Graybill Eric R, Rouhier Matthew F, Kirby Charles E, Hawes John W
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
Arch Biochem Biophys. 2007 Sep 1;465(1):26-37. doi: 10.1016/j.abb.2007.04.039. Epub 2007 May 21.
In this study, the product of the CIT3 gene has been identified as a dual specificity mitochondrial citrate and methylcitrate synthase and that of the CIT1 gene as a specific citrate synthase. Recombinant Cit1p had catalytic activity only with acetyl-CoA whereas Cit3p had similar catalytic efficiency with both acetyl-CoA and propionyl-CoA. Deletion of CIT1 dramatically shifted the ratio of these two activities in whole cell extracts towards greater methylcitrate synthase. Deletion of CIT3 had little effect on either citrate or methylcitrate synthase activities. A Deltacit2Deltacit3 strain showed no methylcitrate synthase activity, suggesting that Cit2p, a peroxisomal isoform, may also have methylcitrate synthase activity. Although wild-type strains of Saccharomyces cerevisiae did not grow with propionate as a sole carbon source, deletion of CIT2 allowed growth on propionate, suggesting a toxic production of methylcitrate in the peroxisomes of wild-type cells. The Deltacit2Deltacit3 double mutant did not grow on propionate, providing further evidence for the role of Cit3p in propionate metabolism. (13)C NMR analysis showed the metabolism of 2-(13)C-propionate to acetate, pyruvate, and alanine in wild-type, Deltacit1 and Deltacit2 cells, but not in the Deltacit3 mutant. (13)C NMR and GC-MS analysis of pyruvate metabolism revealed an accumulation of acetate and of isobutanol in the Deltacit3 mutant, suggesting a metabolic alteration possibly resulting from inhibition of the lipoamide acetyltransferase subunit of the pyruvate dehydrogenase complex by propionyl-CoA. In contrast to Deltacit3, pyruvate metabolism in a Deltapda1 (pyruvate dehydrogenase E1 alpha subunit) mutant strain was only shifted towards accumulation of acetate.
在本研究中，CIT3基因的产物已被鉴定为双特异性线粒体柠檬酸和甲基柠檬酸合酶，而CIT1基因的产物则为特异性柠檬酸合酶。重组Cit1p仅对乙酰辅酶A具有催化活性，而Cit3p对乙酰辅酶A和丙酰辅酶A均具有相似的催化效率。CIT1的缺失显著改变了全细胞提取物中这两种活性的比例，使甲基柠檬酸合酶的比例更高。CIT3的缺失对柠檬酸合酶或甲基柠檬酸合酶的活性影响不大。Δcit2Δcit3菌株没有甲基柠檬酸合酶活性，这表明过氧化物酶体异构体Cit2p可能也具有甲基柠檬酸合酶活性。虽然酿酒酵母野生型菌株不能以丙酸盐作为唯一碳源生长，但CIT2的缺失使细胞能够在丙酸盐上生长，这表明野生型细胞过氧化物酶体中甲基柠檬酸的产生具有毒性。Δcit2Δcit3双突变体不能在丙酸盐上生长，这进一步证明了Cit3p在丙酸盐代谢中的作用。(13)C NMR分析表明，野生型、Δcit1和Δcit2细胞中2-(13)C-丙酸盐代谢生成乙酸盐、丙酮酸盐和丙氨酸，但Δcit3突变体中则不然。丙酮酸代谢的(13)C NMR和GC-MS分析表明，Δcit3突变体中乙酸盐和异丁醇积累，这表明代谢改变可能是由于丙酰辅酶A抑制丙酮酸脱氢酶复合体的硫辛酰胺乙酰转移酶亚基所致。与Δcit3相反，Δpda1（丙酮酸脱氢酶E1α亚基）突变体菌株中的丙酮酸代谢仅向乙酸盐积累方向转变。
