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Dan S Tawfik

Publications and source records attributed to Dan S Tawfik.

At least 19 recordsLinked to original sources
PubMed2006

The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase.

In essence, evolutionary processes occur gradually, while maintaining fitness throughout. Along this line, it has been proposed that the ability of a progenitor to promiscuously catalyze a low level of the evolving activity could facilitate the divergence of a new function by providing an immediate selective advantage. To directly establish a role for promiscuity in the divergence of natural enzymes, we attempted to trace the origins of a bacterial phosphotriesterase (PTE), an enzyme thought to have evolved for the purpose of degradation of a synthetic insecticide introduced in the 20th century. We surmised that PTE's promiscuous lactonase activity may be a vestige of its progenitor and tested homologues annotated as "putative PTEs" for lactonase and phosphotriesterase activity. We identified three genes that define a new group of microbial lactonases dubbed PTE-like lactonases (PLLs). These enzymes proficiently hydrolyze various lactones, and in particular quorum-sensing N-acyl homoserine lactones (AHLs), and exhibit much lower promiscuous phosphotriesterase activities. PLLs share key sequence and active site features with PTE and differ primarily by an insertion in one surface loop. Given their biochemical and biological function, PLLs are likely to have existed for many millions of years. PTE could have therefore evolved from a member of the PLL family while utilizing its latent promiscuous paraoxonase activity as an essential starting point.

Amino Acid Sequence↗
PubMed2006

Robustness-epistasis link shapes the fitness landscape of a randomly drifting protein.

The distribution of fitness effects of protein mutations is still unknown. Of particular interest is whether accumulating deleterious mutations interact, and how the resulting epistatic effects shape the protein's fitness landscape. Here we apply a model system in which bacterial fitness correlates with the enzymatic activity of TEM-1 beta-lactamase (antibiotic degradation). Subjecting TEM-1 to random mutational drift and purifying selection (to purge deleterious mutations) produced changes in its fitness landscape indicative of negative epistasis; that is, the combined deleterious effects of mutations were, on average, larger than expected from the multiplication of their individual effects. As observed in computational systems, negative epistasis was tightly associated with higher tolerance to mutations (robustness). Thus, under a low selection pressure, a large fraction of mutations was initially tolerated (high robustness), but as mutations accumulated, their fitness toll increased, resulting in the observed negative epistasis. These findings, supported by FoldX stability computations of the mutational effects, prompt a new model in which the mutational robustness (or neutrality) observed in proteins, and other biological systems, is due primarily to a stability margin, or threshold, that buffers the deleterious physico-chemical effects of mutations on fitness. Threshold robustness is inherently epistatic-once the stability threshold is exhausted, the deleterious effects of mutations become fully pronounced, thereby making proteins far less robust than generally assumed.

Epistasis, Genetic↗
PubMed2006

Reconstruction of functional beta-propeller lectins via homo-oligomeric assembly of shorter fragments.

The modular nature of protein folds suggests that present day proteins evolved via duplication and recombination of smaller functional elements. However, the reconstruction of these putative evolutionary pathways after many millions of years of evolutionary drift has thus far proven difficult, with all attempts to date failing to produce a functional protein. Tachylecin-2 is a monomeric 236 amino acid, five-bladed beta-propeller with five sugar-binding sites. This protein was isolated from a horseshoe crab that emerged ca 500 million years ago. The modular, yet ancient, nature of Tachylectin-2 makes it an excellent model for exploring the evolution of proteins from smaller subunits. To this end, we generated genetically diverse libraries by incremental truncation of the Tachylectin-2 gene and screened them for functional lectins. A number of approximately 100 amino acid residue segments were isolated with the ability to assemble into active homo-pentamers. The topology of most of these segments follows a "hidden" module that differs from the modules observed in wild-type Tachylectin-2, yet their biophysical properties and sugar binding activities resemble the wild-type's. Since the pentamer's molecular mass is twofold higher than the wild-type (approximately 500 amino acid residues), the structure of these oligomeric forms is likely to also differ. Our laboratory evolution experiments highlight the versatility and modularity of the beta-propeller fold, while substantiating the hypothesis that proteins with high internal symmetry, such as beta-propellers, evolved from short, functional gene segments that, at later stages, duplicated, fused, and rearranged, to yield the folds we recognise today.

Amino Acid Sequence↗
PubMed2006

Enzyme promiscuity: evolutionary and mechanistic aspects.

The past few years have seen significant advances in research related to the 'latent skills' of enzymes - namely, their capacity to promiscuously catalyze reactions other than the ones they evolved for. These advances regard (i) the mechanism of catalytic promiscuity - how enzymes, that generally exert exquisite specificity, promiscuously catalyze other, and sometimes barely related, reactions; (ii) the evolvability of promiscuous functions - namely, how latent activities evolve further, and in particular, how promiscuous activities can firstly evolve without severely compromising the original activity. These findings have interesting implications on our understanding of how new enzymes evolve. They support the key role of catalytic promiscuity in the natural history of enzymes, and suggest that today's enzymes diverged from ancestral proteins catalyzing a whole range of activities at low levels, to create families and superfamilies of potent and highly specialized enzymes.

Animals↗
PubMed2006

The 192R/Q polymorphs of serum paraoxonase PON1 differ in HDL binding, lipolactonase stimulation, and cholesterol efflux.

Serum paraoxonase (PON1) is a HDL-associated enzyme exhibiting potentially antiatherogenic properties. Here, we examined the common PON1-192R/Q human polymorphism. Despite numerous studies, the effect of this polymorphism on the antiatherogenic potential of PON1 is yet unresolved. Our structural model suggests that amino acid 192 constitutes part of the HDL-anchoring surface and active site of PON1. Based on our findings that PON1 is an interfacially activated lipolactonase that selectively binds HDL carrying apolipoprotein A-I (apoA-I) and is thereby greatly stabilized and catalytically activated, we examined the interaction of the PON1-192 isozymes with reconstituted HDL-apoA-I particles. We found that PON1 position 192 is indeed involved in HDL binding. The PON1-192Q binds HDL with a 3-fold lower affinity than the R isozyme and consequently exhibits significantly reduced stability, lipolactonase activity, and macrophage cholesterol efflux. We also observed the lower affinity and stability of the 192Q versus the 192R isozyme in sera of individuals belonging to the corresponding genotypes. The observed differences in the properties of PON1-192R/Q isozymes provide a basis for further analysis of the contribution of the 192R/Q polymorphism to the susceptibility to atherosclerosis, although other factors, such as the overall levels of PON1, may play a more significant role.

Aryldialkylphosphatase↗
PubMed2006

Miniaturising the laboratory in emulsion droplets.

Biochemical and genetic assays can be both miniaturized and parallelized by compartmentalization in living cells. In vitro compartmentalization (IVC) offers an alternative strategy based on partitioning reactions in water droplets dispersed to form microscopic compartments in water-in-oil emulsions. The cell-like volumes of these compartments (as low as one femtolitre), the ability to freely determine and regulate their content and the large number of compartments (>10(10) per millilitre emulsion) have provided the basis for a range of new, ultra-high-throughput, cell-free technologies. This review describes the scope and potential of IVC in areas such as in vitro evolution of proteins and RNAs, cell-free cloning and sequencing, genetics, genomics, and proteomics.

Cell-Free System↗
PubMed2006

Mechanism of hydrolysis and aminolysis of homocysteine thiolactone.

Homocysteine thiolactone (tHcy) is deemed a risk factor for cardiovascular diseases and strokes, presumably because it acylates the side chain of protein lysine residues ("N-homocysteinylation"), thereby causing protein damage and autoimmune responses. We analysed the kinetics of hydrolysis and aminolysis of tHcy and two related thiolactones (gamma-thiobutyrolactone and N-trimethyl-tHcy), and we have thereby described the first detailed mechanism of thiolactone aminolysis. As opposed to the previously studied (thio and oxo)esters and (oxo)lactones, aminolysis of thiolactones was found to be first order with respect to amine concentration. Anchimeric assistance by the alpha-amino group of tHcy (through general acid/base catalysis) could not be detected, and the Brønsted plot (nucleophilicity versus pK(a)) for aminolysis yielded a slope (beta(nuc)) value of 0.66. These data support a mechanism of aminolysis where the rate-determining step is the formation of a zwitterionic tetrahedral intermediate. The beta(nuc) value and steric factors dictate a regime whereby, at physiological pH values (pH 7.4), maximal reactivity of tHcy is exhibited with primary amine groups with a pK(a) value of 7.7; this allows the reactivity of various protein amino groups towards N-homocysteinylation to be predicted.

4-Butyrolactone↗
PubMed2006

Evolution of new protein topologies through multistep gene rearrangements.

New protein folds have emerged throughout evolution, but it remains unclear how a protein fold can evolve while maintaining its function, particularly when fold changes require several sequential gene rearrangements. Here, we explored hypothetical evolutionary pathways linking different topological families of the DNA-methyltransferase superfamily. These pathways entail successive gene rearrangements through a series of intermediates, all of which should be sufficiently active to maintain the organism's fitness. By means of directed evolution, and starting from HaeIII methyltransferase (M.HaeIII), we selected all the required intermediates along these paths (a duplicated fused gene and duplicates partially truncated at their 5' or 3' coding regions) that maintained function in vivo. These intermediates led to new functional genes that resembled natural methyltransferases from three known classes or that belonged to a new class first seen in our evolution experiments and subsequently identified in natural genomes. Our findings show that new protein topologies can evolve gradually through multistep gene rearrangements and provide new insights regarding these processes.

Amino Acid Sequence↗
PubMed2006

The histidine 115-histidine 134 dyad mediates the lactonase activity of mammalian serum paraoxonases.

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 lipoprotein-associated 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 pH-rate profile determined for the lactonase activity of PON1 indicated an apparent pK(a) of approximately 7.4. We thus explored the role of all amino acids in the PON1 active site that are not directly ligated to the catalytic calcium and that possess an imidazolyl or carboxyl side chain (His(115), His(134), His(184), His(285), Asp(183), and Asp(269)). Extensive site-directed mutagenesis studies in which each amino acid candidate was replaced with all other 19 amino acids were conducted to identify the residue(s) that mediate the lactonase activity of PONs. The results indicate that the lactonase activity of PON1 and PON3 and the esterase activity of PON1 are mediated by the His(115)-His(134) dyad. Notably, the phosphotriesterase activity of PON1, which is a promiscuous activity of this enzyme, is mediated by other residues. To our knowledge, this is one of few examples of a histidine dyad in enzyme active sites and the first example of a hydrolytic enzyme with such a dyad.

Animals↗
PubMed2006

The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux.

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by approximately 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.

Animals↗
PubMed2006

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase.

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was approximately 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers.

Animals↗
PubMed2005

Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily.

The amidohydrolase superfamily comprises hundreds of hydrolytic enzymes of the (beta/alpha)8 barrel fold with mono- or binuclear active-site metal centers, and a diverse spectrum of substrates and reactions. Promiscuous activities, or cross-reactivities, between different members of the same superfamily may provide important hints regarding evolutionary and mechanistic relationships. We examined three members: dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP). Of particular interest are PTE, which is thought to have evolved within the last several decades, and PHP, an amidohydrolase superfamily member of unknown function, and the closest known homologue of PTE. We found a diverse and partially overlapping pattern of promiscuous activities in these enzymes, including a significant lactonase activity in PTE, esterase activities in both PTE and PHP, and a weak PTE activity in DHO. Directed evolution was applied to improve the promiscuous esterase activities of PTE and PHP. Remarkably, the most recurrent mutation increasing esterase activity in PTE, or PHP, maps to the same location in their superposed 3D structures. The evolved variants also exhibit newly acquired promiscuous activities that were not selected for, including very weak, yet measurable, paraoxonase activity in PHP. Our results illustrate the mechanistic, structural, and evolutionary links between these enzymes, and highlight the importance of studying laboratory evolution intermediates that might resemble node intermediates along the evolutionary pathways leading to the divergence of enzyme superfamilies.

Amidohydrolases↗
PubMed2005

High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I.

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles. We found that PON1 binds apoA-I HDL with sub-nanomolar affinities (K(d) << 10(-)(9) M) and slow dissociation rates (t(1/2) > 80 min), while binding affinity for other particles was dramatically lower. A truncated form of PON1 lacking the N-terminal helix maintains considerable binding to apoA-I HDL (K(d) = 1.2 x 10(-)(7) M), validating the structural model which indicates additional parts of the enzyme involved in HDL binding. Kinetic inactivation assays revealed the existence of an equilibrium between two forms of PON1 differing in their stability by a factor of 100. Various lipoproteins and detergent preparations shift this equilibrium toward the more stable conformation. Consistent with its highest affinity, only apoA-I HDL is capable of totally shifting the equilibrium toward the stable form. The paraoxonase and arylesterase activities were stimulated by HDL by 2-5-fold as previously reported, almost independently of the apoliporotein content. In contrast, only apoA-I is capable of stimulating the lactonase activity by <or=20-fold to k(cat)/K(M) values of 10(6)-10(7) M(-)(1) s(-)(1), while apoA-IV and apoA-II have almost no effect. Overall, the results indicate the high stability, selectivity, and catalytic proficiency of PON1 when anchored onto apoA-I HDL, toward lactone substrates, and lipophilic lactones in particular.

Apolipoprotein A-I↗
PubMed2005

Structure and kinetics of a transient antibody binding intermediate reveal a kinetic discrimination mechanism in antigen recognition.

Induced fit is a predominant phenomenon in protein-ligand interactions, yet it is invariably attributed without establishing the existence, let alone the structure, of the initial, low-affinity encounter complex. We determined the crystal structure of the encounter complex on the pathway of ligand binding by IgE antibody SPE7. We show that this complex is formed by a wide range of ligands that initially bind with identical affinity. Nonspecific ligands rapidly dissociate, whereupon the antibody isomerizes to a nonbinding isomer. Specific ligand complexes, however, slowly isomerize to give a high-affinity complex. This isomerization involves backbone and side-chain rearrangements of up to 14 A and the formation of specific hydrogen bonds. The postbinding conformational switch, combined with the prebinding isomerization to an energetically favorable nonbinding isomer, results in a "kinetic discrimination" mechanism that mediates selective binding, by a factor of >10(3), between highly related ligands that initially bind with the same affinity. This model may apply to proteins that bind multiple ligands in a specific manner or other proteins that, although capable of binding many ligands, are activated by only a few.

Anthracenes↗