Pirin regulates pyruvate catabolism by interacting with the pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity.
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.
Soo
Po-Chi
PC
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.
Horng
Yu-Tze
YT
Lai
Meng-Jiun
MJ
Wei
Jun-Rong
JR
Hsieh
Shang-Chen
SC
Chang
Yung-Lin
YL
Tsai
Yu-Huan
YH
Lai
Hsin-Chih
HC
D001426:Bacterial Proteins / Q000502:physiology*; D050260:Carbohydrate Metabolism; D002952:Citric Acid Cycle; D003065:Coenzyme A / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000502:physiology; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000378:metabolism*; D012706:Serratia marcescens / Q000378:metabolism*
D001426:Bacterial Proteins; D021122:Protein Subunits; D030481:Pyruvate Dehydrogenase (Lipoamide); D003065:Coenzyme A