An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily

Article abstract of DOI:10.1073/pnas.0903951107

We report a catalytically promiscuous enzyme able to efficiently promote the hydrolysis of six different substrate classes. Originally assigned as a phosphonate monoester hydrolase (PMH) this enzyme exhibits substantial second-order rate accelerations ((k_cat/_KM) /k _w), ranging from 10⁷ to as high as 10¹⁹, for the hydrolyses of phosphate mono-, di-, and triesters, phosphonate monoesters, sulfate monoesters, and sulfonate monoesters. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature, and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half-lives) range from 200 days to 10⁵ years under near neutrality. The substantial rate accelerations for a set of relatively difficult reactions suggest that efficient catalysis is not necessarily limited to efficient stabilization of just one transition state. The crystal structure of PMH identifies it as a member of the alkaline phosphatase superfamily. PMH encompasses four of the native activities previously observed in this superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. The functional links between superfamily activities can be presumed to have played a role in functional evolution by gene duplication.

Full text of DOI:10.1073/pnas.0903951107

An efficient, multiply promiscuous hydrolase
in the alkaline phosphatase superfamily
Bert van Looa,1, Stefanie Jonasa,1, Ann C. Babtiea, Alhosna Benjdiab, Olivier Berteaub,
Marko Hyvönena, and Florian Hollfeldera,2
aDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom; and bInstitut National de la
Recherche Agronomique, Unité Propre de Recherche 910, Unité d’Ecologie et Physiologie du Système Digestif, Domaine de Vilvert,
78352 Jouy-en-Josas, France
Edited by Daniel Herschlag, Stanford University, Stanford, CA, and accepted by the Editorial Board November 20, 2009 (received for review April 24, 2009)
We report a catalytically promiscuous enzyme able to efficiently
promote the hydrolysis of six different substrate classes. Originally
assigned as a phosphonate monoester hydrolase (PMH) this enzyme
exhibits substantial second-order rate accelerations (ðkcat∕KMÞ∕
diesters with higher efficiency, it has been surmised that its ori-
ginal biological role is to hydrolyze phosphodiesters (9). In a clo-
sely related ortholog (RlPMH from Rhizobium leguminosarum;
86% amino acid identity), the unusual formylglycine (fGly) resi-
due, first discovered in type I sulfatases (10), was shown to act as a
nucleophile. fGly results from enzymatic posttranslational oxida-
tion of a cysteine embedded in a recognition sequence (CxPxR)
to an aldehyde (11). On the basis of structural, mutational, and
kinetic studies and in analogy to the structurally related arylsul-
fatases (12), a double displacement mechanism involving a cova-
lent fGly-substrate intermediate was proposed for RlPMH. PMH
is a member of the alkaline phosphatase (AP) superfamily that
encompasses structurally related enzymes known to hydrolyze
phosphate monoesters and diesters and sulfate monoesters
(13). Several members of the AP superfamily show catalytic pro-
miscuity, and in some cases the promiscuous reactions are the
native activities of other superfamily members (14–19).
k
w), ranging from 107 to as high as 1019, for the hydrolyses of phos-
phate mono-, di-, and triesters, phosphonate monoesters, sulfate
monoesters, and sulfonate monoesters. This substrate collection
encompasses a range of substrate charges between 0 and −2, tran-
sition states of a different nature, and involves attack at two dif-
ferent reaction centers (P and S). Intrinsic reactivities (half-lives)
range from 200 days to 105 years under near neutrality. The sub-
stantial rate accelerations for a set of relatively difficult reactions
suggest that efficient catalysis is not necessarily limited to efficient
stabilization of just one transition state. The crystal structure of
PMH identifies it as a member of the alkaline phosphatase super-
family. PMH encompasses four of the native activities previously
observed in this superfamily and extends its repertoire by two
further activities, one of which, sulfonate monoesterase, has not
been observed previously for a natural enzyme. PMH is thus one
of the most promiscuous hydrolases described to date. The func-
tional links between superfamily activities can be presumed to have
played a role in functional evolution by gene duplication.
BcPMH catalyzes the hydrolysis of a total of six different sub-
strate classes, four of which correspond to activities seen in the
AP superfamily. The collection of substrate classes, for which rate
accelerations between 107 and 1019 are observed, encompasses
large variations in charge, the reaction center, size, hydrophobi-
city, reactivity, and nature of the TS of the uncatalyzed reaction.
The range and magnitude of promiscuous activities suggest that
BcPMH is an enzyme in which high reactivity is combined with a
relative lack of specificity, challenging the notion that a very ac-
tive enzyme must be highly specialized. The kinetic and structural
analysis of BcPMH highlights mechanistic and structural features
that promote catalytic promiscuity for hydrolytic reactions.
catalytic promiscuity ∣ evolution ∣ mechanism ∣ sulfatase ∣ superfamily
nzymes are usually seen as highly specific catalysts following
E
the classical rule “one enzyme, one activity.” This view is chal-
lenged by an increasing number of enzymes with broad substrate
specificities or side activities indicating that enzymes are catalyti-
cally more flexible than originally assumed. Some of these
promiscuous enzymes can turn over substrates with different
structures while catalyzing the same chemical reaction involving
the same transition state (substrate promiscuity). Catalytic pro-
miscuity, by contrast, is the ability of an enzyme to catalyze che-
mically distinct reactions by stabilization of different transition
states (TSs) (1). Catalytic efficiencies (kcat∕KM) for the promis-
cuous substrates are often substantially lower (2 to 9 orders of
magnitude) than for the native conversions (1–3). The growing
number of examples of this phenomenon (1–4) has engendered
excitement on a number of fronts. Catalytic promiscuity provides
a functional basis for enzyme evolution by gene duplication. The
initial head-start activity of the duplicated gene copy could
support an immediate selective advantage (1, 2, 5, 6), to be
followed by improvement of the initially weak activity (7). Even
low kcat∕KM values for a promiscuous function can support
a significant selective advantage (8). Mimicking this evolutionary
shortcut could also provide a more efficient route to changing the
function of proteins by directed evolution (5).
Results
BcPMH: A Highly Promiscuous Hydrolase. A highly purified sample of
BcPMH exhibits 107–1012 second-order rate accelerations (ðkcat
∕
KMÞ∕kw) for the hydrolysis of phosphate monoesters 1a/b, phos-
phate triester 3b, sulfate monoesters 5a/b, and sulfonate monoe-
sters 6a/b, in addition to the previously reported hydrolysis of
phosphonate monoesters 4a/b and phosphodiesters 2a/b (Fig. 1
and Table 1). The enzyme performs multiple turnovers with each
substrate (Fig. S1A–F), and saturation kinetics are observed for
all substrate classes except phosphate triester 3b (Fig. S1G–L).
kcat∕Kw values for all reactions, including the two fastest conver-
sions, differ by 6 orders of magnitude. Catalysis is efficient with
TS binding constants (KTS) ranging from 10−6 to 10−18 M.
Author contributions: B.v.L., S.J., A.C.B., A.B., O.B., M.H., and F.H. designed research;
B.v.L., S.J., A.C.B., and A.B. performed research; B.v.L., S.J., A.C.B., M.H., and F.H. analyzed
data; and B.v.L., S.J., A.C.B., M.H., and F.H. wrote the paper.
The authors declare no conflict of interest.
We describe multiple and efficient catalytic promiscuity in an
enzyme from Burkholderia caryophilli PG2952 originally assigned
as a phosphonate monoester hydrolase (BcPMH) when this or-
ganism was identified in a screen for growth on glyceryl glypho-
sate (9). The kcat∕KM for glyceryl glyphosate is relatively low
(9.5 M−1 s−1) and, because BcPMH also hydrolyzes phosphate
This article is a PNAS Direct Submission. D.H. is a guest editor invited by the Editorial Board.
1B.v.L. and S.J. contributed equally to this work
2To whom correspondence should be addressed: E-mail: fh111@cam.ac.uk.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0903951107/DCSupplemental.
2740–2745 ∣ PNAS ∣ February 16, 2010 ∣ vol. 107 ∣ no. 7
www.pnas.org/cgi/doi/10.1073/pnas.0903951107

expected molecular weight of tetrameric BcPMH (∼240 kDa)
(9), copurifying contaminants interacting specifically with
BcPMH are unlikely to be present, because they would have in-
creased the observed mass. Affinity purification of a Strep-tagged
version of the enzyme followed by a size-exclusion step yielded
pure protein that also catalyzed all six reactions with identical
rate parameters as the untagged protein. During size-exclusion
chromatography of the tagged protein, all activities coeluted
(Fig. S3). When the same expression and purification procedures
were followed with an Escherichia coli strain harboring the empty
expression vector, no contaminating activity could be detected
(Fig. S3). These observations collectively suggest that none of
the observed reactions was the result of a contaminant.
Fig. 1. Structures of substrates that are hydrolyzed by BcPMH: 1a: phenyl
phosphate, 1b: p-nitrophenyl phosphate, 2a: diphenyl phosphate, 2b: p-
nitrophenyl ethyl phosphate, 3b: paraoxon, 4a: phenyl phenylphosphonate,
4b: p-nitrophenyl phenylphosphonate, 5a: phenyl sulfate, 5b: p-nitrophenyl
sulfate, 6a: phenyl phenylsulfonate, 6b: p-nitrophenyl phenylsulfonate. Gray
lines indicate the bonds that are broken during hydrolysis.
pH-rate profiles. Catalysis by BcPMH is dependent on its protona-
tion state between pH 6 and 10 (Fig. 2). The pH-rate profiles of
the kcat∕KM values for substrates 1b, 2b, 4b, 5b, and 6b largely
coincide. For the pH range sampled, in which the protonation
states of the substrates do not change, the hydrolysis of these five
compounds could be fitted to a two-pKE model with a pKE1
of 7.0–7.2 (5.8 for sulfate monoester 5b) and a pKE2 of 7.5–8.1.
The fGly residue that results from the oxidation of Cys57
(Fig. 3D) is likely to contribute to catalysis in its deprotonated
state (11), represented by pKE1. The protonated forms of resi-
dues His218 and Lys337 (Fig. 3B) have been suggested to act
as general acids in its close ortholog RlPMH (11), which could
explain the presence of pKE2. The coincidence of the pH-rate
profiles for kcat∕KM for these five substrates suggests that their
conversions are catalyzed by following a similar mechanism as
proposed for phosphonate monoester hydrolysis by RlPMH
(11). Furthermore, the striking similarity between the pH-rate
profiles suggests that these five reactions are catalyzed by the
same active site, because a contaminant would be unlikely to have
a similar pH-rate profile. The enzyme-catalyzed hydrolysis
of phosphotriester 3b shows a pH-rate profile that differs
substantially from that of the other five substrates, suggesting that
hydrolysis of this substrate is promoted in a different way.
In each case hydrolysis involves nucleophilic attack at the
phosphorus or sulfur center, breaking the ester bond to the leaving
group. An alternative addition-elimination (SNAr) mechanism, in
which attack occurs at the nitrophenyl ring instead (Fig. S2A), is
ruled out by isotope-labeling experiments that were analyzed
by mass spectrometry (Fig. S2C). Incubation of substrates 1b–6b
in the presence of enzyme and 18O-labeled water showed no incor-
poration of the label in the p-nitrophenol product.
Thus, all six reactions proceed through an identical reaction
sequence involving nucleophilic attack on the various phosphorus
and sulfur centers (Fig. S2B). The observed activities involve
the making and breaking of bonds to reaction centers with dif-
ferent characteristics via different TSs, so they are genuinely
promiscuous.
All Six Reactions Are Catalyzed by BcPMH. Several lines of evidence
suggest that all six reactions are indeed catalyzed by BcPMH and
not by contaminant(s) in the purified enzyme preparation.
Copurification of all six different activities. BcPMH was extensively
purified by using anion exchange, hydrophobic interaction, and
size-exclusion chromatography (9). Protein-containing fractions
that eluted from the three different columns were assayed for
activity against substrates 1b–6b. All six activities coincided with
the elution of BcPMH during all three purification steps (Fig. S3).
Because all activities eluted from the size-exclusion column at the
Cross-inhibition. Because the kcat values for the hydrolysis of
substrates 1b, 3b, 5b, and 6b are at least 100-fold lower than those
of the phosphodiesterase and phosphonate monoesterase reac-
tion, these four weaker substrates will act as inhibitors of the
faster reactions. Apparent kcat∕KM values for the conversion
of phosphate diester 2b were determined from Michaelis–
Menten curves recorded at various inhibitor concentrations
Table 1. Kinetic parameters for BcPMH wild type at pH 7.5
kcat∕KM
ðkcat∕KMÞ∕
ðkcat∕KMÞ∕
kw
†,
‡
†,
§
†,
¶
∥
Substrate
kcat (s−1
)
KM (mM)
(s−1 M−1
)
kcat∕kuncat
kuncat
(M−1
)
KTS (M)
Phosphate
monoester
Phosphate
diester
Phosphate
triester
Phosphonate
monoester
Sulfate
monoester
Sulfonate
monoester
1a* ð2.1 ꢀ 0.2Þ ꢁ 10−4
0.33 ꢀ 0.03
0.35 ꢀ 0.02
0.071 ꢀ 0.004
0.63 ꢀ 0.04
>2.4
0.63 ꢀ 0.08
22 ꢀ 1
106.2
105.2
1013.7
1013.4
>102.9
109.7
108.7
1017.9
1016.6
105.5
1011.5
1010.4
1019.6
1018.3
107.2
1.9 ꢁ 10−10
2.2 ꢁ 10−9
1.4 ꢁ 10−18
2.8 ꢁ 10−17
3.2 ꢁ 10−6
1b
2a
2b
3b
ð7.7 ꢀ 0.1Þ ꢁ 10−3
2.12 ꢀ 0.02
5.8 ꢀ 0.1
ð3.0 ꢀ 0.2Þ ꢁ 104
ð9.2 ꢀ 0.6Þ ꢁ 103
ð1.6 ꢀ 0.1Þ ꢁ 10−2
>3.7 ꢁ 10−5
4a
4b
1.58 ꢀ 0.04
2.73 ꢀ 0.06
1.23 ꢀ 0.09
0.19 ꢀ 0.02
58 ꢀ 5
ð1.3 ꢀ 0.1Þ ꢁ 103
ð1.5 ꢀ 0.1Þ ꢁ 104
ð1.7 ꢀ 0.3Þ ꢁ 10−3
0.59 ꢀ 0.04
1012.9
1011.2
108.4
107.6
106.7
106.3
1015.8
1014.9
109.6
108.7
1010.0
109.9
1017.6
1016.7
1011.3
1010.5
1011.7
1011.7
1.4 ꢁ 10−16
1.2 ꢁ 10−15
2.5 ꢁ 10−10
1.9 ꢁ 10−9
1.0 ꢁ 10−10
1.1 ꢁ 10−10
5a* ð1.0 ꢀ 0.1Þ ꢁ 10−4
5b
ð4.0 ꢀ 0.1Þ ꢁ 10−2
68 ꢀ 4
6a* ð7.0 ꢀ 0.3Þ ꢁ 10−4
0.51 ꢀ 0.09
0.24 ꢀ 0.03
1.4 ꢀ 0.2
6b
ð1.2 ꢀ 0.1Þ ꢁ 10−2
49 ꢀ 7
*KM was determined as the competitive inhibition constant (Kic) for the conversion of phosphate diester 2b. By using this Kic as the KM value for that
substrate, the kcat could be derived (see SI Text).
†Values for kuncat and kw are listed in Table S1.
‡First-order rate enhancement.
§Catalytic proficiency.
¶Second-order rate enhancement.
∥KTS, the transition state binding constant, was calculated as the inverse of the catalytic proficiency [ðkcat∕KMÞ∕kuncat].
van Loo et al.
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an aldehyde-specific labeling agent (20) showed that Cys57 was
indeed modified to fGly in this enzyme but that the modification
was incomplete (Fig. S5A). Incomplete modification can be ex-
plained by saturation of the (as yet unidentified) E. coli fGly-
generating machinery under overexpression conditions. Coexpres-
sion of BcPMH with the bacterial fGly-generating enzyme from
Mycobacterium tuberculosis (MtbFGE) (21) increased the activity
of the enzyme 3-fold, but unmodified Cys57 was still detected.
To resolve which form of the enzyme—fGly57 or Cys57—
provides the active site nucleophile, BcPMH was expressed under
anaerobic conditions to prevent formylglycine formation (22).
MALDI-TOF analysis of the tryptic digest of the anaerobically
expressed BcPMH confirms an increased fraction of unmodified
Cys57 (Fig. S5B/C). Replacement of fGly57 by Cys57 resulted
in decreased catalytic efficiency for the hydrolysis of substrates
1b, 2b, 4b, and 6b by 4–60-fold in terms of kcat∕KM (Table S2)
and activity towards sulfate monoester 5b could not be detected
for this “mutant.” These data collectively suggest that the fGly
form of BcPMH is mainly responsible for the hydrolysis of these
five substrates observed with the partially modified enzyme pre-
paration. In contrast, the activity toward phosphotriester 3b in-
creased 2-fold, suggesting that the unmodified cysteine form of
BcPMH is mainly responsible for phosphotriester hydrolysis. The
lack of cross-inhibition between phosphotriester 3b and phospho-
diester 2b, as well as the differences in the pH-rate profiles in
comparison to all other substrates, supports this conclusion.
Thus, changing the nucleophile (fGly to Cys) adds the capability
to hydrolyze phosphate triester 3b.
Structure of BcPMH. To obtain insight into the structural features
that enable the catalytic variability of BcPMH, we solved its crys-
tal structure (Fig. 3). Strep-tagged BcPMH yielded orthorhombic
crystals that diffracted to 2.4 Å. Phases were calculated by mo-
lecular replacement using the structure of RlPMH as a search
model [Protein Data Bank (PDB): 2vqr] (11). Statistics for the
data collection and refinement are listed in Table S3.
The α∕β-fold of BcPMH is similar to AP rendering it a member
of the AP superfamily. Furthermore, its structure is almost iden-
tical to RlPMH (PDB: 2vqr, rmsd 0.5 Å over Cα of 511 residues)
and shows high homology to the structure of Pseudomonas aeru-
ginosa arylsulfatase (PDB: 1hdh, rmsd 2.5 Å over Cα of 331 resi-
dues). The enzyme crystallizes as a symmetric homotetramer (a
dimer of dimers, Fig. 3A) and as the extensive oligomerization in-
terfaces suggest [buried surface area calculated with AreaIMol
(23): 5; 498 Å2 for the dimer; 17; 030 Å2 for the tetramer] is
also tetrameric in solution. It elutes from a gel filtration column
Fig. 2. pH-rate profiles of kcat∕KM for all six substrates: (A) phosphate
monoester 1b, (B) phosphate diester 2b, (C) phosphate triester 3b, (D) phos-
phonate monoester 4b, (E) sulfate monoester 5b, and (F) sulfonate monoe-
ster 6b. For details see SI Text.
(Fig. S4B). The Kic was determined by fitting the dependence of
kacaptp∕Kapp on inhibitor concentration (Fig. S4). Inhibition of
M
native phosphodiesterase activity was shown for substrates 1b,
5b, and 6b (Fig. S4) with competitive inhibition constants (Kic)
that were in agreement with the KM for the conversion of these
nonnative substrates (Table 2), suggesting that these reactions are
catalyzed by the same active site. For phosphotriester 3b, no in-
hibition was observed within the sampled concentration range of
the inhibitor consistent with the assumption that this
reaction is catalyzed by following a different pathway or in a dif-
ferent part of the active site than the other five reactions
(see below).
corresponding to a molecular weight of ∼240 kDa (Mprotomer
¼
61 kDa) and shows a hydrodynamic radius in dynamic light
scattering experiments of 110 Å as predicted for the tetramer
by HYDROPRO (24).
The salient feature of the BcPMH structure is the very spa-
cious active site (≈10 × 20 Å2 wide and 15 Å deep) (Fig. 3C), al-
lowing for a range of substrate sizes to be accepted without
steric hindrance. BcPMH is a mononuclear metalloenzyme, yet
the nature of the native metal ion is uncertain. Metal analysis
by microfocused photon-induced x-ray emission (microPIXE)
showed the presence of 0.28 ꢀ 0.04 eq iron and 0.26 ꢀ 0.06 eq
zinc per protein molecule. Modeling the electron density with
Fe and Zn, both at 0.3 occupancy, fitted the electron density well.
However, the presence of other metal ions with occupancies
<10% cannot be excluded. Furthermore, Mn2þ ions are known
to increase enzyme activity (9), and in RlPMH, Mn2þ has been
suggested as the native metal ion (11). Indeed, when BcPMH
was coexpressed with MtbFGE to increase the fGly-modification
efficiency, 0.19 eq Mn, 0.29 eq Ca, 0.40 eq Fe, and 0.36 eq
Zn were detected by microPIXE. When the enzyme was incu-
bated in the presence of excess Mn2þ, all six activities increased
Active site mutation. Mutation of Cys57 to alanine prevents the
generation of the fGly nucleophile and results in an impaired
enzyme with reduced activity for all six substrates, suggesting that
all reactions are catalyzed by BcPMH in the same active site. kcat
values decreased 250–1400-fold whereas KM values were almost
unchanged, resulting in reductions of kcat∕KM in the range of
7–1200-fold (Table 3). The smallest mutational effect was
observed for phosphate triesters, consistent with differences in
catalysis for this substrate.
BcPMH Has a Formylglycine (fGly) in its Active Site. BcPMH contains
the recognition sequence 57-CXPXR-61 in which Cys57 can
potentially be oxidized to the fGly residue. MALDI-TOF spectro-
metric analysis of a tryptic digest of BcPMH in the presence of 1.5–2-fold. Although this result does not exclude that other metal
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van Loo et al.

Table 2. Inhibition of BcPMH catalyzed phosphate diester 2b
hydrolysis by additional substrates
Substrate
KM (mM)
Kic (mM)
1b
3b
5b
6b
0.35 ꢀ 0.02
>2.4
0.32 ꢀ 0.02
>2.4
68 ꢀ 4
56 ꢀ 11
0.24 ꢀ 0.03
0.32 ꢀ 0.05
ions can promote activity, Mn2þ enables access to higher effi-
ciency and does so for all reactions, indicating that the promis-
cuous behavior is not caused by the heterogeneity of the active
site metal ion.
Fig. 3B shows the active site configuration of BcPMH.
Given the limited modification efficiency in E. coli of Cys57 to
fGly57 in the absence of MtbFGE coexpression, modest, but iden-
tifiable, electron density was observed for fGly (fitted to an oc-
cupancy of 20%). The metal ion is coordinated by five ligands in
the form of a distorted pyramid. The coordination of one of the
gem-diol hydroxyl groups of the nucleophile fGly57 to the metal
ion is set up to increase the concentration of the reactive depro-
tonated form. Arg61 and Thr107 form hydrogen bonds to Cys57/
fGly57, stabilizing the fGly hydrate. Arg61 and Tyr105 hydrogen
bond to the metal-coordinating residues. His218, Lys337, and
Asn78 are in a position to bind the substrate. On the basis of
the proposed mechanism for phosphodiester hydrolysis in RlPMH
(11), His218 and Lys337 may provide general acid catalysis for
leaving group departure and charge stabilization as the TS is ap-
proached, whereas Asn78 is involved in substrate binding.
Discussion
BcPMH Combines High Catalytic Efficiency with Low Specificity. This
promiscuous enzyme is capable of hydrolyzing various phosphate
and sulfate esters with high rate accelerations (Table 1). Compar-
ison with the native activities of other enzymes illustrates the high
efficiency of BcPMH as a catalyst. The second-order rates of
PMH for its phosphonate monoester hydrolase activity and the
phosphodiesterase activity, its assumed native function, match
the kcat∕KM of other phosphodiesterases toward model substrates
such as phosphate diester 2b (19, 25, 26).
The small rate constants for the uncatalyzed reactions (kuncat
)
are evidence that the reactions catalyzed by BcPMH are difficult.
The second-order rate enhancement ðkcat∕KMÞ∕kw is a measure
for the extent to which the free energy barrier of the enzymatic
reaction is lowered compared to the uncatalyzed process. These
rate enhancements for BcPMH’s best reaction (1018), but also for
four of its additional substrates (1011–1016), correspond to re-
markable decreases in the energy of activation between 14.4
and 27.2 kcal mol−1, falling in the range observed for the native
reactions of other enzymes (1011–1027) (27).
Metal ions alone accelerate phosphate transfer reactions by up
to 2 orders of magnitude (kcat∕kuncat) (28), and in enzyme models
one metal ion can provide up to 6 orders of magnitude in second-
order rate acceleration [ðkcat∕KMÞ∕kuncat] (29, 30). The larger ac-
celerations observed here for PMH suggest that the active site
provides the potential for additional effects that synergize with
the reactivity of the metal ion. Proximity effects, medium effects,
and acid base catalysis can provide the additional enhancement of
1010-fold for the two best reactions and 103-fold for three of the
other reactions. The weakest catalytic reaction, the hydrolysis of
phosphotriester 3b, shows similar activity to that exhibited by me-
tal complexes, suggesting that provision of a metal ion/nucleophile
combination is chiefly responsible for this activity.
Fig. 3. X-ray structure of BcPMH. (A) BcPMH is tetrameric in the crystal and
in solution. The surface of the active site residues at the bottom of the active
site cavity is orange (Upper Left protomer). The arrow indicates the position
of the cut for C. (B) Active site residues as identified by homology
to RlPMH (11) and P. aeruginosa arylsulfatase. Hydrogen bonds and metal-
binding contacts are indicated by dotted lines. The metal ions identified
in the protein were iron and zinc. (C) A cut through the active site illustrates
the spacious active site opening. (D) Proposed mechanism for all five differ-
ent conversions involving the fGly nucleophile [on the basis of the proposed
mechanism of RlPMH (11)]. Upon nucleophilic attack of fGly on the central
P/S atom (X) the leaving group (ROH ¼ phenolate) is abstracted. For all sub-
strates except 3b, a covalent intermediate formed via fGly is broken down by
cleaving the same C-O bond (red, instead of a second attack at X).
BcPMH is resilient to the removal of a crucial feature of its
catalytic machinery. Mutation of the active site nucleophile
(fGly/Cys) to alanine compromises catalysis of all reactions,
but rate accelerations are still substantial, as has been noted in
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Table 3. Kinetic parameters of the BcPMH Cys57Ala mutant for
substrates 1b–6b
of substrate properties that govern the catalytic efficiency of
PMH, which are in descending order of importance: Substrates
with P centers, negative substrate charge, an associative TS, and
smaller substrate size contribute to higher accelerations. These
data also suggest that specialization for one compound does
not rule out significant catalysis for another.
Ratio
(wt:C57A)
kcat∕KM
C57A mutant
kcat (s−1
)
KM (mM)
kcat∕KM (M−1 s−1
)
1b ð3.1 ꢀ 0.1Þ ꢁ 10−5
1.16 ꢀ 0.08
0.71 ꢀ 0.06
>2.4
ð2.6 ꢀ 0.1Þ ꢁ 10−2
828 ꢀ 70
1178 ꢀ 131
7.0 ꢀ 0.3
467 ꢀ 55
405 ꢀ 97
361 ꢀ 95
2b ð5.6 ꢀ 0.1Þ ꢁ 10−3
7.8 ꢀ 0.7
What Causes the Promiscuous Behavior of BcPMH? Like many of the
promiscuous enzymes described (1–3), BcPMH contains a metal
ion (Fig. 3). Metal-coordinated hydroxides or alkoxides are in-
trinsically highly reactive (3), which may equate to low chemical
selectivity. Furthermore, the diameter of the active site is approxi-
mately twice that of the largest substrate and therefore allows
productive binding of substrates with minimal steric hindrance.
BcPMH is likely to employ covalent catalysis for hydrolysis
of all its substrates, confronting it with the problem of different
covalent intermediates that have to be broken down to allow
multiple turnover. By contrast, imperfect catalysts perform only
one turnover: If the covalent intermediate is not broken down,
substrates become in effect suicide inhibitors. We hypothesize
that the fGly nucleophile provides a solution to this problem:
The breakdown of all intermediates is expected to occur by cleav-
age of the C-O bond in the second step (11, 12) (Fig. 3D, shown in
red). This points to a common second step for the collection of
promiscuous substrates, where the same hemiacetal-ester clea-
vage makes different leaving groups depart. Indeed, for all five
substrates involving fGly, multiple turnover is observed with
400–600,000 reaction cycles per enzyme molecule (Fig. S1).
3b
>5.4 ꢁ 10−6
ð2.3 ꢀ 0.1Þ ꢁ 10−3
31 ꢀ 3
4b ð1.90 ꢀ 0.02Þ ꢁ 10−3 0.061 ꢀ 0.005
5b ð3.1 ꢀ 0.2Þ ꢁ 10−5
6b ð1.9 ꢀ 0.2Þ ꢁ 10−5
21 ꢀ 5
ð1.5 ꢀ 0.3Þ ꢁ 10−3
0.14 ꢀ 0.03 0.13 ꢀ 0.03
mutational studies of a range of enzymes (31). Other catalytic
factors seem to substitute for the removed nucleophile: Com-
bined with binding features in the active site, a water molecule
may occupy the vacant metal ligand position by acting as the re-
placement nucleophile. The rate reduction indicates that the me-
tal-coordinated fGly nucleophile contributes up to a factor of 103
to PMH’s efficiency.
Many enzyme models are only able to catalyze the hydrolysis of
activated (especially p-nitrophenyl) esters and cannot catalyze
more demanding reactions with equal proficiency (32). In con-
trast, BcPMH hydrolyzes the less activated phosphomonoester
1a with a 10-fold higher rate enhancement than 1b despite a bet-
ter leaving group and thus higher intrinsic reactivity. The same
observation holds for substrate pairs 2a/b, 4a/b, 5a/b, and 6a/b.
Furthermore, the enzyme is unable to catalyze reactions that are
thermodynamically less demanding, such as the hydrolysis of
carboxylic esters, lactones, and epoxides. Additionally, the half-
lives of the identified substrates vary between 200 days and
105 years under near neutral conditions. Thus, substrate reactiv-
ity is not sufficient to explain catalysis in PMH. The observation
that catalysis is substantial for less-activated leaving groups
suggests that PMH can confer general acid catalysis facilitating
leaving group departure, consistent with the proposed mechan-
ism (Fig. 3).
Comparison to Other Promiscuous Enzymes. In comparison to re-
ported cases of catalytic promiscuity, BcPMH is among the most
promiscuous hydrolases recorded to date. It can catalyze five
chemically distinct reactions with catalytic efficiencies (kcat∕KM
)
that differ by 5 orders of magnitude. By comparison, the five re-
actions catalyzed by alkaline phosphatase differ by more than 9
orders of magnitude (16, 17, 34). The second-order rate accelera-
tion for the native activity of BcPMH with a ðkcat∕KMÞ∕kw of 1018
is comparable to that of other known promiscuous enzymes (3).
Yet, the simultaneous degrees of freedom of PMH in the identity
of the reaction centers, reaction type, and substrate charge dis-
tinguish this example of a promiscuous enzyme.
The five substrate classes that are converted by the fGly form
of BcPMH are characterized by large differences in their mole-
cular properties. Ground state charges varying from −2 to 0 are
tolerated by BcPMH, as well as large differences in substrate size
(160–350 Å3), hydrophobicity (logD −12 to 2.9), bond length
around the reaction center, and the reaction center itself (phos-
phorus vs. sulfur). In solution the substrates are hydrolyzed via
different mechanisms, as defined by the charge development
in the TS. Phosphate and sulfate monoester hydrolysis proceeds
via a loose, dissociative TS, whereas phosphodiesters, phospho-
triesters, and phosphonate and sulfonate monoesters are hydro-
lyzed via tighter, associative TSs (Fig. S6). An enzyme would be
assumed to provide complementary molecular recognition to the
charge development in the TS. However, this does not seem to
apply to PMH: The dissociative sulfatase and phosphatase reac-
tions are catalyzed with the same proficiencies as the associative
sulfonate hydrolysis, suggesting tolerance in TS recognition. We
conjecture that, similar to AP, the enzymatic TSs are not substan-
tially changed from those of the uncatalyzed reactions as has been
suggested as the most straightforward solution to lower the ther-
modynamic barrier for the promiscuous reaction (33). However,
this hypothesis has yet to be tested.
Coincident pH-rate profiles and Cys57Ala mutant data suggest
that catalysis of the five best reactions follows the same overall
reaction pathway. However, a quantitative analysis shows no
clear correlation of catalytic proficiency with the type of TS, the
amount of negative charge per nonbridging oxygen, substrate
hydrophobicity, or bond lengths. This analysis suggests that the
specificity of PMH is not dictated by any obvious single charac-
teristic of the substrate and that it is relatively indiscriminate
toward the weaker substrates. A closer look reveals a hierarchy
Functional Correlations in the AP Superfamily. The x-ray structure of
BcPMH confirms that it is structurally and mechanistically closely
related to arylsulfatases and also, albeit more distantly, to AP
and nucleotide phosphodiesterase (11, 13). These three enzymes
catalyze at least two reactions besides their native activity that are
the native reactions of another family member (14–19). BcPMH
promotes all native reactions of these three enzymes, making
catalytic promiscuity a widespread feature of this superfamily.
Furthermore, various mutants of BcPMH can catalyze the hydro-
lysis of phosphotriesters and sulfonate monoesters, two activities
that have not been observed in the AP superfamily.
Catalytic promiscuity mirrors the chemical potential of the
conserved active site arrangement in a superfamily and can be
exploited by evolution to modulate specificities leading to differ-
entiated members of a structurally related superfamily. This
observation suggests the definition of “catalytic superfamilies”
on the basis of reactive active site arrangements. Indeed conser-
vation of chemical functionality has been recognized, e.g., in the
enolase superfamily (4). The chemical potential of an active site
such as PMH suggests that a targeted search on the basis of
promiscuously related chemical reactions may help to identify
activities of proteins for which no function has been assigned or
where the assignment is computer-generated (and often incor-
rect). Exploration of other phosphatase superfamilies may reveal
promiscuous sulfatase activities indicating the potential of yet
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www.pnas.org/cgi/doi/10.1073/pnas.0903951107
van Loo et al.

unknown sulfatase types in those families. Likewise, this potential
could be exploited by directed evolution or enzyme design (35).
The chemical flexibility provided by promiscuous enzymes ex-
plains how new activities can be picked up rapidly and enhanced
or even introduced by single mutations (35). Our data illustrate
this potential evolutionary versatility: By acquiring the fGly mod-
ification (instead of Cys), the overall activity of PMH is increased,
the sulfatase activity is introduced, and the phosphotriesterase
activity is lost (Table S2).
BcPMH efficiently catalyzes difficult reactions with different
characteristics by using essentially the same catalytic machinery.
The large rate accelerations bring relatively difficult transforma-
tions (albeit with activated leaving groups) into a range where
they become detectable and potentially evolvable. BcPMH is a
striking example of how an active site can be adapted for six che-
mical tasks, encompassing and extending the chemical repertoire
of its superfamily.
Implications of Catalytic Versatility. Catalytic promiscuity has
been suggested to contribute to enzyme evolution, through the
mechanism of gene duplication followed by specialization of one
of the two copies for the new function (1, 2, 5, 6). The side activ-
ities of BcPMH provide a possibly valuable latent functional
potential that coexists with a presumed main activity, even if there
is no immediate need or even natural substrate for them. The
history of BcPMH discovery provides an example of this poten-
tial: Even though glyceryl glyphosate is an unnatural compound
that the bacterium had never encountered in its original habitat,
its ability to hydrolyze this type of phosphonate monoester, there-
by enabling the bacterium to use it as the sole source of phos-
phorus (9), led to the identification of BcPMH. Although
BcPMH has a moderate kcat∕KM of 9.5 M−1 s−1 against glyceryl
glyphosate, this accidental activity conferred survival. Enzymes
with kcat∕KM values in this region (26, 36), and even as low as
0.3 M−1 s−1, have been shown to confer a selective advantage
(8), although in the latter case they were overexpressed from a
multiple copy plasmid under control of a strong promotor.
The enzyme-catalyzed hydrolysis of sulfonate esters is unprece-
dented, and there are no examples of sulfonate monoesters in
nature, either natural or xenobiotic. However, the observed
catalytic efficiencies (kcat∕KM ¼ 1.4 and 49 M−1 s−1 for 6a and
6b, respectively) indicate that BcPMH is proficient enough for
this unseen substrate that it could be immediately advantageous
to an organism.
Materials and Methods
Details of cloning, mutant construction, protein expression, purification, bio-
physical characterization, protein crystallization, and x-ray structure determi-
nation as well as details on the activity assays are listed in SI Text.
ACKNOWLEDGMENTS. We thank A. J. Kirby, A. Aharoni, and O. Khersonsky for
useful comments on the manuscript. We thank C. Bertozzi for the MtbFGE
gene and Monsanto for the kind gift of plasmid pMON9428. This research
was funded by the Biotechnology and Biological Sciences Research Council
(BBSRC), the Medical Research Council (MRC), the EU networks ENDIRPRO,
and ProSA, and studentships to S.J. (German National Academic Foundation)
and A.B. (BBSRC CASE/GlaxoSmithKline). F.H. is an ERC starting investigator.
1. O’Brien PJ, Herschlag
enzymatic activities. Chem Biol, 6:R91–R105.
2. Khersonsky O, Roodveldt C, Tawfik DS (2006) Enzyme promiscuity: evolutionary and
mechanistic aspects. Curr Opin Chem Biol, 10:498–508.
3. Jonas S, Hollfelder F (2008) Mechanism and Catalytic Promiscuity: Emerging Mechan-
istic Principles for Identification and Manipulation of Catalytically Promiscuous
D
(1999) Catalytic promiscuity and the evolution of new
19. Zalatan JG, Fenn TD, Brunger AT, Herschlag D (2006) Structural and functional com-
parisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phospha-
tase: Implications for mechanism and evolution. Biochemistry, 45:9788–9803.
20. Peng J, Schmidt B, von Figura K, Dierks T (2002) Identification of formylglycine in
sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spec-
trometry. J Mass Spectrom, 38:80–86.
Enzymes. Handbook of Protein Engineering,
Weinheim, Germany), Vol 1, pp 47–79.
, ed Bornscheuer U (Wiley VCH,
21. Carrico IS, Carlson BL, Bertozzi CR (2007) Introducing genetically encoded aldehydes
into proteins. Nat Chem Biol, 3:321–322.
4. Glasner ME, Gerlt JA, Babbitt PC (2006) Evolution of enzyme superfamilies. Curr Opin
Chem Biol, 10:492–497.
5. Aharoni A, et al. (2005) The ‘evolvability’ of promiscuous protein functions. Nat Genet,
37:73–76.
6. Jensen RA (1976) Enzyme recruitment in evolution of new function. Annu Rev
Microbiol, 30:409–425.
7. Hughes AL (1994) The evolution of functionally novel proteins after gene duplication.
Proc Biol Sci, 256:119–124.
22. Benjdia A, Deho G, Rabot S, Berteau O (2007) First evidences for a third sulfatase
maturation system in prokaryotes from E. coli aslB and ydeM deletion mutants. FEBS
Lett, 581:1009–1014.
23. Lee B, Richards FM (1971) The interpretation of protein structures: Estimation of static
accessibility. J Mol Biol, 55:379–400.
24. Garcia De La Torre J, Huertas ML, Carrasco B (2000) Calculation of hydrodynamic prop-
erties of globular proteins from their atomic-level structure. Biophys J , 78:719–730.
25. Shenoy AR, Sreenath N, Podobnik M, Kovacevic M, Visweswariah SS (2005) The Rv0805
gene from Mycobacterium tuberculosis encodes a 30, 50-cyclic nucleotide phosphodies-
terase: biochemical and mutational analysis. Biochemistry, 44:15695–15704.
26. Ghanem E, Li Y, Xu C, Raushel FM (2007) Characterization of a phosphodiesterase
capable of hydrolyzing EA 2192, the most toxic degradation product of the nerve
agent VX. Biochemistry, 46:9032–9040.
27. Wolfenden R (2006) Degrees of difficulty of water-consuming reactions in the absence
of enzymes. Chem Rev, 106:3379–3396.
28. Kuusela S, Lönnberg H (1993) Metal ions that promote the hydrolysis of nucleoside
phosphoesters do not enhance intramolecular phosphate migration. J Phys Org Chem,
6:347–356.
29. Morrow JR, Iranzo O (2004) Synthetic metallonucleases for RNA cleavage. Curr Opin
Chem Biol, 8:192–200.
30. Feng G, Mareque-Rivas JC, Torres Martin de Rosales R, Williams NH (2005) A highly
reactive mononuclear Zn(II) complex for phosphodiester cleavage. J Am Chem Soc,
127:13470–13471.
8. Patrick WM, Matsumura
phosphoribosylpyrophosphate amidotransferase is
I
(2008)
A
study in molecular contingency: Glutamine
promiscuous and evolvable
a
phosphoribosylanthranilate isomerase. J Mol Biol, 377:323–336.
9. Dotson SB, Smith CE, Ling CS, Barry GF, Kishore GM (1996) Identification, character-
ization, and cloning of
a phosphonate monoester hydrolase from Burkholderia
caryophilli PG2982. J Biol Chem, 271:25754–25761.
10. Schmidt B, Selmer T, Ingendoh A, von Figura K (1995) A novel amino acid modification
in sulfatases that is defective in multiple sulfatase deficiency. Cell, 82:271–278.
11. Jonas S, van Loo B, Hyvonen M, Hollfelder F (2008) A new member of the alkaline
phosphatase superfamily with a formylglycine nucleophile: Structural and kinetic
characterisation of
a phosphonate monoester hydrolase/phosphodiesterase from
Rhizobium leguminosarum. J Mol Biol, 384:120–136.
12. Hanson SR, Best MD, Wong C-H (2004) Sulfatases: Structure, mechanism, biological
activity, inhibition, and synthetic utility. Angew Chem Int Ed, 43:5736–5763.
13. Galperin MY, Bairoch A, Koonin EV (1998) A superfamily of metalloenzymes unifies
phosphopentomutase and cofactor-independent phosphoglycerate mutase with
alkaline phosphatases and sulfatases. Protein Sci, 7:1829–1835.
31. Peracchi
A (2001) Enzyme catalysis: Removing chemically ‘essential’ residues by
site-directed mutagenesis. Trends Biochem Sci, 26:497–503.
32. Menger FM, Ladika M (1987) Origin of rate accelerations in an enzyme model: The
p-nitrophenyl ester syndrome. J Am Chem Soc, 109:3145–3146.
33. Zalatan JG, Herschlag D (2006) Alkaline phosphatase mono- and diesterase reactions:
Comparative transition state analysis. J Am Chem Soc, 128:1293–1303.
34. Yang K, Metcalf WW (2004) A new activity for an old enzyme: Escherichia coli bacterial
alkaline phosphatase is a phosphite-dependent hydrogenase. Proc Natl Acad Sci USA,
101:7919–7924.
14. Babtie AC, Bandyopadhyay S, Olguin LF, Hollfelder
F (2009) Efficient catalytic
promiscuity for chemically distinct reactions. Angew Chem Int Ed, 48:3692–3694.
15. Lassila JK, Herschlag D (2008) Promiscuous sulfatase activity and thio-effects in a phos-
phodiesterase of the alkaline phosphatase superfamily. Biochemistry, 47:12853–12859.
16. O’Brien PJ, Herschlag
D (1998) Sulfatase activity of E. coli alkaline phosphatase
demonstrates a functional link to arylsulfatases, an evolutionary related enzyme fa-
mily. J Am Chem Soc, 120:12369–12370.
17. O’Brien PJ, Herschlag D (2001) Functional interrelationships in the alkaline phospha-
tase superfamily: Phosphodiesterase activity of Escherichia coli alkaline phosphatase.
Biochemistry, 40:5691–5699.
35. Toscano MD, Woycechowsky KJ, Hilvert
D (2007) Minimalist active-site redesign:
Teaching old enzymes new tricks. Angew Chem, Int Ed, 46:3212–3236.
36. McLoughlin SY, Jackson C, Liu JW, Ollis DL (2004) Growth of Escherichia coli
coexpressing phosphotriesterase and glycerophosphodiester phosphodiesterase,
using paraoxon as the sole phosphorus source. Appl Environ Microbiol, 70:404–412.
18. Olguin LF, Askew SE, O’Donoghue AC, Hollfelder F (2008) Efficient catalytic promiscu-
ity in an enzyme superfamily: An arylsulfatase shows a rate acceleration of 1013 for
phosphate monoester hydrolysis. J Am Chem Soc, 130:16547–16555.
van Loo et al.
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