X-ray structure of ILL2, an auxin-conjugate amidohydrolase from Arabidopsis thaliana

Abstract

The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 A resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N-(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three-layer alpha beta alpha sandwich architecture and aminopeptidase topology and a smaller satellite domain with two-layer alphabeta-sandwich architecture and alpha-beta-plaits topology. The metal-coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side-chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family.

Figure 1

The X-ray crystal structure of ILL2. (A) Stereo view of a 2mF_o−DF_c electron density map of the refined ILL2 model around Ile92, which adopts an unusual backbone conformation. The map is contoured at 1.3σ level. (B) Ribbon diagram of ILL2 in stereo representation shows that this enzyme consists of two distinct domains. Strands and helices in both the catalytic domain (bottom, red and cyan) and the satellite domain (top, green and yellow) are numbered in order of their appearance in respective domains. The catalytic domain of ILL2 consists of the central 8-stranded β-sheet (red) sandwiched between five and two helices on either side (cyan). The satellite domain (top) is inserted between β-strand 6 and helix E of the catalytic domain.

Figure 2

Comparison of the active sites of ILL2 and its structural homologs. (A) A stereo view the ribbon trace of superimposed ILL2 (red; PDB ID 1xmb) and yxeP from B. subtilis (cyan; PDB ID 1ysj). Catalytic domains of ILL2 and yxeP are highly similar and superimpose with an all-atom rmsd of 0.5 Å. The residues involved in coordination of Metal 1 and Metal 2 (green spheres) in yxeP are shown as cyan sticks. The analogous residues present in the apo-ILL2 structure are also shown (red sticks) and annotated by labels. (B) A stereo view of the ribbon trace of superimposed ILL2 (red) and CPG2 (cyan, PDB ID 1cg2). The figure is given in the same orientation as in Figure 2a to allow for easy comparison of all structures. The residues involved in metal coordination are shown in cyan sticks and are annotated for CPG2. Asp141 of CPG2 serves as a bidentate ligand in metal coordination, replacing Cys137 of ILL2. The double arrow points to the functionally related metal ligand that originates from topologically distinct positions in ILL2 (His139) and CPG2 (His112).

Figure 3

The multiple sequence alignment of selected ILL2 homologs. The multiple sequence alignment of all known IAA-amino acid hydrolases from Arabidopsis thaliana (first seven entries; UniProt codes ILR1_ARATH through ILL6_ARATH), IAR3 homolog from wheat (UniProt code Q66VR4_WHEAT), and several bacterial ILL2 homologs including yxeP protein from Bacillus subtilis, N-acyl-L-amino acid amidohydrolase from Bacillus clausii (gi|56964568), amino acid amidohydrolase from Lactobacillus acidophilus (gi|58337160), N-acetyldiaminopimelate deacetylase from Lactobacillus salivarius (gi|90961445), hippurate hydrolase from Geobacillus kaustophilus (gi|56419585) and hippurate hydrolase from Bacillus halodurans (gi|15615231). The ILL2 sequence is shown in bold letters and accompanied by a ruler indicating every 5^th (:) and 10^th (|) residue. Invariant residues of all the aligned sequences are highlighted in red, other highly conserved residues are colored in blue. The important active site residues are boxed and color-coded as in Figure 4a with metal-coordinating residues in cyan, hydrophobic residues that bind the indole ring of a substrate in orange, a putative general acid-base residue in green, a putative amino acid side chain selectivity filter residue in magenta, and other conserved residues in the vicinity of the active site in gray. Secondary structural elements of ILL2 are shown as cylinders (helices) and arrows (β-strands). The numbering and color-coding scheme of the secondary structural elements corresponds to that in Figure 1b. Purple italics letters highlight the predicted signal peptide sequences of A. thaliana amidohydrolases. Bold green letters highlight residues mutated in loss-of-function mutants of A. thaliana ILR1 and IAR3., Further information about these mutations is given in Table II.

Figure 4

The active site of ILL2 with a docked substrate. (A) A stereo view of the active site of ILL2 with a modeled substrate N-(indole-3-acetyl)-alanine (IAA-Ala; yellow sticks). Important residues of the active site of ILL2 are shown as sticks and are color-coded as follows: metal-coordinating residues (cyan), residues forming a hydrophobic cavity that may bind the indole ring of a substrate (orange), a putative general acid-base residue Glu172 (green) and a putative side-chain selectivity filter residue Leu175 (magenta), other conserved residues (gray) involved in formation of the active site hydrogen bond network. (B) A stereo view of a surface representation of the ILL2 active site with the docked IAA-Ala (yellow sticks). The hydrophobic cavity (orange) accommodates the indole ring of a substrate. The carbonyl group of a scissile peptide bond of the substrate is coordinated by one of the metals in the active site (green spheres) while the C-terminal carboxyl of the substrate points toward the surface of the protein. Leucine 175 (magenta) restricts the size of the amino acid side chain of the substrate that can fit into the active site, providing the basis for the substrate specificity of ILL2.