen III, which is the common intermediate for the synthesis of hemes, chlorophylls, cobalamins, F430, and sirohemes; (ii) conversion of urogen III into cobyrinic acid, including the methylations at C-1, C-2, C-5, C-7, C-12, C-15, C-17, and C-20, the decarboxylation of the acetic side chain at C-12, the loss of C-20, and the introduction of cobalt; (iii) formation of cobinamide from cobyrinic acid by amidation of six of seven peripheral carboxylic groups, the seventh being amidated by (R)-1-amino-2-propanol; (iv) conversion of cobinamide into coenzyme B12
Summary
DNA Fragment Containing Five cob Genes and Identification of Structural Genes Encoding S-Adenosyl-L-Methionine: Uroporphyrinogen III Methyltransferase and Cobyrinic Acid a,c-Diamide Synthase. Cloned genes involved in cobalamin synthesis (cob genes) are valuable tools for the study of the coenzyme B12 biosynthesis at the biochemical and genetic levels. We have reported the cloning of at least 14 different genes, involved in cobalamin biosynthesis in Pseudomonas denitrificans, based on complementation data [9] Of these 14 genes, 12 are involved in the transformation of urogen III into cobinamide. Cobalamin is synthesized by a large number of bacteria, the cloning of cob genes has so far been reported for three organisms-P. denitrificans [9], B. megaterium [8], and S. typhimurium [1] This is the first report of a genetic analysis of cob genes at the molecular level
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