A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes

Abstract

O-Acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate (PLP) dependent sulfide-utilizing enzyme in the L-cysteine and L-methionine biosynthetic pathways of various enteric bacteria and fungi. OAHS catalyzes the conversion of O-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. However, the source of the sulfur has not been identified and no structures of OAHS have been reported in the literature. Here, the crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution is reported. MetY crystallized in space group C2 with two monomers in the asymmetric unit. Size-exclusion chromatography, dynamic light scattering and crystal packing indicate that the biological unit is a tetramer in solution. This is further supported by the crystal structure, in which a tetramer is formed using a combination of noncrystallographic and crystallographic twofold axes. A search for structurally homologous proteins revealed that MetY has the same fold as cystathionine γ-lyase and methionine γ-lyase. The active sites of these enzymes, which are also PLP-dependent, share a high degree of structural similarity, suggesting that MetY belongs to the γ-elimination subclass of the Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.

Figure 1

Proposed pathway for sulfur assimilation in W. succinogenes. MetY catalyses the PLP-dependent condensation of HcyS-COSH and OAH to form HcyS-Hcy. The S atom that originates from sulfate is highlighted in red throughout.

Figure 2

Structure of MetY. (a) Dimer observed in the asymmetric unit. (b) Monomer with secondary-structure elements labelled. (c) Topology diagram with the same labels as in (b). The numbers for the first and last amino-acid residue in each secondary-structure element are shown. (d) The biological tetramer. PLP is represented as spheres. The letters N and C in the monomer and topology diagrams denote the N- and C-termini, respectively.

Figure 3

Model of the active site of MetY. (a) Stereo diagram. (b) Schematic representation. The PLP cofactor shown in cyan and red in (a) and (b), respectively, is manually positioned in the active site based on superposition of structural homologs. Predicted interactions are indicated by the dashed lines. ‘W’ denotes a water molecule.

Figure 4

Structural superposition of the MetY monomer with homologous enzymes. Color coding is as follows: MetY, green; TtOAHS (PDB entry 2cb1), blue; ScCGL (PDB entry 1n8p), magenta; PpMGL (PDB entry 2o7c), gray. The PLP molecule is shown as cyan spheres.

Figure 5

Superposition of the active sites of MetY with those of other γ-lyases. The enzymes and coloring schemes are the same as in Fig. 4 ▶, except that the color of PLP is the same as its corresponding enzyme. Residues marked with an asterisk are from a neighboring monomer.

Figure 6

Sequence alignment of MetY with TtOAHS (PDB entry 2cb1), ScCGL (PDB entry 1n8p) and PpMGL (PDB entry 2o7c). Numbering and secondary-structure assignments are for MetY. Helices are indicated by blue cylinders, strands are indicated by green arrows and the two disordered loops are indicated by black dashed lines. Conserved residues are shown in boxes with white font and red shading. Conservative substitutions are shown in boxes with a red font. The conserved active-site residues are marked with asterisks and other active-site residues of MetY are marked with black circles. The two conserved residues from the disordered loop of an adjacent monomer, which are predicted to bind to the PLP phosphate group, are marked with red triangles.

Figure 7

Mechanistic proposal for MetY-catalyzed sulfur transfer from HcyS-COSH to OAH. The S atom that originates from sulfate is highlighted in red.