No abstract

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.

See also Kanse SM, Etscheid M. Factor VII activating protease (FSAP): caught in the cross-fire between polycations and polyanions. This issue, pp 556-8.

The rimL gene of Escherichia coli K12 encodes an enzyme catalyzing the acetylation of the N-terminal serine of ribosomal protein L12, thereby converting it into L7. Using a mutant strain defective in this acetylation reaction, we cloned the rimL gene into cosmid pHC79 and characterized it at the molecular level. From analysis by SDS-polyacrylamide gel electrophoresis of the proteins synthesized in maxi-cells containing derivatives of the rimL-harboring plasmid into which transposon gamma delta had been inserted at various sites, the product of this gene was identified as a protein with an apparent molecular weight of 20.3 kDa. The nucleotide sequence of the gene and the amino acid sequence deduced from the nucleotide sequence were compared with those of two other ribosomal protein acetylases encoded by the rimI and rimJ genes (Yoshikawa et al. 1987). A considerable degree of overall similarity was seen between rimL and rimJ, but the degree of similarity between rimL and rimI was very low. In addition, a short stretch of similar amino acid sequence was found in all three rim acetylases. The significance of these results with respect to other acetylating enzymes, in particular those involved in the acetylation of aminoglycoside antibiotics is discussed.

Among plant-specific transcription factors, ethylene response factors (ERFs) comprise one of the largest families. ERFs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report on the functional analysis of a transcriptional repressor, NtERF3, with regard to cell death associated with a hypersensitive response (HR), a plant-specific resistance reaction against pathogens. Expression of NtERF3 was upregulated during HR induction by Tobacco mosaic virus (TMV) infection in tobacco plants harboring the resistance N gene to TMV. Transient overexpression of NtERF3 by Agrobacterium-mediated gene delivery induced HR-like cell death in tobacco, associated with the production of reactive oxygen species and ion leakage. Deletion of the ERF-associated amphiphilic repression (EAR) motif from NtERF3 resulted in no induction of cell death, while the deletion had no effect on nuclear localization of the proteins. After virus-mediated gene delivery, similar results also were observed in tobacco without the N gene. In addition to NtERF3, other EAR motif-containing ERFs from tobacco, Nicotiana benthamiana and rice also induced cell death when overproduced in tobacco plants. The results suggested that many ERF genes encoding EAR motif-containing proteins might have the ability to induce cell death when overexpressed.