Lucas Kerry A, Filley Jessica R, Erb Jeremy M, Graybill Eric R, Hawes John W
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
J Biol Chem. 2007 Aug 24;282(34):24980-9. doi: 10.1074/jbc.M701028200. Epub 2007 Jun 18.
The subcellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using (13)C NMR, accumulation of beta-hydroxypropionate from [2-(13)C]propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants, including both monocots and dicots. Examination of coding sequences and subcellular targeting elements indicated that the completed genome of A. thaliana likely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, Arabidopsis mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to beta-hydroxypropionate by a modified beta-oxidation pathway. The chy1-3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of beta-hydroxyisobutyrate from exogenous isobutyrate, but not the accumulation of beta-hydroxypropionate from exogenous propionate. The chy1-3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine. (13)C NMR analysis of Arabidopsis seedlings exposed to [U-(13)C]valine did not show an accumulation of beta-hydroxypropionate. No evidence was observed for a modified beta-oxidation of valine. (13)C NMR analysis showed that valine was converted to leucine through the production of alpha-ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified beta-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine.
植物中支链氨基酸代谢的亚细胞位点一直存在争议，尤其是在缬氨酸分解代谢方面。缬氨酸分解代谢途径中某些步骤的潜在酶在线粒体和过氧化物酶体中均明显存在，但这些同工型的代谢功能尚不清楚。本研究考察了这些酶在异丁酰辅酶A和丙酰辅酶A代谢中的可能功能，这两种物质是缬氨酸以及奇数链和支链脂肪酸代谢的中间产物。利用¹³C核磁共振技术，在拟南芥幼苗以及包括单子叶植物和双子叶植物在内的一系列其他植物中，观察到了[2-(¹³)C]丙酸盐生成β-羟基丙酸盐的积累现象。对编码序列和亚细胞靶向元件的研究表明，拟南芥完整基因组可能编码了在线粒体中将缬氨酸转化为丙酰辅酶A所需的所有酶。然而，拟南芥线粒体可能缺乏丙酰辅酶A代谢的一些关键酶。已知的过氧化物酶体酶可能通过一种修饰的β-氧化途径将丙酰辅酶A转化为β-羟基丙酸盐。chy1-3突变导致过氧化物酶体羟基酰基辅酶A水解酶出现缺陷，消除了外源异丁酸盐生成β-羟基异丁酸盐的积累，但并未消除外源丙酸盐生成β-羟基丙酸盐的积累。chy1-3突变体对过量丙酸盐和异丁酸盐的毒性作用表现出显著增强的敏感性，但对缬氨酸的毒性作用不敏感。对暴露于[U-(¹³)C]缬氨酸的拟南芥幼苗进行¹³C核磁共振分析，未显示β-羟基丙酸盐的积累。未观察到缬氨酸存在修饰的β-氧化的证据。¹³C核磁共振分析表明，缬氨酸通过生成α-酮异戊酸和异丙基苹果酸转化为亮氨酸。这些数据表明，用于异丁酰辅酶A和丙酰辅酶A修饰β-氧化的过氧化物酶体酶可能在除缬氨酸以外的底物代谢中发挥作用。
