Amino acid racemases catalyze the stereoinversion of the chiral C ␣ to produce the D-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this large protein family, bacterial proline racemases have been extensively studied as a model of enzymes acting with a pyridoxalphosphate-independent mechanism. Here we report the crystal structure of the proline racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric ␣͞␤ subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2-carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid͞base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme.B cell mitogen ͉ pyridoxal phosphate-independent proline racemase ͉ epimerases ͉ enzyme-inhibitor complex ͉ titration calorimetry T he vast majority of amino acids found in living cells correspond to the L-stereoisomer at the C ␣ chiral center. However, D-amino acids are often found as constituents of bacterial cell walls (1, 2) and were identified in archaea and higher eukaryotes (3-5). Amino acid racemases and epimerases, which catalyze the L,D-stereochemistry inversion on free amino acids, have been extensively studied in prokaryotic systems (6). In the D3L sense, cells use D-amino acids to feed the large L-amino acid pool in normal amino acid͞protein metabolism; whereas, in the L3D sense, bacteria generate the D-enantiomers widely distributed in bacterial cell walls, in particular D-alanine, D-glutamate, and D,L-diaminopimelate, as peptidoglycan components that function as innate defense against host proteolytic mechanisms (1, 2) All known racemases catalyze the inversion of the chiral center by deprotonation of the C ␣ , followed by reprotonation on the opposite face of the planar carbanionic transition-state species. To overcome the high energetic barrier of this reaction [estimated pK a values for the C ␣ are in the range 21-32 (7, 8)], some racemases evolved to use pyridoxal phosphate (PLP) as cofactor, because formation of an imine PLP-substrate covalent bond greatly acidifies the chiral center by resonance (9). However, a second class of enzymes, which includes proline, aspartate, and glutamate racemases and diaminopimelate epimerase, operates through a twobase mechanism in a cofactor-independent manner (10-13). A foundational paper on the Cl...

Secretory immunoglobulin A (S-IgA) was investigated in human secretions for the presence of natural antibodies (Abs) acting as the first "immune barrier" to infection before induction or boosting of specific responses. These molecules could be the secretory counterpart of the natural Abs in serum that were previously shown by our laboratory to be polyreactive to autoantigens. Significant levels of S-IgA Abs to human actin, myosin, tubulin, and spectrin were detected in 10 saliva and 8 colostrum samples from normal subjects. Computer-assisted analysis of immunoblots of extracts from human muscles showed these Abs to react with a large number of autoantigens. Their polyreactivity was confirmed by cross-inhibition and by immunoblotting studies of affinity-purified natural Abs, assayed against a large variety of surface or secreted antigens from Streptococcus pyogenes. The thiocyanate elution method showed that functional affinities of some natural Abs can be of the same order of magnitude as those of tetanus vaccine antitoxins. Moreover, nonimmune binding of these natural Abs to the gut protein Fv (Fv-fragment binding protein) can enhance their effector functions. This demonstrates that human secretions contain polyreactive auto-Abs which can also react with pathogens. These secretory Abs of "skeleton key" specificities are possibly produced by a primordial B-1-cell-associated immune system and can be involved in a plurispecific mucosal protection against pathogens, irrespective of the conventional immune response.

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