The structure of a putative S-formylglutathione hydrolase from Agrobacterium tumefaciens

Abstract

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 A resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S-formylglutathione hydrolase (AtuSFGH). The three-dimensional structure of AtuSFGH contains the alpha/beta hydrolase fold topology and exists as a homo-dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes C--O bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S-glutathionylation is also proposed.

Figure 1

Crystal structure of AtuSFGH and superposition with eukaryotic SFGHs. (A) Stereo view of the monomer structure of AtuSFGH showing the conserved SFGHs sequence motifs. Motif I: Leu-Ser-Gly-Leu-Thr-Cys (black), Motif II: Pro-Asp-Thr-Ser-Pro-Arg-Gly (blue), Motif III: Gly-Xaa-Gly-Ala-Gly-Phe-Tyr-Xaa-Xaa-Ala-Thr (red), Motif IV: Ile-Xaa-Gly-His-Ser-Met-Gly-Gly-Xaa-Gly-Ala (magenta), Motif V: Asp-His-Ser-Tyr-Tyr-Phe (green), and new Motif VI: Xaa-Gly-Xaa-Xaa-Asp-Xaa-Phe (brown). Ser-Asp-His catalytic triad, the regulatory Cys and Leu/Met from the oxyanion hole are shown as ball-and-stick representations. The oxyanion hole is indicated with a black star. (B) The AtuSFGH dimer structure. The β-sheet is colored red and α-helixes are colored blue. The proposed active site residues are shown as ball-and-stick representations.

Figure 2

Sequence and structural homology of AtuSFGH. The sequence of AtuSFGH was aligned with five S-formylglutathion hydrolases (SFGHs). The sequences are from E. coli (FrmB (P51025) and YeiG (P33018)), A. thaliana (Q8LAS8), Sinorhizobium medical (A6U894), human esterase D (P10768), and S. cerevisiae ScSFGH (YJG8) (P40363). The multiple sequence alignment was guided by a structural alignment of AtuSFGH and ScSFGH (PDB code 1PV1) using SEQUOIA. Highly conserved residues are highlighted in red. The conserved cysteine residue and the residues contributing to the catalytic triad are marked with an asterisk below the alignment. The oxyanion hole residues are denoted with triangles. Proposed residues in substrate and product binding are marked with open circles. The secondary structure elements refer to the structure of AtuSFGH (above) and ScSFGH (below) the sequence alignment. The consensus sequence motifs are denoted with dotted lines below the alignment and have the same color as in Figure 1(A). The figure was prepared with ESPript.