Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB–cysG region of the Escherichia coli K‐12 chromosome

Abstract

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,

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

“…The SUMT internal amino acid sequence, obtained after trypsin digestion, corresponds in Fig. 5 (32), which has been shown to have SUMT activity (32,44). Sequences homologies between these four proteins are reported in Fig.…”

The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

“…The localization of NAR1 in the chloroplast membranes is supported by DNA sequencing analysis, which predicts a protein with six transmembranespanning segments containing a typical chloroplast signal peptide, and the immunodetection of NAR1 in the chloroplast membranes. The predicted amino acid sequence for NAR1 showed a significant homology with the bacterial integral membrane proteins FOCA (Suppmann and Sawers, 1994) and NIRC (Peakman et al, 1990) and with an expressed sequence tag from Arabidopsis of unknown function (Newman et al, 1994). FOCA is proposed to be a formate transporter, and in fact, FOCA mutants have been shown to be defective in formate transport (Suppmann and Sawers, 1994).…”

“…These transit peptide sequences, which are structurally different from those of imported stromal or thylakoid proteins, consist of ‫ف‬ 70 to 90 amino acids, with a slight conservation among them, and the processed mature proteins have an N-terminal alanine (Brink et al, 1995;Weber et al, 1995;Kammerer et al, 1998). The putative mature NAR1 protein (263 amino acids) shows a high identity with putative formate transporters from Escherichia coli (32%) (GenBank accession number P21501; Suppmann and Sawers, 1994) and Methanobacterium phormicium (31%) (GenBank accession number P35839; White and Ferry, 1992) as well as with putative nitrite transporters NIRC from E. coli (26%) (GenBank accession number P11097; Peakman et al, 1990) and Salmonella typhimurium (28%) (GenBank accession number P25926; Wu et al, 1991). A partial expressed sequence tag from Arabidopsis, with unknown function, showed some identity to NAR1 (30%) (GenBank accession number N37972; Newman et al, 1994).…”

The formate channel FocA exports the products of mixed-acid fermentation

“…NirC imports nitrite, NO 2 -, into the cytoplasm. It is commonly found in an operon with the cytoplasmic, assimilatory nitrite reductase NirBD (6) that produces ammonium, NH 4 + , for subsequent assimilation into biomolecules. Enteric bacteria such as Escherichia coli or Salmonella typhimurium also follow this route for the detoxification of peroxynitrite, produced by the inducible NO synthase in macrophages of their eukaryotic hosts as an aspect of the innate immune response (7,8).…”