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.

Recent Developments and Applications of the MMPBSA Method

“…Their analysis showed that BPA-A was the strongest binder to the receptor (Starovoytov et al, 2014 ). Other similar studies include globin, for its catalytic mechanism in the hydrolysis of substituted phenyl hexanoates (Ercan et al, 2014 ), the role of conserved residues in substrate binding to Brassica rapa auxin amidohydrolase (Smolko et al, 2016 ), the effect of hydrophobic interactions in substrate binding to recombinant enzyme carboxylesterase (Shao et al, 2014 ), the substrate-enzyme interactions of endo-1,4-β-xylanase (Zhan et al, 2014 ), azoreductase protein for the biodegradation of azo dyes (Dehghanian et al, 2016 ; Haghshenas et al, 2016 ), cytochrome P450 2A6 for nicotine addiction (Lu et al, 2014 ), Cel48F for producing bioethanol via fiber degradation (Qian M. D. et al, 2016 ), rubisco for biofuel production (Siqueira et al, 2016 ), streptavidin-biotin complex in biochemical sensing (Liu F. J. et al, 2016 ), sorotidine 5-monophosphate decarboxylase for its impressive rate enhancement (Jamshidi et al, 2014 ), tyrosyl-tRNA synthetases for genetic encoding of unnatural amino acids (Ren et al, 2015 ), T7 RNA polymerase for generating RNA labels (Borkotoky et al, 2016 ), AF9 in the YEATS family for the recognition of H3K9ac (Wang Q. et al, 2016 ), cysteine protease 1 precursor from Zea for the hydrolysate of corn gluten meal (Liu et al, 2014 ), folate receptor alpha for producing milk with high folate concentration (Sahoo et al, 2014c ), and both acid amido synthetase (Wang X. et al, 2015 ) and brassinosteroid (Lei et al, 2015 ) for plant growth.…”

Auxin Amidohydrolases – From Structure to Function: Revisited

“…Bacterial peptidases from the M20 family prefer Zn 2+ ions as cofactors, [59] whereas auxin amidohydrolases prefer Mn 2+ . [42,58,60] The crystal structure of AtILL2 was published and metal and substrate binding sites have been proposed. [61] Based on the crystal structure of the AtILL2 apoenzyme (without metal cofactor in the metal binding site) Bitto et al (2009) [61] suggested a model for auxin conjugate hydrolase activity towards the preferred substrate IAA-Ala.…”

“…New insights into the structure-function relationship of the putative PpIAR3 would add to our understanding of the evolution of all auxin amidohydrolases. So far, modeling and subsequent analysis of mutant enzymes has been carried out for a bacterial auxin conjugate hydrolase specific for IAA-Asp, [50] and now a few studies on plant enzyme counterparts have been added to the picture, [42,58] following the publication of the crystal structure of Arabidopsis ILL2 (an IAA specific auxin conjugate hydrolase, Fig. 1.…”

“…B), [61] which is available and has been used for modeling studies. [42,58,61] All auxin amidohydrolases possess N-and C-terminal protein sequences suggesting their localization in endoplasmic reticulum (ER). Recent study proved that IAR3, ILL2 and ILR1 of A. thaliana reside in the ER, indicating that in this compartment the hydrolases regulate the rates of amido-IAA hydrolysis, which results in activation of auxin signalling.…”

“…Auxin amidohydrolases are active in the monomeric form, but, as such a form is highly unstable, the enzymes have a tendency to stabilize by forming polymers in vivo. [42,58] For their activity they require the presence of a reducing agent, such as dithiothreitol, and a metal co-factor. Members of the M20 peptidases generally have two cations in the active site.…”