title: Recombinant production of eight human cytosolic aminotransferases and assessment of their potential involvement in glyoxylate metabolism.
abstract: PH1 (primary hyperoxaluria type 1) is a severe inborn disorder of glyoxylate metabolism caused by a functional deficiency of the peroxisomal enzyme AGXT (alanine-glyoxylate aminotransferase), which converts glyoxylate into glycine using L-alanine as the amino-group donor. Even though pre-genomic studies indicate that other human transaminases can convert glyoxylate into glycine, in PH1 patients these enzymes are apparently unable to compensate for the lack of AGXT, perhaps due to their limited levels of expression, their localization in an inappropriate cell compartment or the scarcity of the required amino-group donor. In the present paper, we describe the cloning of eight human cytosolic aminotransferases, their recombinant expression as His6-tagged proteins and a comparative study on their ability to transaminate glyoxylate, using any standard amino acid as an amino-group donor. To selectively quantify the glycine formed, we have developed and validated an assay based on bacterial GO (glycine oxidase); this assay allows the detection of enzymes that produce glycine by transamination in the presence of mixtures of potential amino-group donors and without separation of the product from the substrates. We show that among the eight enzymes tested, only GPT (alanine transaminase) and PSAT1 (phosphoserine aminotransferase 1) can transaminate glyoxylate with good efficiency, using L-glutamate (and, for GPT, also L-alanine) as the best amino-group donor. These findings confirm that glyoxylate transamination can occur in the cytosol, in direct competition with the conversion of glyoxylate into oxalate. The potential implications for the treatment of primary hyperoxaluria are discussed.
authors: Department of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy.
authors: Stefano
authors: S
authors: Donini
authors: Manuela
authors: M
authors: Ferrari
authors: Chiara
authors: C
authors: Fedeli
authors: Marco
authors: M
authors: Faini
authors: Ilaria
authors: I
authors: Lamberto
authors: Ada Serena
authors: AS
authors: Marletta
authors: Lara
authors: L
authors: Mellini
authors: Michela
authors: M
authors: Panini
authors: Riccardo
authors: R
authors: Percudani
authors: Loredano
authors: L
authors: Pollegioni
authors: Laura
authors: L
authors: Caldinelli
authors: Stefania
authors: S
authors: Petrucco
authors: Alessio
authors: A
authors: Peracchi
mesh_terms: D000818:Animals; D001219:Aspartate Aminotransferases / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology*; D006038:Glyoxylates / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D013552:Swine
chemical_list: D006038:Glyoxylates; D011994:Recombinant Proteins; D001219:Aspartate Aminotransferases; C031150:glyoxylic acid