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.The protein pirin is widely found in mammals, plants, fungi, and also prokaryotic organisms (32). While the cellular functions of pirin show diversity and pirin homologs play important roles in a number of different biological processes, cellular localization of pirin is not restricted to specific compartments. In eukaryotes, pirin was initially isolated through a yeast twohybrid screen from the HeLa cell cDNA library and is localized within cell nuclei; it acts as an interactor with nuclear factor