Soo Po-Chi, Horng Yu-Tze, Lai Meng-Jiun, Wei Jun-Rong, Hsieh Shang-Chen, Chang Yung-Lin, Tsai Yu-Huan, Lai Hsin-Chih
Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, No. 1 Chan-Der Street, Taipei 100, Taiwan, Republic of China.
J Bacteriol. 2007 Jan;189(1):109-18. doi: 10.1128/JB.00710-06. Epub 2006 Sep 15.
The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin(Sm)) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin(Sm) gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin(Sm) gene in S. marcescens CH-1. The S. marcescens CH-1 pirin(Sm) gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin(Sm) mutant. Concomitantly, the cellular NADH/NAD(+) ratio increased in the pirin(Sm) mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin(Sm) gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin(Sm) is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.
吡啉蛋白参与多种生物过程，从原核微生物、真菌、植物到哺乳动物都有其保守形式。在哺乳动物中，它作为转录辅因子或凋亡相关蛋白发挥作用，在植物中则参与种子萌发和幼苗发育。在原核生物中，虽然在蓝细菌中吡啉蛋白受应激诱导，在大肠杆菌中可能作为槲皮素酶发挥作用，但吡啉直系同源物的功能大多仍未明确。我们发现粘质沙雷氏菌的吡啉蛋白（pirin(Sm)）基因编码吡啉蛋白的一个直系同源物。通过蛋白质下拉和细菌双杂交试验，随后进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和电喷雾电离串联质谱分析，结果表明丙酮酸脱氢酶（PDH）E1亚基是与pirin(Sm)基因相互作用的一个组分。功能分析表明，在粘质沙雷氏菌CH - 1中，pirin(Sm)基因抑制了PDH E1亚基活性和PDH酶复合物活性。随后通过插入 - 缺失同源重组对粘质沙雷氏菌CH - 1的pirin(Sm)基因进行了突变。相应地，在粘质沙雷氏菌CH - 1的pirin(Sm)突变体中，PDH E1和PDH酶复合物活性以及细胞ATP浓度分别增加了250%、140%和220%。同时，pirin(Sm)突变体中的细胞NADH/NAD(+)比值增加，表明三羧酸（TCA）循环活性增强。我们的结果表明，pirin(Sm)基因通过与PDH E1亚基相互作用并抑制粘质沙雷氏菌CH - 1中的PDH酶复合物活性，在丙酮酸分解代谢为乙酰辅酶A的过程中发挥调节作用，这表明pirin(Sm)是一种重要的蛋白质，参与决定丙酮酸代谢是走向TCA循环还是发酵途径。
