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J. Biol. Chem., Vol. 277, Issue 23, 20490-20498, June 7, 2002
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From the The Arabidopsis thaliana flavoprotein
AtHAL3a, which is linked to plant growth and salt and osmotic
tolerance, catalyzes the decarboxylation of
4'-phosphopantothenoylcysteine to 4'-phosphopantetheine, a key step in
coenzyme A biosynthesis. AtHAL3a is similar in sequence and structure
to the LanD enzymes EpiD and MrsD, which catalyze the oxidative
decarboxylation of peptidylcysteines. Therefore, we hypothesized that
the decarboxylation of 4'-phosphopantothenoylcysteine also occurs via
an oxidatively decarboxylated intermediate containing an aminoenethiol
group. A set of AtHAL3a mutants were analyzed to detect such an
intermediate. By exchanging Lys34, we found that AtHAL3a is
not only able to decarboxylate 4'-phosphopantothenoylcysteine but also
pantothenoylcysteine to pantothenoylcysteamine. Exchanging residues
within the substrate binding clamp of AtHAL3a (for example of
Gly179) enabled the detection of the proposed aminoenethiol
intermediate when pantothenoylcysteine was used as substrate. This
intermediate was characterized by its high absorbance at 260 and 280 nm, and the removal of two hydrogen atoms and one molecule of
CO2 was confirmed by ultrahigh resolution mass
spectrometry. Using the mutant AtHAL3a C175S enzyme, the product
pantothenoylcysteamine was not detectable; however, oxidatively
decarboxylated pantothenoylcysteine could be identified. This result
indicates that reduction of the aminoenethiol intermediate depends on a
redox-active cysteine residue in AtHAL3a.
Molecular Characterization of the Arabidopsis
thaliana Flavoprotein AtHAL3a Reveals the General Reaction
Mechanism of 4'-Phosphopantothenoylcysteine Decarboxylases*
,, and
Instituto de Biología Molecular y
Celular de Plantas, Universidad Politécnica de Valencia-CSIC,
Camino de Vera s/n, 46022 Valencia, Spain, § Institut
für Organische Chemie, Universität Tübingen, Auf der
Morgenstelle 18, 72076 Tübingen, and ¶ Lehrstuhl
für Mikrobielle Genetik, Universität Tübingen, Auf
der Morgenstelle 15, Verfügungsgebäude,
72076 Tübingen, Germany
*
This work was supported by Deutsche Forschungsgemeinschaft
Grant KU869/6-1 (to T. K.) and by European Molecular Biology
Organization Fellowship ASTF 9878 (to P. H.-A.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence and reprint requests should be
addressed. E-mail: Thomas.Kupke@t-online.de.
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