Ben-David M


Full name : Ben-David Moshe

First name : Moshe

Mail : Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100

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Country : Israel

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References (5)

Title : Catalytic stimulation by restrained active-site floppiness--the case of high density lipoprotein-bound serum paraoxonase-1 - Ben-David_2015_J.Mol.Biol_427_1359
Author(s) : Ben-David M , Sussman JL , Maxwell CI , Szeler K , Kamerlin SC , Tawfik DS
Ref : Journal of Molecular Biology , 427 :1359 , 2015
Abstract : Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168--a key catalytic residue residing >15A from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.
ESTHER : Ben-David_2015_J.Mol.Biol_427_1359
PubMedSearch : Ben-David_2015_J.Mol.Biol_427_1359
PubMedID: 25644661

Title : Catalytic metal ion rearrangements underline promiscuity and evolvability of a metalloenzyme - Ben-David_2013_J.Mol.Biol_425_1028
Author(s) : Ben-David M , Wieczorek G , Elias M , Silman I , Sussman JL , Tawfik DS
Ref : Journal of Molecular Biology , 425 :1028 , 2013
Abstract : Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca(2+). We describe the crystal structures of active-site mutants bearing mutations at position 115. The histidine at this position acts as a base to activate the lactone-hydrolyzing water molecule. Mutations to Trp or Gln indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-A upward displacement towards the enzyme's surface of the catalytic Ca(2+) in the His115 mutants and configurational changes in the ligating side chains and water molecules, relative to the wild-type enzyme. Biochemical analysis and molecular dynamics simulations suggest that this alternative, upward metal mode mediates the promiscuous hydrolysis of organophosphates. The upward Ca(2+) mode observed in the His115 mutants also appears to mediate the wild type's paraoxonase activity. However, whereas the upward mode dominates in the Trp115 mutant, it is scarcely populated in wild type. Thus, the plasticity of active-site metal ions may permit alternative, latent, promiscuous activities and also provide the basis for the divergence of new enzymatic functions.
ESTHER : Ben-David_2013_J.Mol.Biol_425_1028
PubMedSearch : Ben-David_2013_J.Mol.Biol_425_1028
PubMedID: 23318950

Title : Catalytic versatility and backups in enzyme active sites: the case of serum paraoxonase 1 - Ben-David_2012_J.Mol.Biol_418_181
Author(s) : Ben-David M , Elias M , Filippi JJ , Dunach E , Silman I , Sussman JL , Tawfik DS
Ref : Journal of Molecular Biology , 418 :181 , 2012
Abstract : The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.
ESTHER : Ben-David_2012_J.Mol.Biol_418_181
PubMedSearch : Ben-David_2012_J.Mol.Biol_418_181
PubMedID: 22387469

Title : Evolved stereoselective hydrolases for broad-spectrum G-type nerve agent detoxification - Goldsmith_2012_Chem.Biol_19_456
Author(s) : Goldsmith M , Ashani Y , Simo Y , Ben-David M , Leader H , Silman I , Sussman JL , Tawfik DS
Ref : Chemical Biology , 19 :456 , 2012
Abstract : A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show <=340-fold increased rates and catalytic efficiencies of 0.2-5 x 10(7) M(-1) min(-1). Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 x 10(-4) in favor of the R(P) isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S(P) isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.
ESTHER : Goldsmith_2012_Chem.Biol_19_456
PubMedSearch : Goldsmith_2012_Chem.Biol_19_456
PubMedID: 22520752

Title : Directed evolution of hydrolases for prevention of G-type nerve agent intoxication - Gupta_2011_Nat.Chem.Biol_7_120
Author(s) : Gupta RD , Goldsmith M , Ashani Y , Simo Y , Mullokandov G , Bar H , Ben-David M , Leader H , Margalit R , Silman I , Sussman JL , Tawfik DS
Ref : Nat Chemical Biology , 7 :120 , 2011
Abstract : Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by approximately 10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) approximately 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
ESTHER : Gupta_2011_Nat.Chem.Biol_7_120
PubMedSearch : Gupta_2011_Nat.Chem.Biol_7_120
PubMedID: 21217689