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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 Ialpha 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.

SUBMITTER: Sobrado VR

PROVIDER: S-EPMC2323874 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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

Sobrado Verónica R VR Montemartini-Kalisz Marisa M Kalisz Henryk M HM De La Fuente María Candelaria MC Hecht Hans-Jürgen HJ Nowicki Cristina C

Protein science : a publication of the Protein Society 20030501 5

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 amino ...[more]

PMID: 12717026

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Project description:We report the results of an investigation into the catalytic role of highly conserved amide (asparagine, glutamine) and OH-containing (serine, tyrosine) residues in several prenyltransferases. We first obtained the X-ray structure of cyclolavandulyl diphosphate synthase containing two molecules of the substrate analog dimethylallyl (S)-thiolodiphosphate (DMASPP). The two molecules have similar diphosphate group orientations to those seen in other ζ-fold (cis- head-to-tail and head-to-middle) prenyltransferases with one diphosphate moiety forming a bidentate chelate with Mg2+ in the so-called S1 site (which is typically the allylic binding site in ζ-fold proteins) while the second diphosphate binds to Mg2+ in the so-called S2 site (which is typically the homoallylic binding site in ζ-fold proteins) via a single P1O1 oxygen. The latter interaction can facilitate direct phosphate-mediated proton abstraction via P1O2, or more likely by an indirect mechanism in which P1O2 stabilizes a basic asparagine species that removes H+, which is then eliminated via an Asn-Ser shuttle. The universal occurrence of Asn-Ser pairs in ζ-fold proteins leads to the idea that the highly conserved amide (Asn, Gln) and OH-containing (Tyr) residues seen in many "head-to-head" prenyltransferases such as squalene and dehydrosqualene synthase might play similar roles, in H+ elimination. Structural, bioinformatics and mutagenesis investigations indeed indicate an important role of these residues in catalysis, with the results of density functional theory calculations showing that Asn bound to Mg2+ can act as a general (imine-like) base, while Gln, Tyr and H2O form a proton channel that is adjacent to the conventional (Asp-rich) "active site". Taken together, our results lead to mechanisms of proton-elimination from carbocations in numerous prenyltransferases in which neutral species (Asn, Gln, Ser, Tyr, H2O) act as proton shuttles, complementing the more familiar roles of acidic groups (in Asp and Glu) that bind to Mg2+, and basic groups (primarily Arg) that bind to diphosphates, in isoprenoid biosynthesis.

Dataset Information

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

Publications

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

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