Probing the specificity of a trypanosomal aromatic α-hydroxy acid dehydrogenase by site-directed mutagenesis

Javier Vernal, Andras Fiser, Andrej Šali, Miklós Müller, Juan José Cazzulo, Cristina Nowicki

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The aromatic L-α-hydroxy acid dehydrogenase (AHDAH) from Trypanosoma cruzi has over 50% sequence identity with cytosolic malate dehydrogenases (cMDHs), yet it is unable to reduce oxaloacetate. Molecular modeling of the three-dimensional structure of AHADH using the pig cMDH as template directed the construction of several mutants. AHADH shares with MDHs the essential catalytic residues H195 and R171 (using Eventoff's numbering). The AHADH A102R mutant became able to reduce oxaloacetate, while remaining fully active towards aromatic α-oxoacids. The Y237G mutant diminished its affinity for all of the natural substrates, whereas the double mutant A102R/Y237G was more active than Y237G and had similar activity with oxaloacetate and with aromatic substrates. The present results reinforce our proposal that AHADH arose by a moderate number of point mutations from a cMDH no longer present in the parasite.

Original languageEnglish (US)
Pages (from-to)633-639
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume293
Issue number1
DOIs
StatePublished - 2002
Externally publishedYes

Fingerprint

Oxaloacetic Acid
Malate Dehydrogenase
Mutagenesis
Hydroxy Acids
Site-Directed Mutagenesis
Oxidoreductases
Keto Acids
Molecular Conformation
Molecular modeling
Trypanosoma cruzi
Substrates
Point Mutation
Parasites
Swine

Keywords

  • Aromatic amino acid metabolism
  • Aromatic L-α-hydroxy acid dehydrogenase
  • Malate dehydrogenase
  • Trypanosoma cruzi

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Probing the specificity of a trypanosomal aromatic α-hydroxy acid dehydrogenase by site-directed mutagenesis. / Vernal, Javier; Fiser, Andras; Šali, Andrej; Müller, Miklós; Cazzulo, Juan José; Nowicki, Cristina.

In: Biochemical and Biophysical Research Communications, Vol. 293, No. 1, 2002, p. 633-639.

Research output: Contribution to journalArticle

Vernal, Javier ; Fiser, Andras ; Šali, Andrej ; Müller, Miklós ; Cazzulo, Juan José ; Nowicki, Cristina. / Probing the specificity of a trypanosomal aromatic α-hydroxy acid dehydrogenase by site-directed mutagenesis. In: Biochemical and Biophysical Research Communications. 2002 ; Vol. 293, No. 1. pp. 633-639.
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AU - Nowicki, Cristina

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