In Crystallo Capture of a Michaelis Complex and Product-binding Modes of a Bacterial Phosphotriesterase

Jackson, Colin J.; Foo, Jee Loon; Kim, Hye-Kyung; Carr, P.D.; Liu, Jianwei; Salem, Geoffrey; Ollis, David L.

doi:10.1016/j.jmb.2007.10.061

Cited by 85 publications

(102 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Finally, for efficient catalysis, the energy cost of reorganization from E closed S to the transition state E closed S ‡ must be low (3,24). As described previously (22), the distances and angles of the reactants in the enzyme are very similar to those in the computed gas-phase transition state, implying that very little reorganization energy would be required for the transformation of ES into ES ‡ . In summary, the E closed S complex observed in the crystal structure appears very well suited, via electrostatic preorganization, to the reduction of the already relatively low energy barrier for hydrolysis of paraoxon.…”

Section: Resultsmentioning

confidence: 63%

“…Previous structural analyses of the PTEs have identified one, dominant, conformation in the enzyme's crystal, which we refer to as the closed conformation (E closed ). E closed was observed in both the substrate-free enzyme, as well as in the recently solved enzyme-substrate (ES) Michaelis complex of arPTE and the slow substrate 4-methoxylphenyl diethyl phosphate (22), which is structurally very similar to paraoxon. Thus, this is not an example of substrate-induced conformational change.…”

Section: Resultsmentioning

confidence: 92%

“…Third, many PTE variants have been identified through directed evolution experiments (19)(20)(21), as well as natural evolution (14,15), providing a set of enzymes with different catalytic efficiencies and sequence compositions. Finally, crystal structures of several snapshots along the reaction coordinate have been solved (22), providing useful reference structures.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Jackson

Foo

Tokuriki

et al. 2009

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

114

134

View full text Add to dashboard Cite

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies ''open'' and ''closed'' CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.dynamics ͉ enzyme catalysis ͉ evolution ͉ conformational fluctuation A catalyst is defined as a molecule that increases the rate of a chemical reaction by providing an alternative pathway of lower activation energy. A second, sometimes overlooked, requirement of a catalyst is that it is not consumed during the reaction, i.e., it must turn over multiple reactions. With reported rate enhancements of up to 10 17 (1) and turnover rates reaching 10 4 s Ϫ1 (2), enzymes are truly extraordinary catalysts. In addition to efficient rate enhancement of the chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex and product release. So, how does a single protein sequence satisfy these different demands?Our understanding of the mechanisms by which the active sites of enzymes lower the activation energy for various chemical reactions has developed steadily in the past decades and it is clear that the specific organization of chemical groups within the active sites of enzymes provides an electrostatic environment that is markedly different to the conditions in which the uncatalyzed reaction occurs, and results in remarkable rate enhancements (3). There have also been a number of studies showing that transitions between conformational substates (CSs) (4, 5) on a variable energy landscape (6, 7) are an integral part of the full catalytic cycles, or substrate turnover, in many enzymes (7-12). Despite much progress, there is some debate surrounding the exact role that such transitions, or dynamics, play in enzymatic catalysis (7, 13). The deb...

show abstract

Section: Resultsmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 92%

mentioning

confidence: 99%

See 1 more Smart Citation

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Jackson

Foo

Tokuriki

et al. 2009

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

114

134

View full text Add to dashboard Cite

show abstract

“…We also solved a structure of R18 with bound cacodylate, an analogue of the phosphodiester product of paraoxon hydrolysis, (1.50 Å resolution), and a structure of R18 in the absence of any ligand (1.85 Å). The binuclear metal ion centre of PTE at the active site (a-and b-sites) catalyses hydrolysis by stabilizing both the hydroxide nucleophile and the developing negative charge on the TS 23,28,32,33 . The metal ion centre also involves the carboxylation of a lysine residue 34 .…”

Section: Resultsmentioning

confidence: 99%

“…Here, we describe a complete evolutionary trajectory comprising a functional transition from a naturally occurring phosphotriesterase (PTE) from Pseudomonas diminuta that catalyses the hydrolysis of the pesticide paraoxon with high efficiency (k cat /K M ¼ 2.2 Â 10 7 M À 1 s À 1 ) 22,23 , to an arylesterase that catalyses the hydrolysis of 2-naphthyl hexanoate (2NH; Fig. 1).…”

mentioning

confidence: 99%

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

et al. 2012

Self Cite

View full text Add to dashboard Cite

Optimization processes, such as evolution, are constrained by diminishing returns-the closer the optimum, the smaller the benefit per mutation, and by tradeoffs-improvement of one property at the cost of others. However, the magnitude and molecular basis of these parameters, and their effect on evolutionary transitions, remain unknown. Here we pursue a complete functional transition of an enzyme with a 410 9 -fold change in the enzyme's selectivity using laboratory evolution. We observed strong diminishing returns, with the initial mutations conferring 425-fold higher improvements than later ones, and asymmetric tradeoffs whereby the gain/loss ratio of the new/old activity decreased 400-fold from the beginning of the trajectory to its end. We describe the molecular basis for these phenomena and suggest they have an important role in shaping natural proteins. These findings also suggest that the catalytic efficiency and specificity of many natural enzymes may be far from their optimum.

show abstract

Phosphotriesterase

Bigley

Raushel

2004

Handbook of Metalloproteins

View full text Add to dashboard Cite

Phosphotriesterase (PTE) is a member of the amidohydrolase superfamily with the ability to hydrolyze numerous organophosphate substrates including many insecticides and nerve agents. The native active site metal is zinc, but the enzyme is fully active with a variety of metals. The overall fold of PTE is a (β/α) 8 TIM‐barrel with the active site located at the C‐terminal end of the barrel. The active site contains the binuclear metal center with the metal ligands being four histidines, an aspartate, and a carboxylated lysine from the C‐terminal ends of the β‐strands. In addition, the metal center contains a bridging hydroxyl that serves as the nucleophile in the S N 2‐like reaction. The substrate‐binding site is external to the metal center and is made of residues from the loops connecting the C‐terminal end of each β‐strand to the subsequent α‐helix. The best known substrate for PTE is the insecticide paraoxon, which has a kinetic efficiency that approaches the limits of diffusion. PTE has significant potential as an enzyme tool for the detection and decontamination of pesticides and nerve agents. Numerous variants of PTE have been developed by directed evolution with improved kinetic efficiency toward various organophosphates.

show abstract

In Crystallo Capture of a Michaelis Complex and Product-binding Modes of a Bacterial Phosphotriesterase

Cited by 85 publications

References 25 publications

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

Phosphotriesterase

Contact Info

Product

Resources

About