Naslov
Auxin amidohydrolases from Brassica rapa cleave the alanine conjugate of indolepropionic acid as a preferable substrate: a biochemical and modeling approach

Autori
Savić, Bojana ; Tomić, Sanja ; Magnus, Volker ; Gruden, Kristina ; Barle, Katja ; Grenković, Renata ; Ludwig-Müller, Jutta ; Salopek-Sondi, Branka

Izvornik
Plant and cell physiology (0032-0781) 50 (2009), 9; 1587-1599

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Auxin amidohydrolase ; Auxin amino acid conjugate ; Brassica rapa ; Chinese cabbage ; Enzyme kinetics ; Modeling

Sažetak
Two auxin-amidohydrolases, BrIAR3 and BrILL2, from Chinese cabbage (Brassica rapa L. ssp. pekinensis (Lour.) Hanelt) were produced by heterologous expression in E. 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 was measured for BrILL2 and the obtained parameters suggested similar binding affinities for long-chained 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 the root growth bioassay, higher growth inhibition was caused by IPA-Ala and IBA-Ala in comparison to 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 Glu residue from the active site of the enzyme.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA