Methylthioinosine phosphorylase from Pseudomonas aeruginosa. Structure and annotation of a novel enzyme in quorum sensing

Abstract

The PA3004 gene of Pseudomonas aeruginosa PAO1 was originally annotated as a 5'-methylthioadenosine phosphorylase (MTAP). However, the PA3004 encoded protein uses 5'-methylthioinosine (MTI) as a preferred substrate and represents the only known example of a specific MTI phosphorylase (MTIP). MTIP does not utilize 5'-methylthioadenosine (MTA). Inosine is a weak substrate with a k(cat)/K(m) value 290-fold less than MTI and is the second best substrate identified. The crystal structure of P. aeruginosa MTIP (PaMTIP) in complex with hypoxanthine was determined to 2.8 Å resolution and revealed a 3-fold symmetric homotrimer. The methylthioribose and phosphate binding regions of PaMTIP are similar to MTAPs, and the purine binding region is similar to that of purine nucleoside phosphorylases (PNPs). The catabolism of MTA in P. aeruginosa involves deamination to MTI and phosphorolysis to hypoxanthine (MTA → MTI → hypoxanthine). This pathway also exists in Plasmodium falciparum, where the purine nucleoside phosphorylase (PfPNP) acts on both inosine and MTI. Three tight-binding transition state analogue inhibitors of PaMTIP are identified with dissociation constants in the picomolar range. Inhibitor specificity suggests an early dissociative transition state for PaMTIP. Quorum sensing molecules are associated with MTA metabolism in bacterial pathogens suggesting PaMTIP as a potential therapeutic target.

Figure 1

Early and late transition-state mimics as inhibitors for PaMTIP and PfPNP.

Figure 2

Stereoview of the hypoxanthine-omitted electron density map of PaMTIP in complex with hypoxanthine. The residues interacting with hypoxanthine are shown as yellow sticks and overlaid with a 2mF_o-DF_c electron density map (contour at 1 σ). Hypoxanthine carbons are shown as gray sticks and overlaid with mF_o-DF_c electron density map (contour at the 3 σ).

Figure 3

Crystal structure of PaMTIP. (A) A view of the PaMTIP monomer looking toward the catalytic site. The structure is colored from blue (N-terminus) to red (C-terminus). Hypoxanthine is included as a space-filling model to show the position of active site. (B) The monomers of trimeric PaMTIP are shown in blue, green and magenta. Hypoxanthine is included as space-filling models.

Figure 4

Structure-based sequence alignment. Protein are listed by PDB ID. The top 7 sequences are PNPs, the bottom 4 sequences are MTAPs and PaMTIP is 3OZB. The conserved residues in phosphate binding region of PNPs and MTAPs are shaded in light blue and dark blue, respectively. The conserved residues in the purine binding region of PNPs and MTAPs are shaded in light green and dark green, respectively. The conserved Phe and His in ribose-binding region of PNPs are shaded in yellow. The conserved His and small hydrophobic amino acid in the (methylthio)ribose-binding region of MTAPs are shaded in orange. The active site residues conserved in all species are shaded in grey. The Leu of PaMTIP is not conserved to either MTAPs or PNPs and is shaded in pink. The PDB IDs are as follow: 1YR3, E. coli PNP II; 1N3I, Mycobacterium tuberculosis PNP; 3KHS, Grouper Iridoviurs PNP; 2P4S, Anopheles Gambia PNP; 1VMK, Thermotoga maritime PNP; 1A9T, bovine PNP; 1RR6, human PNP; 3OZB, PaMTIP; 1V4N, Sulfolobus tokodaii MTAP; 1K27, human MTAP; 1WTA, Aeropyrum pernix K1 MTAP; 2A8Y, Sulfolobus solfataricus MTAP.

Figure 5

Active sites of human PNP (PDB ID: 1RR6), PaMTIP (PDB ID: 3OZB), and human MTAP (PDB ID: 1K27). (A) The conserved active site residues of human PNP in complex with ImmH are shown as sticks. The hydrogen bonds are indicated as dashed lines. ImmH is a transition state analogue inhibitor of human PNP and a mimic of inosine. (B) The active-site residues of PaMTIP and hypoxanthine (in yellow) are shown as sticks. The residues conserved in PNPs are colored in gray and the residues conserved in MTAPs are colored in orange. The hydrogen bonds are indicated as dashed lines. (C) The conserved active-site residues of human MTAP in complex with MT-ImmA are shown as sticks. The hydrogen bonds are indicated as dashed lines. The water molecule is drawn as a red dot. MT-ImmA is a transition state analogue inhibitor of human MTAP, and a mimic of MTA.

Figure 6

Metabolism of [8-¹⁴C]MTA in P. aeruginosa. P. aeruginosa lysate was incubated with [8-¹⁴C]MTA for 0, 10, and 25 min, respectively. The ¹⁴C-metabolites MTA, MTI, adenine and hypoxanthine were purified using RP-HPLC and quantitated by scintillation counting.