Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.
Purified recombinant sorbose dehydrogenase from Sinorhizobium sp. 97507 exhibited high reactivity for 1,5-anhydro-d-glucitol (1,5-AG) and l-sorbose, but little activity for the other sugars or sugar alcohols tested. Kinetic analysis revealed that its catalytic efficiency (kcat/Km) for l-sorbose and 1,5-AG is 1.8 × 102 and 1.5 × 102 s−1·M−1, respectively.
Ovotransferrin N lobe contains six intrachain disulfides (SS-I/Cys10-Cys45; SS-II/Cys20-Cys36; SS-III/Cys115-Cys197; SS-IV/Cys160-Cys174; SS-V/Cys171-Cys182; SS-VI/Cys228-Cys242) in a single polypeptide chain of 332 amino acid residues. Upon the protein disulfide reduction with dithiothreitol under nondenaturing conditions, the intermediate species with four, three, and two disulfides were generated. The partially disulfide-reduced intermediates were isolated, and the localization of intact disulfides in the intermediates was determined by an indirect end-labeling method. This method included the S-cyanocysteine-specific protein fragmentation, followed by gel electrophoresis and the immunochemical visualization of the C terminus-intact fragments using antiserum raised against a non-cysteine C-terminal fragment (Ser280-Arg332). Results clearly showed that first SS-IV and SS-V, second SS-III, and then SS-VI are cleaved. No reduction was observed for SS-I and SS-II under the employed reducing conditions. The conclusion was confirmed by peptide mapping analyses for the same disulfide intermediates using reverse phase high performance liquid chromatography. Transverse urea gradient gel electrophoresis and visible absorption spectra revealed that the four-disulfide intermediate, but not the three- or two-disulfide intermediate, retains essentially the same iron-binding function as the native protein. By far-UV CD analyses, the residual native conformation of the partially disulfide-reduced intermediates was found to decrease with increased number of the reduced disulfides. Implications of the partially disulfide-reduced intermediates for the disulfide-reductive unfolding pathway in ovotransferrin N lobe are discussed.
To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5′-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p1, p4-di(adenosine) 5′-tetraphosphate, and adenosine-5′-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 μM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively). Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5′-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap4A, P1, P4-di(adenosine) 5ʹ-tetraphosphate; AMP: adenosine-5′-monophosphate; PAR: pyrophosphate amplification rate
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