The effects of substrate orientation on the mechanism of a phosphotriesterase

Jackson, Colin J.; Liu, Jianwei; Coote, Michelle L.; Ollis, David L.

doi:10.1039/b512399b

Cited by 32 publications

(36 citation statements)

References 29 publications

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“…Because the HADs degraded paraoxon but not parathion, it is suggested that both enzymes degrade only organophosphates and not organothiophosphates. Similar trends were observed for SsoPox of Sulfolobus solfataricus and OpdA of A. radiobacter, and this phenomenon is known as the "thiono effect" (22,23).…”

Section: Discussionsupporting

confidence: 63%

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Abe

Yoshida

Suzuki

et al. 2014

Appl Environ Microbiol

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Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) of Burkholderia sp. strain NF100 and organophosphorus hydrolase (OphB) of Burkholderia sp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family.

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Section: Discussionsupporting

confidence: 63%

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Abe

Yoshida

Suzuki

et al. 2014

Appl Environ Microbiol

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“…Electron density in this structure clearly indicated that E open and E closed exist in equilibrium in the crystal; this structure thus provides a key link between E closed seen in wild-type and E open seen in arPTE 8M, because it demonstrates that the same sequence can adopt either CS. Alternate conformations of some of these regions (F132, H254, loop 7) in various wild-type PTE structures have been previously noted (23,25,26), further supporting the notion that E open is not a novel conformation caused by the mutations, but a shift in a preexisting conformational equilibrium. The addition of alternative conformations of these residues improved the accuracy of the model of arPTE 4M (R work /R free decreased from 0.137/0.152 to 0.127/0.144).…”

Section: Resultsmentioning

confidence: 65%

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Jackson

Foo

Tokuriki

et al. 2009

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

115

134

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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...

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“…These metal ions can play structural or catalytic roles, and their incorporation into apoenzyme folding intermediates is an important step in the overall folding pathway (2). One of the most diverse superfamilies of enzymes, which includes a large number of microbial enzymes that are capable of catalyzing the hydrolysis of toxic synthetic compounds (3)(4)(5), is the metal ion-dependent amidohydrolase superfamily (6,7). Unfortunately, many of these proteins form insoluble aggregates or are produced only at very low levels when overexpressed in recombinant systems.…”

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confidence: 99%

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

Jackson

Coppin

Carr

et al. 2014

Appl Environ Microbiol

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Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.A pproximately half of all known proteins contain a metal ion cofactor in their mature form (1). These metal ions can play structural or catalytic roles, and their incorporation into apoenzyme folding intermediates is an important step in the overall folding pathway (2). One of the most diverse superfamilies of enzymes, which includes a large number of microbial enzymes that are capable of catalyzing the hydrolysis of toxic synthetic compounds (3-5), is the metal ion-dependent amidohydrolase superfamily (6, 7). Unfortunately, many of these proteins form insoluble aggregates or are produced only at very low levels when overexpressed in recombinant systems.There is a need to improve heterologous protein expression in order to enable us to characterize proteins in the laboratory or to produce proteins on a large scale for industrial or medical purposes. Heterologous overexpression of proteins is often less than ideal because of protein aggregation or degradation (8). This can arise due to differences between the native folding situation and that which occurs when expression levels are much higher and in a different host, such as a lack of suitable folding chaperones, different pH, high local concentration of the protein, or the absence of a cofactor, which is particularly important for metalloproteins. The technique of laboratory evolution has great potential for improving soluble-protein production and involves subjecting the protein to random mutagenesis and recombination, followed by screening of the many variants that are produced for enhanced expression (9, 10)...

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The effects of substrate orientation on the mechanism of a phosphotriesterase

Cited by 32 publications

References 29 publications

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

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The effects of substrate orientation on the mechanism of a phosphotriesterase

Cited by 32 publications

References 29 publications

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

300-Fold Increase in Production of the Zn 2+ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

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300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization