The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide...

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Biochimica et Biophysica A

The structure of an enzyme–product complex reveals the critical role

of a terminal hydroxide nucleophile in the bacterial

phosphotriesterase mechanism

Colin Jackson, Hye-Kyung Kim, Paul D. Carr, Jian-Wei Liu, David L. Ollis*

Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia

Received 12 April 2005; received in revised form 8 June 2005; accepted 9 June 2005

Available online 13 July 2005

Abstract

A detailed understanding of the catalytic mechanism of enzymes is an important step toward improving their activity for use in

biotechnology. In this paper, crystal soaking experiments and X-ray crystallography were used to analyse the mechanism of the

Agrobacterium radiobacter phosphotriesterase, OpdA, an enzyme capable of detoxifying a broad range of organophosphate pesticides. The

structures of OpdA complexed with ethylene glycol and the product of dimethoate hydrolysis, dimethyl thiophosphate, provide new details

of the catalytic mechanism. These structures suggest that the attacking nucleophile is a terminally bound hydroxide, consistent with the

catalytic mechanism of other binuclear metallophosphoesterases. In addition, a crystal structure with the potential substrate trimethyl

phosphate bound non-productively demonstrates the importance of the active site cavity in orienting the substrate into an approximation of

the transition state.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Phosphotriesterase; Mechanism; Binuclear; Metalloenzyme; Nucleophile

1. Introduction

Organophosphate triesters (OPs) have been used as

pesticides for over 60 years [1]. They function by the

irreversible inhibition of acetylcholinesterase (AChE), thus

preventing nerve signal transduction and causing the death

of affected organisms. Commercially used organophosphate

pesticides are typically phosphothionates, such as parathion

or phosphorothiolate compounds such as dimethoate (Fig.

1). Over the years a number of enzymes capable of

breaking down OPs have been identified. These include the

bacterial phosphotriesterases from Agrobacterium radiobacter

1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbapap.2005.06.008

Abbreviations: AChE, acetylcholinesterase; OpdA, organophosphate

degrading enzyme; PTE, phosphotriesterase; PEG, polyethylene glycol;

EGL, ethylene glycol; DMTP, dimethyl thiophosphate; TMP, trimethyl

phosphate

* Corresponding author. Tel.: +61 2 6125 4733; fax: +61 2 6125 0750.

E-mail address: [email protected] (D.L. Ollis).

(OpdA) [2] and Pseudomonas diminuta (PTE) [3]. Despite a

great deal of work our understanding of the catalytic mechanism

of these phosphotriesterases is still far from complete.

OpdA [2] is very similar to PTE [3] both in terms of

sequence (90% sequence identity) and structure, and it is

assumed that their mechanisms are essentially identical [4].

The interest in the mechanism of these enzymes is

heightened as a result of their potential utility in the

detoxification of organophosphate pesticides and related

chemical warfare agents such as VX and sarin. For this

reason, the structures of OpdA and PTE have been solved

crystallographically, and both adopt an (a/h)8 barrel tertiarystructure with a binuclear metal centre at the active site. A

carboxylated lysine and a hydroxide ion bridge the metals,

while the a-metal (as defined by Benning et al. [5]) is

further coordinated by the residues His55, His57 and

Asp301, and the h-metal by His201 and His230 [4,5]. Both

proteins are active with a variety of metals. Most of the

physical characterisation of the PTE enzyme has been done

cta 1752 (2005) 56 – 64

Fig. 1. The phosphotriesters methyl parathion and trimethyl phosphate, and the phosphorothiolate dimethoate.

C. Jackson et al. / Biochimica et Biophysica Acta 1752 (2005) 56–64 57

with Zn2+ in the active site, but both proteins are more

active toward phosphothionates with Co2+ [4,6]. Studies of

PTE complexed with substrate analogues have provided

some insight into the catalytic mechanism. For instance, in

the case of a PTE-diisopropyl methyl phosphonate complex,

the inhibitor was found to be coordinated to the more

solvent-exposed h-metal [7]. However, in some of these

structures, the substrate analogues are assumed to have

bound in incorrect orientations [7,8]. It has been proposed

that the attacking nucleophile in the reaction is the bridging

hydroxide. The alternative proposal involving attack by a

water molecule bound to the a-metal has not been made, in

part because ligands have not been observed to bind in a

position appropriate for attack of the substrate.

Kinetic studies of PTE have demonstrated that hydroly-

sis proceeds through an Sn2-type catalytic mechanism [9].

The a-metal has been shown to determine the strength of

the attacking nucleophile while the h-metal affects sub-

strate binding [10]. That study also demonstrated that PUO

bond hydrolysis in substrates such as parathion and

paraoxon, with leaving groups that have pKa values below

the pH of the reaction, is catalysed at diffusion-limited

rates. This result is complemented by theoretical studies of

paraoxon hydrolysis that have demonstrated that no

intermediate is formed during hydrolysis in the gas phase,

and nucleophilic attack occurs in concert with departure of

the leaving group [11]. In contrast, characterisation of PUS

bond hydrolysis has been less thorough. The most detailed

studies to date have shown that there are significant

mechanistic differences, and that the catalytic rate of

PUS bond hydrolysis in phosphorothiolates such as

demeton and dimethoate is considerably slower than the

rate of PUO bond hydrolysis in phosphothionates such as

parathion and coumaphos [4,12]. Theoretical calculations

have also shed light on these results, demonstrating that a

stable pentacoordinate intermediate is formed during PUS

bond hydrolysis in an analogue of VX [13].

The previously reported structure of OpdA was crystal-

lised at pH 5.5 and had a sulfite ion bound tridentately at the

active site, displacing the bridging hydroxide [4]. We have

found new crystallisation conditions that produce a more

physiologically relevant structure of the protein with Co2+ in

the active site. We have soaked these new OpdA crystals in

separate solutions containing the rapidly hydrolysed sub-

strate parathion, the slowly hydrolysed substrate dimethoate,

or the potential substrate trimethyl phosphate. The structures

of these complexes have led to an alternative explanation of

the catalytic mechanism utilised by OpdA. We suggest that

the hydroxide nucleophile is terminally coordinated to the

a-metal and that the active site surface has an important role

to play orienting the substrate for hydrolysis.

2. Materials and methods

2.1. Strains, plasmids and chemicals

The plasmid used to express OpdA was the same as that

previously described [4]. Growth media was supplemented

with ampicillin (100 Ag/mL). Methyl parathion and dime-

C. Jackson et al. / Biochimica et Biophysica Acta 1752 (2005) 56–6458

thoate were purchased from Chem Service (Aus). Trimethyl

phosphate (TMP) was purchased from Sigma. The purity of

the organophosphates was >95% as stated by the manu-

facturers. Molecular biology reagents were purchased from

New England Biolabs or Roche and other chemicals

purchased from Sigma.

2.2. Purification and crystallisation of OpdA

For crystallisation purposes, the enzyme was expressed,

purified and treated as previously described, including

addition of Co2+ to the storage buffer (50 mM HEPES, pH

7.0, 1 mM CoCl2) to ensure saturation of the metal binding

sites [4]. Crystals were formed using vapour diffusion of

hanging drops, grown from a mixture of 5 AL protein

solution and 5 AL of reservoir solution, consisting of 20%

PEG 3350, 0.2 M NaNO3, pH 6.6, and grew within 2

weeks.

2.3. Crystal soaking experiments

Crystals of OpdA were transferred to a cryoprotectant

solution identical to the reservoir solution, in which the PEG

3350 concentration was increased to 40% with 1 mM

dimethoate for less than one min, or either 5 mM dimethoate,

150 mM trimethyl phosphate (TMP), or 10 mM parathion for

10 min before flash cooling at �173 -C.

2.4. Data collection

Diffraction data of the crystals were collected on a

Rigaku R-axis IIC, using Cu Ka radiation. Crystals were

transferred to a cryoprotectant solution, with or without

substrate, and flash-cooled to �173 -C in a stream of

nitrogen gas. All data reduction was performed using

DENZO and SCALEPACK [14].

2.5. Structure determination

Crystals were isomorphous to those previously solved

[4]. Initial protein phases were calculated using the refined

OpdA structure [4] as a model, omitting the bound SO3�

molecule from the active site. Refinement was undertaken

using the program REFMAC [15], as implemented in the

CCP4 suite of programs [16]. Structures of ethylene glycol

(EGL), trimethyl phosphate (TMP) and dimethyl thiophos-

phate (DMTP) were created using the monomer library

sketcher as implemented in the CCP4 suite of programs

[15], and XPLO2D [17].

In the case of the crystals soaked in dimethoate for less

than 1 min, or soaked in parathion for 10 min, inspection of

difference Fourier maps indicated that a water was

coordinated terminally to the h-metal. An ethylene glycol

molecule was modelled into the positive density observed

terminal to the a-metal and refined at full occupancy,

without residual density, but with B-factors ¨3 times higher

than the coordinated metal. The Co2+ ions present in the

original model at full occupancy refined in all structures

with reasonable B-factors.

Difference Fourier maps of crystals soaked in dime-

thoate for 10 min showed density at the active site

corresponding to a tetrahedral molecule dually bound to

the active site metals. Assignment of the sulfur and

hydroxyl groups was made on the basis of the greater

electron density seen for the sulfur, and the longer PjS

bond. A molecule of the product, dimethyl thiophosphate

(DMTP) (Fig. 3), was modelled into the density and refined

at full occupancy.

Difference Fourier maps of crystals soaked in TMP

displayed positive density corresponding to a tetrahedral

molecule bound terminally to the a-metal. Trimethyl

phosphate was modelled into this density and refined at full

occupancy, without residual density, and with B-factors ~3

times that of the coordinated metal, accommodating each

methyl side chain and showing no sign of reaction with the

water molecule terminally bound to the h-metal.

3. Results and discussion

3.1. Quality of the structures

Crystal structures of OpdA soaked in dimethoate,

parathion or trimethyl phosphate were refined. The stereo-

chemical correctness of each structure was checked with

the programs PROCHECK [18] and SFCHECK [19]. The

Ramachandran plot [20] showed that all residues were in

the most favoured or additionally allowed regions with the

exception of Glu159 that has been an outlier in Ram-

achandran plots of all known PTE structures [5] and is

found in a type IIV reverse turn [5,21] at the dimer axis,

remote from the active site (5). Parameters relating to the

stereochemistry were all within normal limits when tested

with PROCHECK (Table 1).

3.2. Active site and metal ligation

A schematic of the active site of OpdA is shown in Fig. 2.

As indicated in this figure the metal coordinating ligands are

similar to those reported for PTE-Zn2+ [5], although the

ligands are arranged in distorted octahedra around both

metals in the case of OpdA-Co2+. At the a-metal, the

carboxylated lysine, K169, and D301 are coordinated

axially, while the bridging hydroxide, H55, H57 and either

an ethylene glycol (EGL) molecule, the PUOH group of

dimethyl thiophosphate (DMTP), or the PjO group of

trimethyl phosphate (TMP) comprise the equatorial ligands.

The bond length between the PUOH group of DMTP and

the metal is 0.5 A shorter than that to the EGL or TMP

molecules in the other structures (Fig. 3), suggesting it is

more tightly coordinated. The coordination of the h-metal is

also octahedral in the EGL and TMP complexes: the

Fig. 2. A comparison of the metal coordination between PTE–Zn2+ (lower)

and OpdA–Co2+ (upper in complex with EGL/TMP, middle in complex

with DMTP). The octahedral coordination of the metals in OpdA and the

trigional bipyramidal coordination of the metals in PTE are shown.

Table 1

Data collection and refinement statistics

DMTP

(<1 min)

DMTP

(10 min)

TMP

(10 min)

Data collection

Space group P3121

a =109.2,

c =63.2 A

P3121

a =109.0,

c =62.3 A

P3121

a =109.0,

c =62.4 A

No. of observations 252561 193584 205450

No. of unique reflections 42577 34142 36356

Completeness (%) 96.6 92.7 91.5

<I/r(I)> 25.7 18.9 19.4

Rscal (%) (overall/outer shell) 4.0/14.2 4.7/16.7 4.4/22.6

Refinement

Resolution range (A) 28.0–1.75 25.0–1.85 19.6–1.80

Reflections in working set 40420 32517 34612

Reflections in test set 2157 1625 1742

R/Rfree (%) (overall/

outer shell)

17.1/19.6 16.7/19.4 18.8/21.4

No. of protein atoms 2511 2511 2511

No. of water molecules 330 317 322

No. of Co2+ ions 2 2 2

No. of dimethylthiophosphate

molecules

0 1 0

No. of ethylene

glycol molecules

2 0 0

No. of trimethyl phosphate

molecules

0 0 1

R.m.s deviation from target bonds

Lengths (A) 0.014 0.013 0.012

Angles (-) 1.345 1.320 1.319

Mean B-factors (A2)

Main chain 15.3 13.8 13.2

Side chain 17.4 15.8 15.2

Metals 15.7 14.2 13.9

Ligands 39.6 17.2 36.6

Waters 28.4 26.6 26.6

Ramachandran plot (%)

Most favoured region 90.1 90.1 89.4

Additionally allowed 9.6 9.6 10.3

Generously allowed 0.4 0.4 0.4

Disallowed 0 0 0


carboxylated lysine, K169, and a water molecule are

coordinated axially, while the bridging hydroxide, H201,

H230 and a water/hydroxide are the equatorial ligands. In

the case of the OpdA–DMTP complex, the h-metal is

coordinated in a trigonal bipyramidal arrangement, with

the equatorial water ligand replaced by the sulfur of

DMTP, and the movement of R254 displacing the axial

water (Fig. 3).

Ethylene glycol was observed bound at the a-metal of

OpdA structures soaked in 1 mM dimethoate for less than 1

min, suggesting that at least 10 min are required for the

substrate to permeate the crystal. EGL was also observed

bound at the a-metal of the OpdA crystal soaked in

parathion. The ethylene glycol molecules are present either

as a result of degradation of the PEG 3350 used in the

crystallisation, or as an ordered section of a PEG chain.

The B-factor of the ethylene glycol molecule was ¨3 times

higher than that of the metal, indicating that the molecules

were not tightly coordinated. Two water molecules were

observed coordinated to the h-metal, R254 and D233, as

shown in Fig. 3.

The structure of OpdA refined from crystals soaked in

TMP was essentially identical to the apo-enzyme structure,

with the exception that EGL was replaced by TMP. There

was no sign of any interaction between the terminally

coordinated water at the h-metal and the electrophilic

phosphorous centre of TMP. The distance between the two

atoms was 3.8 A (Figs. 4 and 5). The B-factors of the atoms

within TMP were 40T1 A2. This was significantly higher

than the B-factor of the single coordinating metal, which

was 15.1 A2. This initially suggested that the ligand was

present at low occupancy, although when the occupancy

was lowered, positive density arose at the site of the TMP

Fig. 4. The non-productive binding of trimethyl phosphate to OpdA,

contrasted with methyl parathion manually modelled in the active site of

OpdA based on the coordination of the product DMTP. The leaving group

pocket formed by W131, F132, H230, H201, R254, D301 and L271 causes

the substrate to tilt toward the nucleophile upon binding at the h-metal. This

also highlights the change in the molecular surface as a consequence of the

movement of R254.

Fig. 3. The coordination of ethylene glycol (EGL, top) and dimethyl

thiophosphate (DMTP, bottom) to the binuclear metal centre of OpdA.

Differences in metal coordination and the orientation of R254 are shown.

To make the changes clear, side chains ligated to the metals are not shown.


molecule, and the B-factors could not be reduced to a

comparable value to the coordinating metal. We therefore

propose that the site is fully occupied and that the B-factors

reflect genuine mobility, most probably a result of rapid

exchange and rotation, consistent with the electron density

maps that display poor density around the methyl sidechains

(Fig. 6). The high B-factors and apparent mobility of the

ligand are consistent with the nature of its terminal

coordination by a single cobalt atom. The alternative

explanation for the poor density around the methyl side-

chains would be that OpdA hydrolyzed all of the TMP,

followed by all of the product, DMP, within 10 min of

crystal soaking. This can be ruled out, as OpdA does not

catalyse the hydrolysis of TMP.

Dimethoate was soaked into crystals of OpdA for 10 min,

and although the intermediate in the hydrolysis had already

decomposed, the product dimethyl thiophosphate (DMTP) is

present, shown in Fig. 3. DMTP is bound in a bidentate

fashion, the PjS group bound to the more exposed h-metal,

and the PUOH group bound at the a-metal. The greater

electron density of the sulfur, and the longer PjS bond made

assignment of the PjS and PUOH groups unequivocal.

Another significant difference to the other structures is the

conformation of R254. In the absence of dimethoate, it is

oriented away from the active site; however, upon binding of

dimethoate, its conformation changes, displacing the two

water molecules seen in the apo-enzyme structure. This

results in the NH1 moiety of R254 forming a hydrogen bond

to a side chain oxygen of the substrate/product, presumably

aiding orientation or stabilising the molecule (Fig. 3). The B-

factor of the molecule was comparable to that of its

coordinating ligands: the B-factors of the metal coordinated

oxygen and sulfur, and the oxygen coordinated to R254 were

16.6, 14.6 and 17.4 A2, respectively. The B-factors of the

coordinating metals and NH1 group were 13.4, 15.1 and 15.7

A2, respectively. This suggests there was little exchange of

DMTP within the crystal and that it was tightly coordinated.

The tight coordination of DMTP contrasts with that of TMP

and is consistent with coordination by the two cobalt atoms

and R254. An OMITelectron density map is shown in Fig. 6.

Fig. 5. Tilting of the substrate is required to move the nucleophile and electrophile into an approximation of the transition state.


This shows the bound ligand is certainly DMTP, and that the

larger sulfur atom is clearly coordinated to the h-metal.

The metal coordination in the OpdA–Co2+ crystals

resembles that of PTE–Zn2+ even though the two molecules

have different numbers of ligands [5] (Fig. 2). The geometry

of the coordination sphere in PTE is best described as

distorted trigonal bipyramid. At the a-metal, PTE lacks the

EGL ligand found in OpdA and as a consequence has

equatorial bond angles closer to those required for trigonal

symmetry. For example, the angle formed between the

ligating nitrogen of H55, the a-metal and the ligating

nitrogen of H57 is 116- in PTE while in the OpdA–EGL

complex the same angle is only 103-.

3.3. The mechanism of OpdA

The structures of these OpdA-product/substrate com-

plexes have some implications for the mechanism of the

protein. The observation of equatorial ligands at the a-metal

(EGL and TMP) suggests that water/hydroxide could bind at

this site, and the structure of the OpdA–DMTP complex

suggests such a terminal metal-hydroxide is the attacking

nucleophile in the reaction, as shown in Fig. 5. The

bidentate binding of DMTP to the two metals is most likely

a consequence of dimethoate initially binding at the h-metal

via the PjS group, nucleophilic attack by a hydroxide

terminally coordinated to the a-metal and rapid departure of

the leaving group, producing DMTP dually bound to the

two metals (Scheme 1). The roles of the two metals are

therefore straightforward: the substrate binds at the h-metal

and the phosphorous is made more electrophilic, and a water

molecule binds at the a-metal and is converted to a metal-

hydroxide nucleophile. This proposal is consistent with the

results of a previous mechanistic study of PTE [10]; namely,

the strength of the nucleophile is determined by the a-metal,

and the nature of the h-metal affects substrate binding. The

bridging hydroxide may therefore exist as a necessary

structural feature of the active site, as its distance to the

phosphorous of the bound substrate is likely to be ~4 A,

making it an unsuitable nucleophile. This is consistent with

previous studies of inorganic complexes demonstrating that

the alignment of a bridging hydroxide and a terminally

bound phosphate ester is not favourable to nucleophilic

attack [22]. Further reinforcing this, TMP showed no sign of

interaction with the terminally bound water: if the bridging

hydroxide were the nucleophile, hydrolysis should be

possible from either metal as the bridging hydroxide is

essentially equidistant. Although TMP has a very poor

leaving group, which will require protonation, this should

not prevent interaction of the electrophilic phosphorous with

an hydroxide nucleophile and formation of a pentacoordi-

nate intermediate, which we see no indication of in the

crystal structure.

In contrast to crystals soaked in dimethoate, no product

was observed in the active site of crystals soaked with

parathion. Significant differences between the hydrolysis of

phosphotriesers and phosphorothiolates have been described

previously [4,12] and although the evidence presented here

is insufficient to make any definite conclusions relating to

the nature of this difference, it appears that the formation, or

nature, of a dually bound product is important. A

comparison of the B-factors of EGL, TMP and DMTP

gives some indication of this: DMTP had similar B-factors

to its surrounding ligands, indicating that it was tightly

coordinated to both metals and R254. In contrast, EGL and

TMP had B-factors ¨3- to 4-fold higher than the sole

coordinating atom, the a-metal, indicting that their coordi-

nation to the metal was weak enough to allow significant

Fig. 6. The active site of OpdA in the presence of trimethyl phosphate and

dimethyl thiophosphate. OMIT electron density maps were calculated from

models refined in the absence of the bound ligands. 2mFO–DFC electron

density is shown contoured at 1 j (brown), mFO–DFC electron density is

contoured at 3 j (red). Some sidechain and solvent molecules have been

omitted for clarity.

Scheme 1. A mechanistic scheme for the catalysed hydrolysis of dimethoate

by the binuclear metal active site of OpdA.


mobility or rapid exchange. The formation of a dually

bound product, which is stable enough, and its departure

slow enough, to be observed in a complex with the enzyme

is inconsistent with the diffusion limited hydrolysis of

parathion. If no dually bound product forms during para-

thion hydrolysis, or the dually bound product is in some

manner less tightly coordinated, this weak coordination

would permit the rapid departure of the products and the

extremely high catalytic rates observed during catalysis,

consistent with the diffusion limited nature of its turnover

[10]. Equally, the ~1000 fold slower turnover of the

phosphorothiolates [4], and the uncompetitive inhibition of

PUO bond hydrolysis in phosphotriesters by phosphoro-

thiolate substrates [12], may be a result of slow departure of

the dually bound product seen in Fig. 3. At this time, our

understanding of these differences is inconclusive and will

require further work.

3.4. The role of the active site surface

The structure of the OpdA–DMTP complex provided a

clear indication of the position at which substrates will bind

within the active site. Modelling the substrate methyl

parathion into the active site demonstrates that the shape of

the active site pockets constrains the binding of substrate to

the h-metal, and the pocket formed by W131, F132, H201,


H230, R254, D301 and L271 forces the substrate to tilt

toward the nucleophile to accommodate the leaving group

(Fig. 4). In this figure, the structure of methyl parathion [23]

was modelled into the active site by superimposing the

coordinated PjS group to the coordination of DMTP. The

orientation of this molecule in the active site is consistent with

results from molecular dynamics in which the theoretical

binding of the analogue paraoxon at the active site was

calculated [24], and is the only reasonable orientation

possible. This demonstrates that the only way the substrate

can bind is by tilting toward the a-metal, significantly

decreasing the distance between the nucleophile and electro-

philic centre and effectively forcing the reactants (substrate

and nucleophile) into an approximation of transition state

(Figs. 4 and 5). The predicted distance from the nucleophile

to the phosphorous from this modelling is 3.1 A, which is

approaching the 2.7 A distance observed in the geometry of

the transition state of the substrate paraoxon in the gas phase

[11]. This is therefore an excellent example of the ability of

enzymes to catalyse reactions by lowering the energy

required to reach the transition state.

The structure of the OpdA–TMP complex was surprising in

thatallpreviousdatahaveindicatedthattheh-metal isresponsible

for substrate binding. However, this complex demonstrates that

substrates preferentially bind at the a-metal when they are

sufficiently small. When substrates are larger, courtesy of a

bulkier leaving group, they are unable to bind at thea-metal and

are forced to bind at the h-metal. The molecular surface of the

active site is displayed in Fig. 4, illustrating the necessity for

substrateswithlargeleavinggroupstobindat theh-metal.TMPis

thereforeessentiallyboundnon-productively inOpdA,as there is

no indication of any interaction between the nucleophile and the

electrophilic phosphorous centre. This demonstrates is that if

substrates bind terminally, and the metal S/OjP and metal-

hydroxide bonds are parallel, the distance of the terminal

nucleophile to the electrophilic phosphorous is too great (3.8 A)

to allow attack (Fig. 5).

3.5. Comparison with other binuclear metallophosphatases

These results demonstrate that the catalysis of phospho-

triester hydrolysis by the binuclear metal site of the

bacterial phosphotriesterases is consistent with that of

other binuclear metal centre phosphoesterases, such as the

phosphomonoesterase purple acid phosphatase [25,26], and

the phosphodiesterases 5V nucleotidase [27] and Mre11

nuclease [28], despite the lack of sequence homology.

Nucleophilic attack by a terminally bound and aligned

water, deprotonated by a transition metal, therefore appears

to be a functionally conserved mechanism used throughout

this diverse family of phosphatases.

3.6. Summary

Important aspects of the catalytic mechanism of this

potentially very useful enzyme have been explained, such

as the identity of the attacking nucleophile and the role of

the active site surface. The mechanism is now consistent

with that of other binuclear metallophosphoesterases,

suggesting the binuclear active site provides a scaffold

upon which the hydrolysis of a broad range of phosphate

esters is possible depending on the structure of the

surrounding cavity.

4. Supporting information available

X-ray coordinates have been deposited in the Research

Collaboration for Structural Bioinformatics, Rutgers Uni-

versity, New Brunswick, N. J. and will be released upon

publication.

Acknowledgements

We are grateful for the helpful discussion from reviewers.

This work has been supported by an Australian Research

Council Discovery-Project Grant DP 0342678.

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The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide...

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