title: The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability.
abstract: Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate.
authors.affiliation: Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia. salopek@irb.hr.
authors.forename: Ana
authors.initials: A
authors.lastname: Smolko
authors.affiliation: Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, 10000 Zagreb, Croatia. ivo.piantanida@irb.hr.
authors.forename: Filip
authors.initials: F
authors.lastname: Šupljika
authors.affiliation: Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia. salopek@irb.hr.
authors.forename: Jelena
authors.initials: J
authors.lastname: Martinčić
authors.affiliation: Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, 10000 Zagreb, Croatia. ivo.piantanida@irb.hr.
authors.forename: Nina
authors.initials: N
authors.lastname: Jajčanin-Jozić
authors.affiliation: Division of Physical Chemistry, Ruđer Bošković Institute, 10000 Zagreb, Croatia.
authors.forename: Marina
authors.initials: M
authors.lastname: Grabar-Branilović
authors.affiliation: Division of Physical Chemistry, Ruđer Bošković Institute, 10000 Zagreb, Croatia.
authors.forename: Sanja
authors.initials: S
authors.lastname: Tomić
authors.affiliation: Institute of Botany, Technische Universität Dresden, 01062 Dresden, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Ludwig-Müller
authors.affiliation: Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, 10000 Zagreb, Croatia. ivo.piantanida@irb.hr.
authors.forename: Ivo
authors.initials: I
authors.lastname: Piantanida
authors.affiliation: Division of Molecular Biology, Ruđer Bošković Institute, 10000 Zagreb, Croatia. salopek@irb.hr.
authors.forename: Branka
authors.initials: B
authors.lastname: Salopek-Sondi
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D029741:Brassica rapa / Q000201:enzymology*; D002151:Calorimetry; D003545:Cysteine / Q000737:chemistry*; D004795:Enzyme Stability; D007210:Indoleacetic Acids / Q000378:metabolism*; D013058:Mass Spectrometry; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed
chemical_list: D007210:Indoleacetic Acids; D000581:Amidohydrolases; D003545:Cysteine