通过改换大肠杆菌腺苷酸代谢途径来增加胞内能荷水平

原核生物细胞能量和物质代谢的途径是一个很复杂的网络,改变代谢途径中的基因会对能量和物质流产生怎样的影响,仍然不是很清楚.以往的文献和研究已经将大肠杆菌的腺嘌呤核苷酸补救合成途径研究的很透彻.使用HPLC对删除了add基因的大肠杆菌细胞内腺苷类核苷酸分析表明,在腺嘌呤核苷酸补救途径中单一基因的途径操作不能有效改变腺嘌呤类核苷酸的代谢流向.实验中通过删除大肠杆菌JM83株中的add基因(编码腺苷脱氨酶[EC:3.5.4.4][1,2]),deoD基因(编码嘌呤核苷磷酸酶[EC:2.4.2.1][3,4]),amn基因(编码AMP核苷酶[EC:3.2.2.4][5])并引入外源ado1基因(来自酵母编码腺苷激酶[EC:2.7.1.20][6,7,8]),构建了菌株J991 (add-,deoD-,amn-,ado1 ,JM83),将其在含腺苷的LB培养基培养,使用HPLC分析其胞内腺苷类能量物质发现,ATP,ADP,AMP胞内含量都有所增加,分别都比对照JM83菌株提高一倍左右,大大加强了腺苷转化AMP的代谢流量,实现了改变物质代谢流向并使ATP积累的目的.该菌种实现了高产ATP代谢通路的构建,为下游生物工程发酵提供了较野生菌更高效的菌种,有望通过发酵工程优化培养,大幅提高ATP产量.同时,"尝试改变AMP的浓度而非直接针对ATP调节代谢途径,达到ATP积累的目"这一思路为同类研究提供参考.最后也表明在腺嘌呤核苷酸补救代谢途径中,为达到物质代谢流改变的目的,多基因联合操作较之单基因敲除更为有效.     

Consequences of the salvage of purine compounds on the proliferation of rat T-lymphocytes with normal or inhibited purine de novo synthesis

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 μM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 μM. Guanine, guanosine and 2′-deoxyguanosine at concentrations up to 500 μM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 μM adenine, adenosine, 2′-deoxyadenosine or 100 μM 2′-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2′-deoxyguanosine toxicity was dependent and 2′-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.     

A Review Comparing Deoxyribonucleoside Triphosphate (dNTP) Concentrations in the Mitochondrial and Cytoplasmic Compartments of Normal and Transformed Cells

The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double membrane of the mitochondria. Perturbed homeostasis of mitochondrial dNTP pools is associated with a set of severe diseases collectively termed mitochondrial DNA depletion syndromes. The degree of interaction of the mitochondrial dNTP pools with the corresponding dNTP pools in the cytoplasm is currently not clear. We reviewed the literature on previously reported simultaneous measurements of mitochondrial and cytoplasmic deoxyribonucleoside triphosphate pools to investigate and quantify the extent of the influence of the cytoplasmic nucleotide metabolism on mitochondrial dNTP pools. We converted the reported measurements to concentrations creating a catalog of paired mitochondrial and cytoplasmic dNTP concentration measurements. Over experiments from multiple laboratories, dNTP concentrations in the mitochondria are highly correlated with dNTP concentrations in the cytoplasm in normal cells in culture (Pearson R = 0.79, p = 3 × 10^?7) but not in transformed cells. For dTTP and dATP there was a strong linear relationship between the cytoplasmic and mitochondrial concentrations in normal cells. From this linear model we hypothesize that the salvage pathway within the mitochondrion is only capable of forming a concentration of approximately 2 μM of dTTP and dATP, and that higher concentrations require transport of deoxyribonucleotides from the cytoplasm.     

Immucillins as Antibiotics for T‐Cell Proliferation and Malaria

The genetic deficiency of human PNP causes a specific immunodeficiency by inducing apoptosis in dividing T‐cells. Powerful inhibitors of PNP have been designed from the experimental determination of the transition state structure of PNPs. The Immucillins are transition state analogue inhibitors with K _d values as low as 7 pM. In the presence of deoxyguanosine the Immucillins kill activated human T‐cells but not other cell types. The Immucillins are orally available and of low toxicity to mice. Immucillins also inhibit PNP from Plasmodium falciparum. Parasites cultured in human erythrocytes are killed by purine starvation in the presence of Immucillins and can be rescued by hypoxanthine.     

N-acetylamino acid utilization by kidney aminoacylase-1

Mammalian aminoacylase-1 (Acy1) participates in the breakdown of N-acetylated amino acids during intracellular protein catabolism. Acy1 is most abundantly expressed in the kidney tubular epithelium. Lately, Acy1 deficiency was identified in children with increased urinary excretion of several N-acetylamino acids. Here we report detailed N-acetylamino acid specificity profiles for human and porcine Acy1 based on steady state kinetic measurements. We found that LLC-PK1 cells, a model of the porcine kidney proximal tubular epithelium, robustly express Acy1. For the first time, we demonstrate uptake and utilization of N-acteylleucine and -methionine in replacement of the free amino acid, respectively, in cultured epithelial cells. Our data are consistent with a specific role of kidney Acy1 in the salvage of amino acids originating from systemic degradation of N-acetylated proteins.     

New evidence for cofactor's amino group function in thiamin catalysis by transketolase

Transketolase from Saccharomyces cerevisiae exhibits a rarely reported activity with a methylated analogue of the native cofactor, 4′-methylamino-thiamin diphosphate. We demonstrated the kinetic stability of the dihydroxyethyl carbanion/enamine intermediate to be dependent on the functionality of the 4′-aminopyrimidine moiety of thiamin diphosphate [R. Golbik, L.E. Meshalkina, T. Sandalova, K. Tittmann, E. Fiedler, H. Neef, S. König, R. Kluger, G.A. Kochetov, G. Schneider, G. Hübner, Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisae, FEBS J. (2005) 272 1326-1342]. This paper extends these investigations of the function of the coenzyme’s aminopyrimidine in transketolase catalysis exemplified for the 4′-monomethylamino-thiamin diphosphate analogue. Here, we report near UV circular dichroism data and NMR-based analysis of reaction intermediates that give evidence for a strong destabilisation of the carbanion/enamine of DHE-4’-monomethylamino-thiamin diphosphate on the enzyme. A new negative band in near UV circular dichroism arising during turnover is attributed to the conjugate acid of the carbanion/enamine intermediate, an assignment additionally corroborated by ¹H NMR-based intermediate analysis. As opposed to the kinetically stabilized carbanion/enamine intermediate in transketolase when reconstituted with the native cofactor, DHE-4′-monomethylamino-thiamin diphosphate is rapidly released from the active centers during turnover and accumulates in the medium on a preparative scale.