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Journal of Bacteriology, November 2007, p. 7626-7633, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00830-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Phenylpyruvate Decarboxylase, Involved in Auxin Production of Azospirillum brasilense{triangledown}

Stijn Spaepen,1 Wim Versées,2,3 Dörte Gocke,4 Martina Pohl,4 Jan Steyaert,2,3 and Jos Vanderleyden1*

Centre of Microbial and Plant Genetics and INPAC, K.U. Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium,1 Department of Ultrastructure, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium,2 Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, 1050 Brussel, Belgium,3 Institute of Molecular Enzyme Technology, Heinrich-Heine University Düsseldorf, Research Centre Jülich, 52426 Jülich, Germany4

Received 29 May 2007/ Accepted 23 August 2007

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).

* Corresponding author. Mailing address: Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. Phone: 32 16321631. Fax: 32 16321963. E-mail: jozef.vanderleyden{at}biw.kuleuven.be

{triangledown} Published ahead of print on 31 August 2007.

Journal of Bacteriology, November 2007, p. 7626-7633, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00830-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Versees, W., Spaepen, S., Wood, M. D. H., Leeper, F. J., Vanderleyden, J., Steyaert, J. (2007). Molecular Mechanism of Allosteric Substrate Activation in a Thiamine Diphosphate-dependent Decarboxylase. J. Biol. Chem. 282: 35269-35278 [Abstract] [Full Text]  


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