Molecular characterization of the Arabidopsis thaliana flavoprotein AtHAL3a reveals the general reaction mechanism of 4'-phosphopantothenoylcysteine decarboxylases.
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 Lys(34), 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 Gly(179)) 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 CO(2) 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.
Hernández-Acosta
Pilar
P
Instituto de Biologia Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, 46022 Valencia, Spain.
Schmid
Dietmar G
DG
Jung
Günther
G
Culiáñez-Macià
Francisco A
FA
Kupke
Thomas
T
D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; C404262:AtHAL3 protein, Arabidopsis; D017931:DNA Primers; D010940:Plant Proteins; D002262:Carboxy-Lyases; C022373:Phosphopantothenoyl-cysteine decarboxylase