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

Abstract:

:The aromatic l-alpha-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 alpha-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.

authors

Vernal J,Fiser A,Sali A,Müller M,Cazzulo JJ,Nowicki C

doi

10.1016/S0006-291X(02)00270-X

subject

Has Abstract

pub_date

2002-04-26 00:00:00

pages

633-9

issue

1

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(02)00270-X

journal_volume

293

pub_type

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