Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Aharoni, Amir; Gaidukov, Leonid; Yagur, Shai; Toker, Lilly; Silman, Israel; Tawfik, Dan S.

doi:10.1073/pnas.2536901100

Cited by 267 publications

(229 citation statements)

References 31 publications

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“…Our results suggest that to alter the substrate specificity of CEs, either a detailed knowledge of the structure of the enzyme is necessary or a selection system for evolution of a desired catalytic activity is required. 35,36 As mentioned above, the hCE1m6 protein that contains eight amino-acid substitutions can metabolize CPT-11 with approximately the same efficiency as hiCE and about 9-fold less than rCE (based on the k cat /K m values). However, the converse eight mutations generated in the rCE protein did not completely abolish the CPT-11-converting ability of this CE.…”

Section: Discussionmentioning

confidence: 93%

An improved human carboxylesterase for enzyme/prodrug therapy with CPT-11

et al. 2008

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CPT-11 is a potent antitumor agent that is activated by carboxylesterases (CE) and intracellular expression of CEs that can activate the drug results in increased cytotoxicity to the drug. As activation of (1-piperidino)-1-piperidino]carbonyloxycamptothecin) by human CEs is relatively inefficient, we have developed enzyme/prodrug therapy approaches based on the CE/CPT-11 combination using a rabbit liver CE (rCE). However, the in vivo application of this technology may be hampered by the development of an immune response to rCE. Therefore, we have developed a mutant human CE (hCE1m6), based on the human liver CE hCE1, that can activate CPT-11 approximately 70-fold more efficiently than the wild-type protein and can be expressed at high levels in mammalian cells. Indeed, adenoviral-mediated delivery of hCE1m6 with human tumor cells resulted in up to a 670-fold reduction in the IC 50 value for CPT-11, as compared to cells transduced with vector control virus. Furthermore, xenograft studies with human tumors expressing hCE1m6 confirm the ability of this enzyme to activate CPT-11 in vivo and induce antitumor activity. We propose that this enzyme should likely be less immunogenic than rCE and would be suitable for the in vivo application of CE/CPT-11 enzyme/prodrug therapy.

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Section: Discussionmentioning

confidence: 93%

An improved human carboxylesterase for enzyme/prodrug therapy with CPT-11

et al. 2008

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“…(ii) The cross-linking between pac-PC and PON1/rHDL with pac-PC (human apoA-I concentration of 70 M) in 0.5 ml of 50 mM PBS buffer at pH 7.0 was incubated with recombinant Histagged PON1 (rPON1, 30 M, in 0.5 ml of 50 mM Tris buffer with 1 mM CaCl 2 , pH 7.0) that was expressed and purified from Escherichia coli, as described previously (60). The mixture was incubated at 37°C for 1 h. This mixture was then transferred into small glass vials on ice and directly exposed to a 365-nm UV light for 1 min.…”

Section: Methodsmentioning

confidence: 99%

Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction

Gu¹,

Huang²,

Levison³

et al. 2016

Journal of Biological Chemistry

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However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry-taggable phospholipid probe, which, when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu-9, Tyr-185, and Tyr-293) were identified through covalent crosslinks with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu-9, Tyr-185, and Tyr-293), coupled with functional studies, reveals their importance in PON1 binding to HDL and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into the PON1-HDL interaction. More generally, the new photoactivatable and affinity-tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes or lipoproteins.

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“…PON1 and PON3 reside on high-density lipoprotein (HDL; 'good cholesterol') and are involved in the prevention of artherosclerosis (Draganov & La Du, 2004). Directed evolution via 'family shuffling' and screening resulted in the production of mammalian PON1 variants that express in a soluble and active form in E. coli (Aharoni et al, 2004). This in turn permitted purification, subsequent crystallization and solution of the threedimensional structure of one such variant to 2.2 Å resolution ( Fig.…”

Section: Directed Evolutionmentioning

confidence: 99%

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value

Banci

Bertini

Cusack

et al. 2006

Acta Crystallogr D Biol Cryst

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The EC `Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, ∼170 structures have been determined to date. Here, the impact of high‐throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

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Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Cited by 267 publications

References 31 publications

An improved human carboxylesterase for enzyme/prodrug therapy with CPT-11

An improved human carboxylesterase for enzyme/prodrug therapy with CPT-11

Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value

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