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.

Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

“…Since hTAT showed transamination activity toward glutamate, phenylalanine as well as tyrosine (Sivaraman and Kirsch, 2006), kinetic parameters of mTAT toward these three amino acids were also tested and calculated (Table 1). Based on the mTATsubstrate specificity, mTAT behaves in a manner similar to hTAT, in that it has a narrow substrate specificity compared to tcTAT (Nowicki et al, 2001; Sobrado et al, 2003; Sivaraman and Kirsch, 2006; Donini et al, 2009). TcTAT, on the other hand, has broad substrate specificity; in particular, it has an affinity to alanine, which differs considerably from tyrosine, the primary substrate of mTAT and hTAT (Nowicki et al, 2001).…”

Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

“…His 6 -tagged (mouse) mmNit1 (lacking the targeting peptide sequence) and mmNit2 were expressed as reported earlier (5). The human cytosolic transaminases, glutamine transaminase K (KYAT1; previously CCBL1), aspartate aminotransferase (GOT1), and phosphoserine aminotransferase (PSAT1) were expressed as described previously (25). The coding sequences of the yeast Nit1 and Nit2 orthologs (encoded by the NIT2 and NIT3 genes, respectively), and of the bacterial homologs shown in Table 1, with the exception of ecYafV and blYbeM, were PCR-amplified from genomic DNA, cloned in the pET28a vector, and expressed as recombinant proteins (N-terminal His6 tag) in E. coli BL21(DE) cells.…”

“…HPLC analysis of reaction mixtures in which OA and glyoxylate-which has the advantage of binding quite weakly to glutamine transaminase K, limiting the phenomenon of substrate inhibition observed with larger α-keto acid substrates (25,26)-were incubated overnight with KYAT1 indicated the appearance of two peaks corresponding to dOA (SI Appendix, Figs. S7B and S8).…”

Molecular Identification of Hydroxylysine Kinase and of Ammoniophospholyases Acting on 5-Phosphohydroxy-l-lysine and Phosphoethanolamine

“…It catalyzes the pyruvate-dependent conversion of D-␤-aminoisobutyrate, a breakdown product of thymine, to methylmalonate semialdehyde (4). Unlike AGXT2, AGXT2L1 and AGXT2L2 did not act as transaminases (5).…”