Active site organization of bacterial type I fatty acid synthetase

Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits.     

Decreased intracellular free magnesium in erythrocytes of spontaneously hypertensive rats

Using 31p-NMR (the phosphorus nuclear magnetic resonance) spectroscopy, we measured intracellular free Mg levels in the erythrocytes of untreated (n = 7) and diltiazem-treated spontaneously hypertensive rats (SHR) (n = 8), and compared them with age-matched Wistar-Kyoto rats (WKY) (n = 10). The intracellular free Mg levels were significantly (p less than 0.01) decreased in untreated SHR compared with those in control WKY. A successful antihypertensive treatment with diltiazem increased the intracellular free Mg levels compared with untreated SHR (p less than 0.05). Furthermore, an inverse correlation was observed between intracellular free Mg levels and blood pressure levels in all groups (r = -0.48, p less than 0.01, n = 25). These observations suggest that abnormalities of intracellular Mg metabolism may be, in part, related to the development or the maintenance of hypertension in SHR.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Summary The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.     

Molecular cloning and expression ofThiobacillus ferrooxidans chromosomal ribulose bisphosphate carboxylase genes inEscherichia coli

The genes coding ford-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium,Thiobacillus ferrooxidans, were cloned into anEscherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase.Escherichia coli carrying pTR11 did not show any CO₂-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of atac promoter, conferred ribulose bisphosphate-dependent CO₂-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized inE. coli revealed that it had a hexadecameric form like the native enzyme ofT. ferrooxidans.     

Study of the specific heme orientation in reconstituted hemoglobins

NMR studies of the recombination reaction of apohemoglobin derivatives with natural and unnatural hemes and of the heme-exchange reaction for reconstituted hemoglobin have revealed that the heme is incorporated into the apoprotein with stereospecific heme orientations dependent upon the heme peripheral 2,4-substituents and the axial iron ligand(s). Heme orientations also depend on whether recombination occurs at the alpha or beta subunit and on whether or not the complementary subunit is occupied by the heme. In the recombination reaction with the azido complex of deuterohemin, the alpha subunit of the apohemoglobin preferentially combines with the hemin in the "disordered" heme orientation, whereas protohemin is inserted in either of two heme orientations. Mesohemin inserts predominantly in the "native" heme orientation. For the beta subunit, specific heme orientation was also encountered, but the specificity was somewhat different from that of the alpha subunit. It was also shown that the specific heme orientation in both subunits is substantially affected by the axial heme ligands. These findings imply that apohemoglobin senses the steric bulkiness of both the porphyrin 2,4-substituents and the axial iron ligands in the heme-apoprotein recombination reaction. To gain an insight into the effect of the protein structure, the heme reconstitution reaction of semihemoglobin, demonstrating that the heme orientation in the reconstituted semihemoglobin with the azido-deuterohemin complex was in the native form, was also examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of endogenous calcium ion and its functional and structural regulation in horseradish peroxidase

The endogenous calcium ion (Ca2+) in horseradish peroxidase (HRP) was removed to cause substantial changes in the proton NMR spectra of the enzyme in various oxidation/spin states. The spectral changes were interpreted as arising from the substantial alterations in the heme environments, most likely the heme proximal and distal sides. The comparative kinetic and redox studies revealed that these conformational changes affect the reduction process of compound II, resulting in the decrease of the enzymatic activity of HRP. It is also revealed from the ESR spectrum and the temperature dependences of the NMR and optical absorption spectra of the Ca2+-free enzyme that the heme iron atom of the Ca2+-free enzyme is in a thermal spin mixing between ferric high and low spin states, in contrast to that of the native enzyme. These results show that Ca2+ functions in maintaining the protein structure in the heme environments as well as the spin state of the heme iron, in favor of the enzymatic activity of HRP.