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Plant and Cell Physiology Advance Access originally published online on July 14, 2009
Plant and Cell Physiology 2009 50(9):1587-1599; doi:10.1093/pcp/pcp101
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Auxin Amidohydrolases from Brassica rapa Cleave the Alanine Conjugate of Indolepropionic Acid as a Preferable Substrate: A Biochemical and Modeling Approach

Bojana Savic1, Sanja Tomic1,4, Volker Magnus1, Kristina Gruden2, Katja Barle2, Renata Grenkovic1, Jutta Ludwig-Müller3 and Branka Salopek-Sondi1,*

1Ruder Boskovic Institute, PO Box 180, HR-10002 Zagreb, Croatia
2National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia
3Institute of Botany, Technische Universität Dresden, D-01062 Dresden, Germany

*Corresponding author: E-mail, salopek{at}irb.hr; Fax, +385-1-4561-177.


  Abstract

Two auxin amidohydrolases, BrIAR3 and BrILL2, from Chinese cabbage [Brassica rapa L. ssp. pekinensis (Lour.) Hanelt] were produced by heterologous expression in Escherichia coli, purified, and screened for activity towards N-(indol-3-ylacetyl)–L-alanine (IAA-Ala) and the long-chain auxin–amino acid conjugates, N-[3-(indol-3-yl)propionyl]–L-alanine (IPA-Ala) and N-[4-(indol-3-yl)butyryl]–L-alanine (IBA-Ala). IPA-Ala was shown to be the favored substrate of both enzymes, but BrILL2 was approximately 15 times more active than BrIAR3. Both enzymes cleaved IBA-Ala and IAA-Ala to a lesser extent. The enzyme kinetics were measured for BrILL2 and the obtained parameters suggested similar binding affinities for the long-chain auxin–amino acid conjugates (IPA-Ala and IBA-Ala). The velocity of the hydrolyzing reaction decreased in the order IPA-Ala > IBA-Ala > IAA-Ala. In a root growth bioassay, higher growth inhibition was caused by IPA-Ala and IBA-Ala in comparison with IAA-Ala. Neither these conjugates nor the corresponding free auxins affected the expression of the BrILL2 gene. A modeling study revealed several possible modes of IPA-Ala binding to BrILL2. Based on these results, two possible scenarios for substrate hydrolysis are proposed. In one the metal binding water is activated by the carboxyl group of the substrate itself, and in the other by a glutamate residue from the active site of the enzyme.

Keywords: Auxin amidohydrolase - Auxin–amino acid conjugate - Brassica rapa - Chinese cabbage - Enzyme kinetics - Modeling

Abbreviations: Bal, β-alanine (3-aminopropionic acid); DTT, dithiothreitol; IBA, indolebutyric acid [4-(indol-3-yl)butyric acid]; IPA, indolepropionic acid [3-(indol-3-yl)propionic acid]; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria–Bertani; MD, molecular dynamics; NTA agarose, nitrilotriacetic acid agarose; PMSF, phenylmethylsulfonyl fluoride; qRT-PCR; quantitative real-time PCR; TLC, thin-layer chromatography.

4This author performed the modeling and any correspondence regarding this should be addressed to him: E-mail, sanja.tomic{at}irb.hr.

(Received April 29, 2009; Accepted July 7, 2009)
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