Involvement of conserved asparagine and arginine residues from the N‐terminal region in the catalytic mechanism of rat liver and Trypanosoma cruzi tyrosine aminotransferases

Abstract

Rat liver and Trypanosoma cruzi tyrosine aminotransferases (TATs) share over 40% sequence identity, but differ in their substrate specificities. To explore the molecular features related to these differences, comparative mutagenesis studies were conducted on full length T. cruzi TAT and N-terminally truncated rat TAT recombinant enzymes. The functionality of Arg315 and Arg417 in rat TAT was investigated for comparison with the conserved Arg292 and Arg386 in aspartate and bacterial aromatic aminotransferases (ASATs and ARATs). The rat TAT Arg315Lys variant remained fully active indicating that, as in T. cruzi TAT and contrary to subfamily I␣ aminotransferases, this residue is not critical for activity. In contrast, the Arg417Gln variant was inactive. The catalytic relevance of the putative rat TAT active site residues Asn54 and Arg57, which are strictly conserved in TATs (Asn17 and Arg20 in T. cruzi TAT) but differ in ASATs and ARATs, was also explored. The substitutions Arg57Ala and Arg57Gln abolished enzymatic activity of these mutants. In both variants, spectral studies demonstrated that aromatic but not dicarboxylic substrates could efficiently bind in the active site. Thus, Arg57 appears to be functionally equivalent to Arg292 of ASATs and ARATs. Asn54 also appears to be involved in the catalytic mechanism of rat TAT since its exchange for Ser lowered the k cat /K m ratios towards its substrates. Mutation of the analogous residues in T. cruzi TAT also lowered the catalytic efficiencies (k cat /K m ) of the variants substantially. The results imply that the mamalian TAT is more closely related to the T. cruzi TAT than to ASATs and ARATs.Keywords: Aromatic aminotransferases; rat liver tyrosine aminotransferase; heterologous expression; site directed mutagenesis; Trypanosoma cruzi Aminotransferases belong to the ␣-family of vitamin B 6 -dependent enzymes (Alexander et al. 1994), which play important roles in amino acid biosynthesis and degradation and carbohydrate metabolism in both prokaryotes and eukaryotes. These enzymes possess varied substrate specificities that mainly involve the three 2-oxoacids generated during glycolytic and respiratory cycles. Based on extensive structural, kinetic and site-directed mutagenesis studies on aspartate aminotransferases (ASATs), a ping-pong Bi-Bi mechanism involving several intermediates was proposed for the reversible transamination reaction. Furthermore, the substrate recognition mechanism of ASATs is the best understood among aminotransferases (Kirsch et al. 1984;Arnone et al. 1985;Hayashi 1995).At the structural level, two different aminotransferases belonging to subfamily I␣, the highly specific mammalian

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).…”

“…However the enzyme has the highest activity in the liver. In hTAT, the first 38 amino acids may not be involved in enzyme dimerization, and are not required in the active site stability and enzyme-substrate interactions (Sobrado et al, 2003). This N-terminal fragment, however, is required for being targeted by the ubiquitin-proteosome pathway (Gross-Mesilaty et al, 1997), which degrades proteins to small peptides (Ciechanover et al, 2000).…”

Biochemical and structural characterization of tyrosine aminotransferase suggests broad substrate specificity and a two‐state folding mechanism in Leishmania donovani

“…by site‐directed mutagenesis found that R57A and R57Q mutations in rat tyrosine aminotransferase abolished its activity completely. Mutation in analogous residues of T. cruzi TAT , N17S, and R20A/Q led to lowered catalytic efficiency of mutated variant significantly . Therefore, the authors hypothesize that the N‐terminal region of LdTAT that has few identical residues may hold the reason for co‐substrate specificity.…”

Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy

“…S2). Arg-417 is one of the residues responsible for the interaction of Tyr with mouse and rat TyrAT (Sobrado et al, 2003;Mehere et al, 2010), and Arg-390 of PsTyrAT might perform the same function. As reported for other investigations of plant aminotransferases, the in vitro activity of PsTyrAT was not affected by the addition of exogenous PLP.…”

“…Although Tyr, Phe, and Trp are primarily involved in protein synthesis, a vast array of secondary metabolites are also derived from these aromatic amino acids (Tzin and Galili, 2010). Although the biochemical and structural characterization of tyrosine aminotransferase (TyrAT) in mammals and fungi is well established (Blankenfeldt et al, 1999;Sobrado et al, 2003;Schneider et al, 2008;Mehere et al, 2010), considerably less is known about these enzymes in plants. TyrAT is regulated by coronatine, wounding, and methyl jasmonate (MeJA) and has been implicated as the initial enzyme in tocopherol biosynthesis in Arabidopsis (Arabidopsis thaliana) plants (Lopukhina et al, 2001;Holländer-Czytko et al, 2005) and Amaranthus caudatus and Chenopodium quinoa cell cultures (Antognoni et al, 2009).…”