PubMed · 14092544
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G WAKISAKA, S KONO. 1963. [ASPARTATES].. https://pubmed.ncbi.nlm.nih.gov/14092544/
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The proteomic response of a threonine-overproducing mutant of Escherichia coli was quantitatively analysed by two-dimensional electrophoresis. Evidently, 12 metabolic enzymes that are involved in threonine biosynthesis showed a significant difference in intracellular protein level between the mutant and native strain. The level of malate dehydrogenase was more than 30-fold higher in the mutant strain, whereas the synthesis of citrate synthase seemed to be severely inhibited in the mutant. Therefore, in the mutant, it is probable that the conversion of oxaloacetate into citrate was severely inhibited, but the oxidation of malate to oxaloacetate was significantly up-regulated. Accumulation of oxaloacetate may direct the metabolic flow towards the biosynthetic route of aspartate, a key metabolic precursor of threonine. Synthesis of aspartase (aspartate ammonia-lyase) was significantly inhibited in the mutant strain, which might lead to the enhanced synthesis of threonine by avoiding unfavourable degradation of aspartate to fumarate and ammonia. Synthesis of threonine dehydrogenase (catalysing the degradation of threonine finally back to pyruvate) was also significantly down-regulated in the mutant. The far lower level of cystathionine beta-lyase synthesis in the mutant seems to result in the accumulation of homoserine, another key precursor of threonine. In the present study, we report that the accumulation of important threonine precursors, such as oxaloacetate, aspartate and homoserine, and the inhibition of the threonine degradation pathway played a critical role in increasing the threonine biosynthesis in the E. coli mutant.
Ser-L223 is close to ubiquinone (Q(B)) in the B-branch of the bacterial photosynthetic reaction center (bRC) from Rhodobacter (Rb) sphaeroides. Therefore, the presence of a hydrogen bond (H bond) between the two was naturally proposed from the crystal structure. The hydrogen bonding pattern of Q(B) from the light-exposed structure was studied by generating hydrogen atom coordinates based on the CHARMM force field. In the Q(B) neutral charge state (Q(B)(0)), no H bond was found between the oxygen of the OH group from Ser-L223 and the carbonyl oxygen of Q(B) that is distal to the non-heme iron. In the reduced state (Q(B)(-)), however, Ser-L213 was found to form an H bond with Q(B) only when Asp-L213 is protonated by more than 0.75 H(+). This indicates the significance of the protonation of Asp-L213 in forming an H bond between Ser-L223 and Q(B). We found that the driving force to form the H bond between Ser-L223 and Q(B) is enhanced by the positively charged Arg-L217. The calculated Q(B) redox potentials with or without this H bond discriminated two ET rates, which are close to the faster and slower time phases observed in UV-Vis and FTIR studies. Together with the calculated redox potential of the quinones, this H-bond formation could play a key role in conformational gating for the ET process from Q(A) to Q(B).
The use of a rapid and sensitive assay for N-acetylaspartate (NAA) in urine or eluates from dried urine on filter paper to make a chemical diagnosis of Canavan disease (CD) is described. It involves a simplified urease pretreatment for sample preparation and gas chromatography-mass spectrometry (EI, scanning mode) with or without stable isotope dilution. Significant improvements in the recovery of NAA and the GC-MS data-handling device made the assay without stable isotope dilution sensitive and quantitative enough to diagnose CD: Its coefficient of variation (CV) was below 12%. The CV obtained with stable isotope dilution was below 9%. One patient with CD had an abnormal NAA level that was more than 6 S.D. above the mean of the age-matched controls. This diagnostic procedure is accurate for screening and for the chemical diagnosis of CD, with a good cost:benefit ratio. The urinary NAA levels of the healthy controls decreased significantly with age. This change should be considered in making a chemical diagnosis of this disease.