Cellular and biochemical characterization of two closely related triosephosphate isomerases fromTrichomonas vaginalis

Abstract

SUMMARYThe glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report thatTrichomonas vaginaliscontains 2 fully functionaltpigenes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located inα-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed thattpi2transcript is approximately 3·3-fold more abundant thantpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement anEscherichia colistrain carrying a deletion of its endogenoustpigene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM fromSaccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. AsT. vaginalisdepends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicatedtpicopy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.