Molecular and biochemical characterization of an alanine racemase from Trypanosoma cruzi.

PAES, LS; PEREIRA, CA; SILBER, ARIEL

Foz de Iguazu

Congreso; XXVII Meeting of the Brazilian Society of Protozoology. XXXVIII Annual Meeting on Basic Research in Chagas? Disease.; 2011

SBPZ

The involvement of L-amino acids in a quantity of relevant biological processes in Trypanosomacruzi was extensively demonstrated. However, little is known about the functions of D-aminoacids on the biology of the parasite. The first amino acid racemase isolated from T. cruzi wasthe proline racemase, which was shown to be a potent mitogen and to be involved in theinfectivity of the parasite (Reina San Martin et al., 2000, Chamond et al., 2003). In the T. cruzigenome database we identified a gene encoding a putative alanine racemase (Alr) (EC 5.1.1.1).Alrs are pyridoxal 5?-phosphate (PLP) dependent enzymes which catalyze the racemization ofalanine (interconversion between L-and D-alanine). In the present work, the ORF encoding Alrwas amplified from T.cruzi genomic DNA and cloned into the pET19b vector using NdeI andBamHI enzyme sites. This construction was used for protein expression in E. coli BL21 cells.The recombinant Alr containing a 6xHis-tag showed high levels of expression as a soluble andactive protein. Subsequently, the Alr was purified by affinity chromatography, and showedracemization activity in both directions (conversion of D into L and L into D alanine). Therecombinant protein was used to produce anti-TcAlar polyclonal monospecific antibodies.Western blot analysis showed that the anti-Alar antibody recognized both, the recombinant andnative proteins (a single polypeptide of the 42 KDa in T.cruzi epimastigotes extracts). Since theAlr is an enzyme not present in the mammalian host, it constitutes a promising target for drugs.In this sense, the effects on T. cruzi viability of a set of Alr inhibitors are being evaluated.Supported by:Fapesp, CNPq, INBEQMEDI, USP.