酿酒酵母中NAD激酶同功酶对偶数位不饱和脂肪酸β-氧化的影响

The Effect of NAD Kinase Homologues on the β-oxidation of Unsaturated Fatty Acids with the Double Bond at an Even Position in Saccharomyces cerevisiae

ATP-NAD激酶利用ATP,催化NAD磷酸化,生成NADP,而ATP-NADH激酶则催化NAD和NADH发生磷酸化。酿酒酵母细胞内存在三种NAD激酶同功酶Utr1p、Pos5p和Yef1p,它们都是ATP-NADH激酶,对细胞内NADP(H)的供应起到重要作用。酵母偶数位双键不饱和脂肪酸的β-氧化依赖于过氧化物酶体基质内的NADPH。通过构建NAD激酶基因的单、双基因缺失株,并验证它们和对照菌株对不饱和脂肪酸的氧化、利用能力,证实NAD激酶同功酶,尤其是Pos5p,对过氧化物酶体基质内NADP(H)的供应起着重要作用,并推测NADP可以从过氧化物酶体膜外转运至过氧化物酶体基质内。

ATP-NAD kinase phosphorylates NAD to produce NADP by using ATP, whereas ATP-NADH kinase phosphorylates both NAD and NADH. Three NAD kinase homologues, namely, Utrlp, Pos5p and Utrlp, exist in the yeast Saccharomyces cerevisiae, which were all confirmed as ATP-NADH kinases and found to be important to supply NADP（H） for yeast cells. In S. cerevisiae, fatty acid β-oxidation is restricted to peroxisomes and peroxisomal NADPH is required for β-oxidation of unsaturated fatty acids with the double bonds at even positions. Single and double gene disruption strains of NAD kinase genes, i.e., utrl, pos5, yefl, utrlyefl, utrlpos5 and yeflpos5 were constructed by PCR-targeting method. The utilization ability of these mutants for unsaturated fatty acids with the double bonds at even or uneven positions was examined, with wild type BY4742 as positive control cell, and fatty-acyl-CoA oxidase gene deletion mutant （fox1） and peroxisomal NADP-dependent isocitrate dehydrogenase isoenzymes gene deletion mutant （idp3） as negative control cells. The results indicated that the NAD kinase homologues, especially Pos5p, were critical for supplying NADP and then NADPH in peroxisomal matrix. NADP, which was supplied mainly by Utrlp, Pos5p and Yeflp, particularly by Pos5p, was proposed to be able to transfer from outside of peroxisome into peroxisomal matrix and then converted to NADPH by Idp3p.