The DNA sequence and derived amino-acid sequence of a 5618-base region in the 74-min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH-dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG.Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino-acid sequence with those of database proteins. A directly repeated sequence, which includes the motif -Cys-Xaa-Xaa-Cys-, is suggested as the binding site for either oneThe nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino-acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33-amino-acid peptide of unknown function which includes 8 phenylalanyl residues.The product of the nirC gene is a highly hydrophobic protein with regions of amino-acid sequence similar to cytochrome oxidase polypeptide 1.Two genes essential for the major NADH-dependent nitrite reductase activity are located in the 74-min region of the Escherichiu coli K-12 chromosome [l]. They are nirB, the structural gene for the nitrite reductase apoprotein, and cysG, which is required for sirohaem synthesis. The prosthetic groups of nitrite reductase are FAD, an iron-sulphur cluster and sirohaem which is also found in the NADPH-dependent sulphite reductase. Mutants defective in the cysG gene are unable to grow without cysteine, or to reduce nitrite rapidly to ammonia [2,. The cysG product has recently been purified and characterized (C. Roessner, personal communication). It catalyses two methylation reactions in the conversion of uroporphyrinogen I11 into sirohaem.We have previously located the promoter, transcription and translation start points of the nirB gene and reported the DNA sequence and derived amino-acid sequence of the first 89 bases of nirB and its 5'-regulatory region [4]. We now report Correspondence to J. A. Cole,

A new activity of Escherichia coli and yeast phenylalanyl-tRNA synthetases, the conversion adenosine 5' -triphosphate into diadenosine 5' ,5"' -P(1) ,P(4) -tetraphosphate, is reported. This activity is followed by (31)P NMR and chromatography on poly(ethylenimine)-cellulose. It is revealed by the addition of ZnCl2 to a reaction mixture containing the enzyme, ATP-Mg(2+), L-phenylalanine, and pyrophosphatase It reflects the reaction enzyme-bound phenylalanyl adenylate with ATP instead of PPi and strongly depends on the hydrolysis of pyrophosphate in the assay medium. The zinc dependence of this reaction parallels that of the inhibition of tRNA(phe) aminoacylation which is described in the accompanying paper [Mayaux, J. F., & Blanquet, S. (1981) Biochemistry (preceding paper in this issue)]. In the presence of an unlimiting pyrophosphatase activity, diadenosine tetraphosphate synthesis by E. coli and yeast phenylalanyl-tRNA synthetases occurs at maximal rates of 0.5 and 2 s-1, respectively (37 degrees C, pH 7.8, 150 mM KC1, 5 mM ATP, 10 mM MgCl2, 2 mM L-phenylalanine, and 80 muM ZnCl2). Under identical experimental conditions, E coli isoleucyl-, methionyl-, and tyrosyl-tRNA synthetases produce small amounts of diadenosine tetraphosphate at rates 2 or 3 orders of magnitude lower than that achieved by phenylalanyl-tRNA synthetase. In the case of E. coli phenylalanyl-tRNA synthetase, it is shown that the diadenosine tetraphosphate synthetase activity is accompanied by a diadenosinetetraphosphatase activity. This activity, actually supported by phenylalanyl-tRNA synthetase, is responsible for the appearance of ADP in the assay medium. It requires also the presence of both ZnCl2 and L-phenylalanine. The formation of ADP from diadenosine tetraphosphate and its reaction with enzyme-bound aminoacyl adenylate account for the appearance in the reaction mixture of diadenosine 5' ,5"' -P(1) ,P(3)-triphosphate, after that of diadenosine tetraphosphate. The significance of these findings in the context of the role of diadenosine tetraphosphate in controlling cellular growth is discussed.

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...

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