Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPase-like homology model

Yeung, David T.; Josse, Denis; Nicholson, James D.; Khanal, Akhil; McAndrew, Christopher W.; Bahnson, Brian J.; Lenz, David E.; Cerasoli, Douglas M.

doi:10.1016/j.bbapap.2004.08.002

Cited by 73 publications

(79 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutagenesis experiments supported the suggested mechanism, although it was later found that these mutants were probably misfolded and therefore inactive (13). Moreover, Yeung et al (14) recently reported that the H115W mutant of human PON1 retains activity with paraoxon. They therefore postulated that His 115 is important for substrate binding and specificity, but does not directly participate in catalysis (15).…”

Section: Serum Paraoxonases (Pons)mentioning

confidence: 64%

See 1 more Smart Citation

The Histidine 115-Histidine 134 Dyad Mediates the Lactonase Activity of Mammalian Serum Paraoxonases

Khersonsky

Tawfik

2006

Journal of Biological Chemistry

156

View full text Add to dashboard Cite

Serum paraoxonases (PONs) are calcium-dependent lactonases that catalyze the hydrolysis and formation of a variety of lactones, with a clear preference for lipophilic lactones. However, the lactonase mechanism of mammalian PON1, a high density lipoproteinassociated enzyme that is the most studied family member, remains unclear, and other family members have not been examined at all. We present a kinetic and site-directed mutagenesis study aimed at deciphering the lactonase mechanism of PON1 and PON3. The pHrate profile determined for the lactonase activity of PON1 indicated an apparent pK a of ϳ7. Serum paraoxonases (PONs) 2 constitute a family of calcium-dependent mammalian enzymes that have been recently defined as lipophilic lactonases. PON1 is the best studied member of the family, with other members being PON2 and PON3 (1, 2). PON1 catalyzes the hydrolysis of multiple substrates: lactones, thiolactones, carbonates, esters, and phosphotriesters, including paraoxon, from which its name is derived. However, only after a few decades of research, it became apparent that PON1 and the other PONs are in fact lactonases (3-6), catalyzing both the hydrolysis (4, 6) and formation (7) of a variety of lactones. Structurereactivity studies (6) and laboratory evolution experiments (3) indicate that the native activity of PON1 is lactonase. The other activities, e.g. arylesterase and phosphotriesterase (paraoxonase), are merely promiscuous and are not shared by other family members, e.g. PON2 and PON3. PON1 activation by binding to high density lipoprotein particles carrying apoA-I also indicates high specificity toward lactone substrates and, in particular, lipophilic lactones that display k cat /K m values of 10 6 -10 7 M Ϫ1 s Ϫ1 (5). The physiological substrates of PONs are still unknown, but they are likely to include lactones consumed as food ingredients (8) or derivatives of fatty acid oxidation processes, e.g. 5-hydroxyeicosatetraenoic acid lactone (4,8), that reside in high density lipoprotein, low density lipoprotein, or macrophage cells. PON1 is composed of 354 amino acids. The enzyme has two calciumbinding sites: the higher affinity calcium is required for the structural integrity, whereas the lower affinity calcium is involved in catalysis (9). Early mechanistic studies using chemical labeling and site-directed mutagenesis identified several residues that are involved in the phosphotriesterase and esterase activities of human PON1 (10, 11). However, because these studies were conducted before the three-dimensional structure of PON1 was known, it was largely unclear whether these amino acids are indeed in the PON1 active site or whether they are involved in substrate binding, Ca 2ϩ binding, or catalysis. Recently, a crystal structure of a recombinant PON1 (rePON1) variant (G2E6) was solved at a resolution of 2.2Å, providing the first structure of a PON family member (12). This variant was directly evolved from rabbit PON1. It is expressed in a soluble and active form in Escherichia coli and exhibits enzymatic pr...

show abstract

Section: Serum Paraoxonases (Pons)mentioning

confidence: 64%

“…The mutation of His 115 , which is important for hydrolysis of lactones and esters (14,15), did not have much effect on the phosphotriesterase activity of PON1. The mutations of His 184 caused a Ͼ10-fold decrease in this activity, but there is no other evidence that ascribes a role for His 184 in catalysis.…”

Section: Non-detrimental Effect Of Mutations On Pon1mentioning

confidence: 99%

The Histidine 115-Histidine 134 Dyad Mediates the Lactonase Activity of Mammalian Serum Paraoxonases

Khersonsky

Tawfik

2006

Journal of Biological Chemistry

156

View full text Add to dashboard Cite

show abstract

“…Notably, the active sites of DFPase and PON possess a conserved arrangement of amino acids around the catalytic calcium and, like DFPase, PON also shows activity against selected organophosphate triesters (22,23).…”

mentioning

confidence: 99%

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement

Blum

Mustyakimov

Rüterjans

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calciumdependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca-O-H angle is 53°, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.enzyme mechanism ͉ H/D exchange

show abstract

“…Furthermore, CD spectra of purified tag-free E3 showed a broad minimum centered around 210 nm with a crossover point of 205 nm (Figure 2-8), in a good agreement with known β-fold proteins such as diisopropylfluorophosphatase 53,56 . And compared with wild type huPON1, E3 has 70 the negative peak shifted from 217 to 210 nm, indicating less composition of α-helices 57 . The enzymatic kinetics of tag-free E3 was also measured toward C4-HSL and C6-HSL.…”

Section: Generation Of Soluble Expressed Hupon2 Variantsmentioning

confidence: 91%

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Wang

Mulchandani

et al. 2016

ACS Chem. Biol.

View full text Add to dashboard Cite

Many pathogenic bacteria utilize quorum sensing (QS) systems to regulate the expression of their virulence genes and promote the formation of biofilm, which renders pathogens with extreme resistance to conventional antibiotic treatments. As a novel approach for attenuating antibiotic resistance and in turn fighting chronic infections, enzymatic inactivation of QS signaling molecules, such as N-acyl homoserine lactones (AHLs), holds great promises. Instead of using bacterial lactonases that can evoke immune response when administered, we focus on the human paraoxonase 2 (huPON2). However, insolubility when heterologously overexpressed hinders its application as anti-infection therapeutics. In this study, huPON2 was engineered for soluble expression with minimal introduction of foreign sequences. On the basis of structure modeling, degenerate linkers were exploited for the removal of hydrophobic helices of huPON2 without disrupting its folding structure and thus retaining its enzymatic function. High soluble expression levels were achieved with a yield of 76 mg of fully human PON2 variants per liter of culture media. Particularly, two clones, D2 and E3, showed significant quorum quenching (QQ) bioactivities and effectively impeded Pseudomonas aeruginosa swimming and swarming motilities, signs of an early stage of biofilm formation. In addition, by correlating QQ with luminescence signal readouts, quantitative analysis of QQ toward natural or non-natural AHL-regulator combinations suggested that D2 and E3 exhibited strong lactone hydrolysis activities toward five AHLs of different side chain lengths and modifications widely utilized by a variety of biomedically important pathogens.

show abstract

Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPase-like homology model

Cited by 73 publications

References 37 publications

The Histidine 115-Histidine 134 Dyad Mediates the Lactonase Activity of Mammalian Serum Paraoxonases

The Histidine 115-Histidine 134 Dyad Mediates the Lactonase Activity of Mammalian Serum Paraoxonases

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Contact Info

Product

Resources

About