The tyrosine aminotransferase from Trypanosoma rangeli : sequence and genomic characterization Esteban J. Bontempi a; *, Gabriela A. Garc| ¨a a , Alejandro Buschiazzo b;1 , Jan Henriksson c;2 , Carlos A. Pravia a , Andre ¨s M. Ruiz a , Ulf Pettersson c , Viviana Pszenny a a Instituto Nacional de Parasitolog| ¨a `Dr. M. Fatala Chaben', Av. Paseo Colo ¨n 568 (1063) and Av. Ve ¨lez Sars¢eld 563 (1281), A.N.L.I.S. `Dr. Carlos G. Malbra ¨n', Buenos Aires, Argentina b Instituto de Investigaciones Biotecnolo ¨gicas, Universidad Nacional de Gral. San Mart| ¨n, INTI, Edi¢cio 24, San Mart| ¨n, Buenos Aires, Argentina c Department of Medical Genetics and Pathology, Uppsala University, Uppsala, Sweden Received 6 March 2000; received in revised form 15 June 2000; accepted 23 June 2000 Abstract The complete sequence and genomic characterization of the tyrosine aminotransferase (TAT) gene from Trypanosoma rangeli is reported. The gene was found to be organized in a tandem multicopy gene array. A homologous mRNA species (2.5 kb) was identified in the epimastigote form of the parasite. From the deduced amino acid sequence, the gene encodes a protein of 420 amino acids with a predicted molecular mass of 46.4 kDa and a theoretical pI of 6.23. A high sequence identity was found with the Trypanosoma cruzi, human and rat enzymes. All the essential residues for TAT enzymatic activity are conserved, as well as a pyridoxal-phosphate attachment site typical of class-I aminotransferases. The recombinant enzyme was recognized by a monoclonal antibody against the T. cruzi enzyme. Additionally, the recombinant protein showed enzymatic activity when incubated with L-tyrosine and 2-oxoglutaric acid as substrates. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Tyrosine aminotransferase ; Tandemly repeated gene ; Trypanosoma rangeli ; Trypanosoma cruzi 1. Introduction Tyrosine aminotransferase (TAT) activity, involved in the initial step of tyrosine catabolism, has been described in several organisms (mammals, Crithidia fasciculata, Tet- rahymena pyriformis, etc.), but few TATs accounting for that activity have been fully characterized. TAT activity was found in Trypanosoma cruzi (Tc) [1], the causative agent of Chagas' disease, and the enzyme puri¢ed [2] and the gene sequenced [3]. In spite of the high similarity to the human and mouse counterparts (77%), several dif- ferences in enzymatic properties were seen [2]. Trypanoso- ma rangeli (Tr) is coendemic with Tc in many areas, and they share a similar morphology, common epitopes (pro- ducing serological cross-reactions) and one of the insect vectors (Rhodnius prolixus). Previous results demonstrated TAT activity in Tr whole homogenate (unpublished re- sults) and also suggested the presence of a TAT gene. A heterologous TAT probe from Tc hybridized to Tr chro- mosomes larger than 1.7 megabases in 16 di¡erent Tr stocks [4]. Here we describe the sequence and genomic characterization of Tr TAT, and report that the recombi- nant protein is enzymatically active. 2. Materials and methods 2.1. Growth of parasites Tr epimastigotes of LDG strain and clone D1-D2- Duarte strain N2378 were grown in BHT medium (brain^heart^tryptose) supplemented with 10% fetal calf serum (Sigma) at 28³C. 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII:S0378-1097(00)00293-7 * Corresponding author. Tel.: +54 (11) 4331-4019; Fax: +54 (11) 4331-7432; E-mail: ejbon@yahoo.com 1 Present address : Unite ¨ de Biochimie Structurale, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France. 2 Present address : Department of Research and Development, Eurona Medical AB, Uppsala, Sweden. FEMS Microbiology Letters 189 (2000) 253^257 www.fems-microbiology.org Downloaded from https://academic.oup.com/femsle/article-abstract/189/2/253/524779 by guest on 20 May 2020