The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5], is cumulatively repressed by arginine and the pyrimidines. We describe the structure of the control region of carAB and the sequence of the carA gene. Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites. The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine. Attenuation control does not appear to be involved in the expression of carAB. A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed. The translational start of carA is atypical; it consists of a UUG or AUU codon.

Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. Thiobacillus thioparus strain THI115 can grow on thiocyanate (SCN(-)) as its sole energy source. Previously, we showed that SCN(-) is first converted to COS by thiocyanate hydrolase in T. thioparus strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H2S and CO2, the second step of SCN(-) assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the β-class carbonic anhydrases (β-CAs). Although the crystal structure including the catalytic site resembles those of the β-CAs, CO2 hydration activity of COSase is negligible compared to those of the β-CAs. The α5 helix and the extra loop (Gly150-Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The k(cat)/K(m) value, 9.6 × 10(5) s(-1) M(-1), is comparable to those of the β-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, in vitro and in vivo. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of β-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS.

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Previous genetic and biochemical studies indicate that the carB gene of Escherichia colicodes for the large subunit of carbamoyl-phosphate synthetase (EC 6.3.5.5). We have determined the nucleotide sequence of a 4-kilobase-pair cloned fragment of E. coli DNA with genetic determinants for carB. The DNA sequence is a 3,219-nucleotide-long reading frame. The polypeptide encoded by this reading frame has been verified to be the large subunit of carbamoyl-phosphate synthetase. The gene product is similar to the large subunit in its molecular weight, amino acid composition and amino-terminal residue, and carboxyl-terminal sequence. The amino acid sequence derived from the nucleotide sequence shows a highly significant homology between the amino-and carboxyl-terminal halves of the protein. We propose that the carB gene was formed by an internal duplication of a smaller ancestral gene.

In order to identify the dominant contributors to estrogenic activity in environmental waters, a comprehensive fractionation method using silica gel column chromatography, combined with recombinant yeast assay for detecting estrogenic activity and with gas chromatography-mass spectrometry for quantifying endocrine disruptors and natural estrogens, was developed. The method was applied to the municipal sewage treatment plant (STP) secondary effluent discharged to the Tamagawa River in Tokyo, Japan, where endocrine disruption was observed in wild carp. The instrumental analysis demonstrated that averaged concentrations of nonylphenol, bisphenol A, estrone (E1), and 17beta-estradiol (E2) were 564 +/- 127, 27 +/- 19, 33 +/- 11, and 4.6 +/- 3.0 ng/L, respectively. Based on the concentration and relative potency of these compounds, the natural estrogens E1 and E2 represented more than 98% of the total estrogen equivalent concentration (EEQ) in the STP effluent, while the contribution of phenolic compounds to total EEQ was less than 2%. Estrogenic activities associated with the dissolved phase of the effluent samples were detected by a recombinant yeast assay. By using silica gel column chromatography, the dissolved phase was separated into several fractions that were subjected to the bioassay. The polar fractions exhibited estrogenic activity. The greatest estrogenic activity was found in a polar fraction containing E1 and E2 and represented 66 to 88% of the total estrogenic activities estimated from the bioassay data. These results lead to the conclusion that E1 and E2 were the dominant environmental estrogens in the STP effluent, but a significant contribution to estrogenic activities stems from unidentified components in the effluents.