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...
SummaryPolyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas' disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcP-RACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans -splicing mechanism. Here, we demonstrate that overexpression of Tc PRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that Tc PRAC is a potential target for drug design as well as for immunomodulation of parasiteinduced B-cell polyclonal activation.
There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.
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