The Structure and Function of Paraoxonase-1 and Its Comparison to Paraoxonase-2 and -3

Abstract

Serum paraoxonase-1 (PON1) is the most studied member of the group of paraoxonases (PONs). This enzyme possesses three enzymatic activities: lactonase, arylesterase, and paraoxonase activity. PON1 and its isoforms play an important role in drug metabolism as well as in the prevention of cardiovascular and neurodegenerative diseases. Although all three members of the PON family have the same origin and very similar amino acid sequences, they have different functions and are found in different locations. PONs exhibit substrate promiscuity, and their true physiological substrates are still not known. However, possible substrates include homocysteine thiolactone, an analogue of natural quorum-sensing molecules, and the recently discovered derivatives of arachidonic acid-bioactive δ-lactones. Directed evolution, site-directed mutagenesis, and kinetic studies provide comprehensive insights into the active site and catalytic mechanism of PON1. However, there is still a whole world of mystery waiting to be discovered, which would elucidate the substrate promiscuity of a group of enzymes that are so similar in their evolution and sequence yet so distinct in their function.

Keywords: PON1; PON2; PON3; arylesterase; atherosclerosis; kinetic; lactonase; organophosphate; oxidative stress; paraoxonase; structure.

Figure 1

PON1 substrates from left to right: phenyl acetate, paraoxon, dihydrocoumarin and homocysteine thiolactone.

Figure 2

Presumed proton transfer through a histidine (H115/H134) dyad for water molecule activation, which is followed by tetrahedral intermediate (adduct) formation at the carbonyl groups of aryl esters or (thio)lactones. Although the negative charge of the elusive high-energy tetrahedral intermediate is electrostatically stabilized by the catalytic calcium ion, the C-O bond of this short-life species is subsequently cleaved, and the hydrolytic product is formed.

Figure 3

The generally accepted addition-elimination mechanism of PON1-catalyzed ester hydrolysis in the reaction pathway between the enzyme-substrate (ES) and enzyme-product (EP) complexes. The phosphonate-based ligand as a transition state analogue (TSA) should mimic the transition states (TS1 and TS2) near the tetrahedral intermediate (INT).

Figure 4

Paraoxonase 1 structure and insight into its active site. (A,B) Wild-type protein (protein database (PDB) id. 1V04 [28]). (C) Wild-type protein with bound inhibitor 2HQ (PDB id. 3SRG [30]). The active site residues are presented as backbone and marked by type and sequence number. Inhibitor is shown in magenta.

Figure 5

Paraoxonase 1 structural insight into active site of mutated proteins. Introduced mutations are related to protein specificity, activity or evolution. (A–C) Protein mutants related to enzymatic activity. (A) A mutant exhibiting only OP activity (H115W; PDB id. 4HHO [46]). (B) A mutant exhibiting convergence between OP and lactonase activity (H115Q and H134Q; PDB id. 4HHQ [46]). (C) A mutant affecting HDL binding and active site geometry (K192Q; PDB id. 4Q1U [47]). (D–F) Mutations related to the discovery of evolutionary trajectories. (D) L69S, H115W, and F222S (PDB id. 6G82 [49]); (E) L69S, H134R, F222S, and T332S (PDB id. 6GMU [49]); (F) L69S, H115W, H134R, F22S, and T332S (PDB id. 6H0A [49]). Mutated residues are in magenta.

Figure 6

Sequence alignment of recombinant PON1 (rePON1) and human PON1 (hPON1). The rePON1 protein sequence used for structural studies is from the protein database—PDB id: 1V04. The hPON1, hPON2, and hPON3 sequences are from the UniProt database—P27169, Q15165, and Q15166, respectively. In consensus line (*) is annotating conserved sequence, (.) is annotating conservative mutations and ( ) is annotating nonconservative mutations. Multiple sequence alignment was performed by T-Coffee, and Boxshade was used for alignment presentation. Secondary structure annotation, Ca²⁺ binding sites (both structural and catalytic ion), and active site residues are adapted from (as marked by different colors) [28]. The point mutations that were also evaluated by structural studies are annotated according to PDB id sequences: 4HHO, 4HHQ [46], 4Q1U [47], 6G82, 6GMU, and 6H0A [49].