title: Bifunctional activity of deoxyhypusine synthase/hydroxylase from Trichomonas vaginalis.
abstract: The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ((3)H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity.
authors.affiliation: Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México (UACM), San Lorenzo #290, Col. Del Valle, CP 03100, Mexico City, Mexico.
authors.forename: Laura Itzel
authors.initials: LI
authors.lastname: Quintas-Granados
authors.affiliation: Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México (UACM), San Lorenzo #290, Col. Del Valle, CP 03100, Mexico City, Mexico.
authors.forename: Bertha Isabel
authors.initials: BI
authors.lastname: Carvajal Gamez
authors.affiliation: Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México (UACM), San Lorenzo #290, Col. Del Valle, CP 03100, Mexico City, Mexico.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Villalpando
authors.affiliation: Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN 2508, Col. San Pedro Zacatenco CP 07360, Mexico City, Mexico.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ortega-Lopez
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN 2508, Col. San Pedro Zacatenco CP 07360, Mexico City, Mexico.
authors.forename: Rossana
authors.initials: R
authors.lastname: Arroyo
authors.affiliation: Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México (UACM), San Lorenzo #290, Col. Del Valle, CP 03100, Mexico City, Mexico.
authors.forename: Elisa
authors.initials: E
authors.lastname: Azuara-Liceaga
authors.affiliation: Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México (UACM), San Lorenzo #290, Col. Del Valle, CP 03100, Mexico City, Mexico. Electronic address: elizbethalvarezsanchez@yahoo.com.mx.
authors.forename: María Elizbeth
authors.initials: ME
authors.lastname: Álvarez-Sánchez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014246:Trichomonas vaginalis / Q000201:enzymology* / Q000235:genetics
chemical_list: D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase
keywords: Bifunctional activity; Deoxyhypusine synthase; Eukaryotic translation initiation factor 5A; Eukaryotic-translation-initiation-factor-5A response element; Trichomonas vaginalis