Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4. , respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.Numerous signaling pathways in higher organisms, such as apoptosis, inflammation, blood clotting, and others, involve proteolytic events as mediators of signal initiation, transmission, and termination. Substrate specificity of the involved proteases and a tight regulation of their activation and inhibition are essential regulatory mechanisms of temporal and spatial control in proteolytic signaling. Serpins (serine protease inhibitors) represent a large class of polypeptide serine protease inhibitors that are involved in regulation of a wide spectrum of proteasemediated processes (1, 2). They fold into a metastable native structure with an exposed substrate-like reactive center loop (RCL) 3 (3) and, unlike the small polypeptide inhibitors from the Kunitz or Kazal family, they do not act as reversible competitive inhibitors of the target proteases but rather as stoichiometric suicide inactivators with a unique inhibition mechanism driven by conformational change. Upon cleavage of RCL by the target protease and formation of the covalent acyl-enzyme reaction intermediate, the serpin fold undergoes a major conformational rearrangement, and the RCL is inserted as the middle strand of the beta sheet A to form a six-stranded anti-parallel ␤ sheet at the core of the cleaved serpin structure (4). This conformational change creates a steric clash with the protease and the resulting distortion inactivates the enzyme and traps it as a covalent serpin-protease adduct (5).Serpins are widely distributed in higher eukaryotic organisms and are also found in some viruses where they appear to modulate virus-host interactions and viral infectivity (1). Thirty-four serpins identified in the human genome belong to nine different phylogenetic clades in the currently adopted serpin classification (2). Notably, some members of the serpin ...