Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase 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

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.The cobalamin biosynthetic pathway probably involves 20 to 30 different enzymatic steps, consisting of (i) formation of uroporphyrinogen III (urogen 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 (for reviews on cobalamin synthesis, see references 3, 4, 18, 28, and 39). Only one enzymatic activity involved in the transformation of urogen III to coenzyme B12 has been purified (7), and no biosynthetic intermediate has been purified along the precorrin-3-to-cobyrinic-acid pathway. 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. These genes should enable the identification of enzymatic activities and biosynthetic intermediates of the pathway and facilitate the understanding of the nature of biochemical and genetic regulation mechanisms operative in the cob regulon.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. The other two complement Cob mutants blocked in the conversion of cobinamide into cobalamin and * Corresponding author. are implicated in the last four steps of the cobalamin biosynthetic pathway (18). All 14 cloned genes are grouped on the P. denitrificans genome in four genomic regions, corre. sponding to complementation groups A, B, C, and D (9). In contrast, most of the cob genes in Salmonella typhimuriumand Bacillus megaterium are clustered (23,24, 45). We report the genetic analysis and nucleotide sequence of a 5.4-kilobase-pair (kb) fragment from complementation group C. Part of this fragment is...

Homology of pyridoxal‐5′‐phosphate‐dependent aminotransferases with the cobC (cobalamin synthesis), nifS (nitrogen fixation), pabC (p‐aminobenzoate synthesis) and malY (abolishing endogenous induction of the maltose system) gene products

“…The cobC gene product has been reported to participate with the cobD gene product in the transformation of cobyrinic acid to cobinamide, a segment of the biosynthetic pathway leading to cobalamine (Crouzet et al, 1990). Further details about its function are not known.…”

“…It measures the structural relatedness Crouzet et al, 1990Reidl and Boos, 1991Evans et al, 1991Jacobson et al, 1989Mulligan and Haselkorn, 1989Beynon et al, 1988Slock et al, 1990 Table 2. Reciprocal proNe analysis of COW, pabC, nifl and maEY proteins and animotransferase subgroups.…”

The CobII and CobIII regions of the cobalamin (vitamin B12) biosynthetic operon of Salmonella typhimurium

“…Genetic studies indicate that the CobII region is composed of four complementation groups. Correlation of the genetic map with available DNA sequence data for the CobI and CobIII regions of the operon suggests that three open reading frames are included in the CobIII region (5,(7)(8)(9)11).…”

Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii

“…We described the purification of Pseudomonas denitrificans S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) (6); the cloning of its structural gene, named cobA (8); and its nucleotide sequence (13). P. denitrificans SUMT exhibits two properties which have been suggested to play a regulatory role in cobalamin biosynthesis (6).…”