Structure of Staphylococcus aureus 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase

Abstract

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) and plays a key role in four metabolic processes: biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. The absence of the nucleosidase in mammalian species has implicated this enzyme as a target for antimicrobial drug design. MTAN from the pathogenic bacterium Staphylococcus aureus (SaMTAN) has been kinetically characterized and its structure has been determined in complex with the transition-state analogue formycin A (FMA) at 1.7 A resolution. A comparison of the SaMTAN-FMA complex with available Escherichia coli MTAN structures shows strong conservation of the overall structure and in particular of the active site. The presence of an extra water molecule, which forms a hydrogen bond to the O4' atom of formycin A in the active site of SaMTAN, produces electron withdrawal from the ribosyl group and may explain the lower catalytic efficiency that SaMTAN exhibits when metabolizing MTA and SAH relative to the E. coli enzyme. The implications of this structure for broad-based antibiotic design are discussed.

Figure 1

Structures of SAH, MTA and FMA. Note that for ease of comparison, the numbering of atoms in the purine base of the inhibitor FMA is based on the numbering convention of MTA and not the IUPAC standard.

Figure 2

Structure of SaMTAN and a comparison to EcMTAN. (a) Stereo diagram of the quaternary structure of SaMTAN. Superpositions of (b) the monomeric structures, (c) the adenine and ribose subsites and (d) the 5-alkythio-binding subsites of SaMTAN–FMA (red) and EcMTAN–FMA (blue). The initial σ_A-weighted F _o − F _c electron-density map found in the SaMTAN–FMA structure is shown contoured at 3σ with the refined coordinates of the protein and ligand. This figure was generated using PyMOL (DeLano, 2002 ▶).

Figure 3

Inhibitor conformation and interactions. Schematics of the interactions between the ligand and the protein in the active sites of (a) SaMTAN–FMA, (b) monomer A of EcMTAN–FMA and (c) monomer B of EcMTAN–FMA. Residues donated by a neighbouring subunit are shaded in a grey box and marked with an asterisk. Dotted lines represent protein–protein, protein–ligand or protein–solvent hydrogen bonds, with distances given in angstroms. (b) and (c) are modified from Lee et al. (2003 ▶) and Lee et al. (2001 ▶). (d) Superposition of formycin A from the active sites of the S. aureus (red) and E. coli (blue) enyzmes. The figure was generated using VMD (Humphrey et al., 1996 ▶).

Figure 4

Sequence alignment of E. coli and selected pathogenic MTA/SAH nucleosidases. The primary sequences of MTAN from the following bacterial pathogens are aligned: Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphyloccocus haemolyticus, Vibrio alginoyticus, Shigella flexneri, Shigella dysenteriae, Shigella sonnei, Salmonella typhi, Vibrio parahaemolyticus, Salmonella choleraesuis, Haemophilus influenzae, Vibrio cholerae, Vibrio vulnificus, Listeria monocytogenes, Pasteurella multocida, Bacillus licheniformis, Yersina pestis, Yersinia pseudotuberculosis, Bacillus cereus, Bacillus anthracis, Haemophilus ducreyi, Neisseria gonorrhoeae, Streptococcus faecalis, Neisseria meningitides, Shewanella putrefaciens, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pneumoniae and Helicobacter pylori. The sequence numbering and secondary-structural elements are based on Staphylococcus aureus. Residues that are strictly conserved are displayed as white characters in a red box, while residues that have been conservatively substituted are displayed as red characters. Residues that are similar across groups are framed in blue. Residues in the active site are marked with a green asterisk. This figure was prepared using ESPript (Gouet et al., 1999 ▶). The multiple sequence alignment was generated using ClustalW (Combet et al., 2000 ▶).