Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense.
Azospirillum brasilense belongs to the plant growth-promoting rhizobacteria with direct growth promotion through the production of the phytohormone indole-3-acetic acid (IAA). A key gene in the production of IAA, annotated as indole-3-pyruvate decarboxylase (ipdC), has been isolated from A. brasilense, and its regulation was reported previously (A. Vande Broek, P. Gysegom, O. Ona, N. Hendrickx, E. Prinsen, J. Van Impe, and J. Vanderleyden, Mol. Plant-Microbe Interact. 18:311-323, 2005). An ipdC-knockout mutant was found to produce only 10% (wt/vol) of the wild-type IAA production level. In this study, the encoded enzyme is characterized via a biochemical and phylogenetic analysis. Therefore, the recombinant enzyme was expressed and purified via heterologous overexpression in Escherichia coli and subsequent affinity chromatography. The molecular mass of the holoenzyme was determined by size-exclusion chromatography, suggesting a tetrameric structure, which is typical for 2-keto acid decarboxylases. The enzyme shows the highest kcat value for phenylpyruvate. Comparing values for the specificity constant kcat/Km, indole-3-pyruvate is converted 10-fold less efficiently, while no activity could be detected with benzoylformate. The enzyme shows pronounced substrate activation with indole-3-pyruvate and some other aromatic substrates, while for phenylpyruvate it appears to obey classical Michaelis-Menten kinetics. Based on these data, we propose a reclassification of the ipdC gene product of A. brasilense as a phenylpyruvate decarboxylase (EC 4.1.1.43).
Spaepen
Stijn
S
Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium.
Versées
Wim
W
Gocke
Dörte
D
Pohl
Martina
M
Steyaert
Jan
J
Vanderleyden
Jos
J
D000595:Amino Acid Sequence; D015806:Azospirillum brasilense / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D002262:Carboxy-Lyases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007210:Indoleacetic Acids / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007210:Indoleacetic Acids; D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C109058:phenylpyruvate decarboxylase