Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from Sphingobium sp. TCM1

Article abstract of DOI:10.1021/acs.biochem.8b00145

The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is notable for its ability to hydrolyze organophosphates that are not substrates for other enzymes. In an attempt to determine the catalytic properties of Sb-PTE for hydrolysis of chiral phosphotriesters, we discovered that multiple phosphodiester products are formed from a single substrate. For example, Sb-PTE catalyzes the hydrolysis of the R_P-enantiomer of methyl cyclohexyl p-nitrophenyl phosphate with exclusive formation of methyl cyclohexyl phosphate. However, the enzyme catalyzes hydrolysis of the S_P-enantiomer of this substrate to an equal mixture of methyl cyclohexyl phosphate and cyclohexyl p-nitrophenyl phosphate products. The ability of this enzyme to catalyze the hydrolysis of a methyl ester at the same rate as the hydrolysis of a p-nitrophenyl ester contained within the same substrate is remarkable. The overall scope of the stereoselective properties of this enzyme is addressed with a library of chiral and prochiral substrates.

Full text of DOI:10.1021/acs.biochem.8b00145

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Multiple Reaction Products from the Hydrolysis of
Chiral and Prochiral Organophosphate Substrates by
the Phosphotriesterase from Sphingobium sp. TCM1
Andrew N. Bigley, Tamari Narindoshvili, Dao Feng Xiang, and Frank M. Raushel
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00145 • Publication Date (Web): 07 Mar 2018
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Page 1 of 6
Biochemistry
1
2
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8
9
Multiple Reaction Products from the Hydrolysis of Chiral and
Prochiral Organophosphate Substrates by the Phosphotriesterase
from Sphingobium sp. TCM1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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0
Andrew N. Bigley, Tamari Narindoshvili, Dao Feng Xiang, and Frank M. Raushel*
Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
Supporting Information Placeholder
organophosphates of potential interest as substrates are chiral
entities that include chemical warfare agents and antiviral pro-
drugs. While the stereochemical properties of Sb-PTE have
ABSTRACT: The phosphotriesterase from Sphingobium sp.
TCM1 (Sb-PTE) is notable for its ability to hydrolyze organo-
phosphates that are not substrates for other enzymes. In an at-
tempt to determine the catalytic properties of Sb-PTE for hy-
drolysis of chiral phosphotriesters we discovered that multiple
phosphodiester products are formed from a single substrate. For
6-8
been determined previously for a chiral thiophosphate substrate,
the selectivity for hydrolysis of chiral organophosphate com-
3
pounds has not been addressed. Therefore, determination of the
stereochemical preferences for hydrolysis of chiral organophos-
phate esters such as compound 1 (Scheme 1) was attempted by
monitoring the release of p-nitrophenol at 400 nm as a function
example, Sb-PTE catalyzes the hydrolysis of the (R )-
P
enantiomer of methyl cyclohexyl p-nitrophenyl phosphate with
exclusive formation of methyl cyclohexyl phosphate. However,
6
of time. It was observed that the time course for hydrolysis of
the enzyme catalyzes hydrolysis of the (S )-enantiomer of this
P
(
R /S )-1 catalyzed by Sb-PTE proceeds to ~70% of completion,
P P
substrate to an equal mixture of methyl cyclohexyl phosphate
and cyclohexyl p-nitrophenyl phosphate products. The ability of
this enzyme to catalyze the hydrolysis of a methyl ester at the
same rate as the hydrolysis of a p-nitrophenyl ester contained
within the same substrate is remarkable. The overall scope of
the stereoselective properties of this enzyme is addressed with a
library of chiral and prochiral substrates.
based on the expected amount of p-nitrophenol produced (Fig-
ure 1A). For comparison, the hydrolysis reaction was also initi-
ated with the H257Y/L303T variant of Pd-PTE (YT-Pd-PTE).
9
This variant of Pd-PTE does not show any stereoselectivity
toward the hydrolysis of (R /S )-1, and the reaction proceeds to
P
P
completion with the hydrolysis of both enantiomers when com-
pared to the base-catalyzed hydrolysis with NaOH.
The recently described phosphotriesterase from Sphingobium
sp. TCM1 (Sb-PTE) possesses a rather broad substrate profile
and is able to hydrolyze insecticides, plasticizers and flame
retardants that are not typically substrates for other enzymes of
1
A
0.8
0
0
.6
.4
1,2
this class. While Sb-PTE uses a binuclear metal center that is
nearly identical in structure to that found in the phosphotriester-
ase from Pseudomonas diminuta (Pd-PTE) this enzyme is able
to catalyze the hydrolysis of simple phenyl esters contained
within organophosphate substrates at nearly the same rate as the
0.2
0
0
500
1000
1500
2-4
hydrolysis of p-nitrophenyl esters. In contrast, the catalytic
Time (sec)
activity of Pd-PTE is diminished by ~5 orders of magnitude
when p-nitrophenol is substituted with phenol as the leaving
group.
1
5
B
0
.8
.6
0
0.4
0.2
0
0
500
1000
1500
Time (sec)
Figure 1. Time courses for the hydrolysis of racemic and
separate enantiomers of 1. (A) Hydrolysis of 60 ꢀM (R /S )-1
Scheme 1. The (R )- and (S )-enantiomers of compound 1 and
the generic template for compounds tested as substrates for Sb-
PTE (Tables 1 and 2).
P
P
P
P
by 1.0 M NaOH (black), 4.5 nM YT-Pd-PTE (blue) and 1.7 ꢀM
Sb-PTE (red). (B) Hydrolysis of 60 ꢀM of isolated enantiomers
of compound 1: (S )-1 by 1.0 M NaOH (black); (S )-1 by 4.5
P
P
Given the significant substrate diversity of Sb-PTE, a more in
depth evaluation of its catalytic properties is warranted. Many
nM YT-Pd-PTE (blue); (S )-1 by 1.7 ꢀM Sb-PTE (red); and
P
(R )-1 by 1.7 ꢀM Sb-PTE (yellow).
P
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Biochemistry
Page 2 of 6
When the two enantiomers of 1 are hydrolyzed separately,
the reaction catalyzed by Sb-PTE proceeds to completion with
ther than the more probable attack on phosphorus. If this were
-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the case, then the oxygen from the attacking H O/OH would
2
(
R )-1, but the (S )-enantiomer continues to only ~50% of the
ultimately be found in the methanol product rather than in the
corresponding phosphodiester product. To test this possibility,
P
P
expected amount of p-nitrophenol (Figure 1B). Addition of YT-
Pd-PTE or NaOH did not result in any further formation of p-
nitrophenol, indicating that more than one organophosphate
diester product is formed. To determine what products are
formed from the hydrolysis of 1 by Sb-PTE, ~1 mg of substrate
18
the reaction was conducted in 50% O-labeled water and the
products analyzed by mass spectrometry.
16
When (R )-1 is hydrolyzed by Sb-PTE in 100% O-labeled
P
water, the only products detected by mass spectrometry are p-
nitrophenol and methyl cyclohexyl phosphate (Figure 3A).
3
1
was hydrolyzed and the products analyzed by P NMR spec-
troscopy. As anticipated, hydrolysis with YT-Pd-PTE yields one
phosphodiester product with a single resonance at 1.77 ppm.
When (S )-1 is hydrolyzed with Sb-PTE the product cyclohexyl
P
p-nitrophenyl phosphate is also detected (Figure 3B). When the
(
Figure 2A). Similarly, the reaction of Sb-PTE with (R )-1 re-
18
P
same reaction is conducted in 50% O-labeled water, the two
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
sults in a single product resonance at 1.77 ppm (Figure 2B), but
phosphodiester products appear as “doublets” separated by two
mass units. This finding demonstrates that the mechanism for
hydrolysis of the methyl ester is identical to that for the hydroly-
sis of the p-nitrophenyl ester with nucleophilic attack at phos-
phorus.
when the (S )-enantiomer is hydrolyzed by Sb-PTE two reso-
P
nances of approximately equal intensity are observed with
chemical shifts of 1.77 ppm and -5.06 ppm (Figure 2C). The
second resonance does not correspond to the unreacted sub-
strate, which has a chemical shift of -5.68 ppm (Figure S6).
Sb-PTE has previously been shown to slowly hydrolyze tribu-
tyl phosphate but not triethyl phosphate, leading to the initial
expectation that the second phosphodiester product is due to the
6
10
4
138.01859
6
6
6
A
3 10
2
31
hydrolysis of the cyclohexyl ester. When the P NMR spec-
1
trum of the reaction products is acquired with H spin coupling,
193.06220
2
1
10
10
the resonance at 1.77 ppm exhibits 8 resonances due to proton
coupling with the three hydrogens from the methyl group and
one hydrogen from the cyclohexyl group (inset to Figure 2C).
3
However, the resonance at -5.06 ppm is a simple doublet ( J
=
PH
0
7
.4 Hz). This unambiguously identifies the new product as re-
100
150
200
250
m/z
300
350
350
350
400
400
400
sulting from the hydrolysis of the methyl ester within (S )-1.
P
6
6
6
5
2
10
A
B
B
138.0185
1
.5 10
193.06213
300.06249
1
10
5 10
0
100
150
200
250
m/z
300
6
6
6
6
4
10
C
3 10
1
38.01854
C
2
1
10
10
193.06213
195.06638
300.06249
3
02.0667
0
1
00
150
200
250
300
m/z
Figure 3. Negative ion mass spectra of the reaction products for
hydrolysis of 1 by Sb-PTE. (A) Reaction products from (R )-1.
P
31
Figure 2. P NMR spectra of the enzyme-catalyzed hydrolysis
products of compound 1. (A) The product formed from the hy-
drolysis of (R /S )-1 by YT-Pd-PTE. (B) The product formed
(B) Reaction products from (S )-1. (C) Hydrolysis products
P
18
from (S )-1 when reaction is conducted in 50% O labeled wa-
P
ter.
P
P
from the hydrolysis of (R )-1 by Sb-PTE. (C) The two products
P
The ability of Sb-PTE to catalyze the hydrolysis of more than
one ester contained within the same substrate was further probed
by the synthesis and characterization of a small library of chiral
and prochiral compounds as potential substrates. To address
whether the substrate and product profiles are based, in part, on
the stereochemical arrangement of specific substituents con-
formed from the hydrolysis of (S )-1 by Sb-PTE. The inset to
P
1
panel C shows the H spin coupled spectrum.
The finding that Sb-PTE catalyzes the hydrolysis of the me-
thyl ester at nearly the same rate as hydrolysis of the p-
nitrophenyl ester within (S )-1 is most unusual. This observation
P
led to the initial proposal that an alternative chemical mecha-
nism might be operable for hydrolysis of the methyl ester. We
tained within (S )-1, Sb-PTE was subsequently tested with two
P
prochiral substrates: methyl di-p-nitrophenyl phosphate (2) and
dimethyl p-nitrophenyl phosphate (3). With both compounds,
two phosphodiester products are formed due to the concurrent
speculated that hydrolysis of the methyl substituent might arise
-
via a nucleophilic attack of H O/OH on the methyl carbon ra-
2
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Biochemistry
hydrolysis of the methyl and p-nitrophenyl esters (Table 1).
enantiomers, conformational restrictions or limited reactivity of
the cyclohexyl ester may hinder the hydrolysis of this ester,
whereas the more reactive phenyl ester is the preferred leaving
1
2
3
4
5
6
7
8
9
However, it is not yet known whether there is a stereochemical
preference for hydrolysis of either the proR or proS substituents
in compounds 2 and 3. With both substrates the methyl ester is
hydrolyzed about 10-12% as fast as the p-nitrophenyl ester,
demonstrating that the enhanced ability of Sb-PTE to hydrolyze
methyl esters is not limited to a specific stereochemistry or sub-
strate.
group in compounds such as (S )-5.
P
The specificity for the methyl group was further probed by
examination of the hydrolysis reaction with compounds 11
(paraoxon) and 6, which contain ethyl rather than methyl esters.
Hydrolysis of the ethyl ester was observed. With (R )-6, hydrol-
P
Table 1. Compounds tested for catalytic activity by Sb-PTE.
ysis of the p-nitrophenyl ester results in the only phosphodiester
product obtained, but with (S )-6, hydrolysis of the ethyl ester is
P
% Ester Hydro-
observed at a 6-fold lower rate relative to the hydrolysis of the
p-nitrophenyl ester. These data demonstrate that other primary
alcohols can be cleaved, but Sb-PTE appears to prefer the hy-
drolysis of methyl esters in comparison with bulkier alkyl
groups.
Ester Substituents
lyzed
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
X
Y
Z
X
Y
Z
(R )-1
P
Cy
pNP
Me
<1
>99
<1
(
SP)-1
Me
Me
Me
pNP
pNP
pNP
Cy
pNP
Me
44
12
10
56
88
90
<1
*
Table 2. Kinetic constants for Sb-PTE with compounds 1-11.
k /K
2
cat
m
3
*
-1
-1 -1
Compound
kcat (s )
K (mM)
m
(M s )
(
RP)-4
iPr
Me
Ph
pNP
pNP
pNP
pNP
pNP
pNP
pAP
pAP
Ph
Me
iPr
Me
Ph
7
7
89
93
4
3
4
(
RP)-1
6 ± 1
1.1 ± 0.2
4.7 ± 0.3 x 10
2.1 ± 0.3 x 10
(S )-4
<1
<1
92
<1
<1
<1
<1
<1
(S
P
)-1
2
1.11 ± 0.05 0.053 ± 0.007
P
5
9 ± 1
9.1 ± 0.4
5 ± 2
0.08 ± 0.02
0.76 ± 0.05
2.3 ± 0.9
1.1 ± 0.3 x 10
(
RP)-5
<1
8
>99
<1
4
3
1.2 ± 0.1 x10
2.1 ± 0.1 x 10
2.6 ± 0.4 x 10
1.0 ± 0.1 x 10
2.5 ± 0.7 x 10
4.1 ± 0.5 x 10
5.1 ± 0.9 x 10
1.1 ± 0.2 x 10
3.2 ± 0.7 x 10
1.4 ± 0.2 x 10
nd
(S )-5
Me
Cy
Et
P
3
4
5
3
4
4
3
3
4
(
RP)-4
(
RP)-6
Et
<1
14
<1
50
5
>99
86
(S )-4
2.05 ± 0.09 0.036 ± 0.006
P
(S )-6
Cy
Me
Cy
Me
P
(R )-5
6.3 ± 0.3
0.6 ± 0.1
0.061 ± 0.007
0.25 ± 0.07
P
(
RP)-7
Cy
Me
Cy
>99
50
(S
RP)-6
(S )-6
P
)-5*
(
2.5 ± 0.1
0.059 ± 0.007
0.054 ± 0.009
0.31 ± 0.06
(S )-7
P
2.8 ± 0.2
P
(
RP)-8
95
(
RP)-7
SP)-7
RP)-8
0.36 ± 0.03
0.38 ± 0.03
(S )-8
Me
Me
Me
Et
Ph
Cy
Cy
Cy
Bn
Et
77
nr
nr
7
4
19
nr
nr
*
P
(
0.12 ± 0.02
9
nr
nr
93
(
0.38 ± 0.01 0.006 ± 0.002
1
1
0
1
Bn
(S )-8
nd
nr
nd
P
pNP
9
nr
nr
nr
Cy, cyclohexyl; Me, methyl; Et, Ethyl; iPr, isopropyl; Ph,
phenyl; Bn, benzyl; pNP, p-nitrophenyl; pAP, p-acetylphenyl.
1
0
nr
nr
4
11
6
.5 ± 0.2
0.083 ± 0.009
7.9 ± 0.9 x 10
*The differentiation for hydrolysis of the prochiral substituents
was not determined. The initial substrate concentration was 3.0
mM and hydrolyzed with 1.7 ꢀM Sb-PTE. nr, not reactive.
*Determined by p-nitrophenol release which is <1% of the to-
tal product. Substrates were varied from 2.4 to 1500 ꢀM using
+
The limits of the substrate-based product selectivity were fur-
ther probed by examination of the role of the cyclohexyl group
via substitution with either an isopropyl (4) or phenyl (5) sub-
7.4 to 740 nM Sb-PTE as catalyst in 50 mM HEPES/K , pH 8.5,
o
30 C. nd, not determined; nr, not reactive.
The role of the p-nitrophenyl group was addressed by substi-
tution with p-acetylphenyl (7), phenyl (8) or cyclohexyl (9)
esters. Compound 9 was not hydrolyzed by Sb-PTE, suggesting
that an aromatic and/or electron withdrawing substituent is re-
quired as part of the substrate. The replacement of the p-
nitrophenyl ester by a p-acetylphenyl ester did not alter the ratio
of the observed products for either (R )-7 or (S )-7 relative to
stituent. With (R )-4, Sb-PTE hydrolyzes any one of the three
P
possible ester groups. Hydrolysis of the isopropyl ester proceeds
at nearly twice the rate as that of the methyl ester. However,
when (S )-4 is hydrolyzed by Sb-PTE, cleavage of the isopropyl
P
ester bond is not observed and ~7% of the observed products are
formed from hydrolysis of the methyl ester.
P
P
Similar to the hydrolysis of (R )-1, Sb-PTE catalyzes hy-
P
what is observed for the two enantiomers of 1. The substitution
of the p-nitrophenyl ester with an unsubstituted phenyl ester in
drolysis of (R )-5 only with the release of p-nitrophenol. Some-
P
what surprisingly, hydrolysis of (S )-5 produces essentially no
P
(R )-8 results in a small amount of hydrolysis of the cyclohexyl
P
p-nitrophenol. Hydrolysis of (S )-5 results primarily in the re-
P
ester, but no cleavage of the methyl ester. However, with (S )-8
P
lease of phenol with about 8% of the product due to the hydrol-
ysis of the methyl ester. These results suggest that the physical
bulk of the cyclohexyl group may limit formation of the proper
enzyme-substrate complex that leads to the formation of alterna-
the primarily hydrolysis event is with the methyl ester and there
is substantial hydrolysis of the cyclohexyl ester. Remarkably,
only 4% of the phosphodiester products are due to the cleavage
of the phenyl ester. These data suggests that while an aromatic
substituent is required for the reaction to proceed, a simple phe-
tive products from the (R )-enantiomers. With the (S )-
P
P
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Biochemistry
Page 4 of 6
nyl ester significantly induces the formation of alternative reac-
tion products from both enantiomers as in (R )-8 and (S )-8.
family of enzymes where multiple products can be formed from
13
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
P
P
a common carbocation intermediate. Similarly, two reaction
products could be formed from the enzyme catalyzed hydrolysis
of an asymmetrical organophosphate diester, although such
examples have apparently not been reported to the best of our
knowledge.
It is likely that an aromatic substituent is required to facilitate
the hydrolysis of alkyl esters by Sb-PTE. This requirement may
be due in part to the enhancement of the electrophilic character
of the phosphorus core for nucleophilic attack via the inductive
effect of the phenyl esters. Alternatively, the aromatic substitu-
ent may be required to facilitate proper alignment of the sub-
strate within the active site. To distinguish between these possi-
bilities methyl dibenzyl phosphate (10) was constructed and
evaluated as a potential substrate for Sb-PTE. No products were
detected by NMR spectroscopy for the hydrolysis of 10 suggest-
ing that activation of the phosphorus core is required by at least
one of the three ester groups before a simple alkyl ester can be
hydrolyzed by this enzyme at a significant rate. The kinetic
constants for the hydrolysis of all compounds are presented in
Table 2. It should be noted that these kinetic constants were
measured by following the rate of hydrolysis of the phenyl or
substituted phenyl ester and thus do not reflect the total enzy-
matic activity of Sb-PTE with these compounds.
Much work remains to be done to elucidate the structural ba-
sis for formation of the alternate products as well as discovering
how Sb-PTE can so effectively deliver a proton to the leaving
group to facilitate the reaction. The differential products ob-
tained with isolated enantiomers suggest that Sb-PTE might be a
useful platform for protein engineering where the catalytic
properties of the enzyme can be fine tuned not just in terms of
stereoselectivity, where one enantiomer is the highly preferred
substrate for hydrolysis, but also where any one of three ester
functional groups in a given chiral or prochiral substrate is
forced to be hydrolyzed.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Supplementary information includes materials and methods
used, as well as additional NMR and MS data.
With (S )-1, Sb-PTE catalyzes the hydrolysis of roughly
P
equal amounts of methyl and p-nitrophenyl esters but the pK of
a
p-nitrophenol and methanol differ by approximately 9 pH units.
The transition state energy differences for hydrolysis of the
methyl ester versus the p-nitrophenyl ester is expected to be
substantial. With all of the compounds synthesized and evaluat-
ed for this investigation we were unable to detect any hydrolysis
of the methyl ester by 1 M NaOH or Pd-PTE. This result is
consistent with a βlg of -0.43 determined previously for the hy-
drolysis of organophosphate triesters by hydroxide and -2.2 for
ꢀ AUTHOR INFORMATION
Corresponding Author
*
raushel@tamu.edu
ORCID
Frank M. Raushel: 0000-0002-5918-3089
5
hydrolysis by (Zn/Zn)-Pd-PTE. To further explore the limits of
Notes
methyl ester hydrolysis by Sb-PTE the substrate profile for (S )-
P
The authors declare no competing financial interests.
Organophosphate compounds are generally toxic and should be
used with due care.
7
was measured over the pH range 6-10 and the reaction was
conducted at temperatures ranging from 4 to 38 ˚C (Figures S7
and S8). The product ratios are not affected by temperature and
thus the activation energies are similar. Over the pH range of 7-
ꢀ ACKNOWLEDGMENT
1
0 there is a small drop in the percentage of the methanol prod-
uct formed from ~56% at pH 7 to ~41% at pH 10. This decrease
is to be expected given the apparent requirement for protonation
of the product as the phosphorus oxygen bond is broken during
the transition state. The rather small effect on pH (and appar-
This work was supported in part by the National Institutes of
Health (GM 116894).
ꢀ
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10,11
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12
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11
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(
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