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Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating Pyruvate Dehydrogenase Activity ^,

Po-Chi Soo,^1, Yu-Tze Horng,^1, Meng-Jiun Lai,³ Jun-Rong Wei,¹ Shang-Chen Hsieh,¹ Yung-Lin Chang,¹ Yu-Huan Tsai,¹ and Hsin-Chih Lai^1,2*

Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China,¹ Department of Laboratory Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China,² Department of Laboratory Medicine and Biotechnology, College of Medicine, Tzu Chi University, Hualien, Taiwan, Republic of China³

Received 18 May 2006/ Accepted 28 August 2006

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.

Published ahead of print on 15 September 2006.

Supplemental material for this article may be found at http://jb.asm.org/.

These two authors contributed equally to this work.