Reaction of HppE with substrate analogues: Evidence for carbon-phosphorus bond cleavage by a carbocation rearrangement

Article abstract of DOI:10.1021/ja403441x

(S)-2-Hydroxypropylphosphonic acid ((S)-2-HPP) epoxidase (HppE) is an unusual mononuclear non-heme iron enzyme that catalyzes the oxidative epoxidation of (S)-2-HPP in the biosynthesis of the antibiotic fosfomycin. Recently, HppE has been shown to accept (R)-1-hydroxypropylphosphonic acid as a substrate and convert it to an aldehyde product in a reaction involving a biologically unprecedented 1,2-phosphono migration. In this study, a series of substrate analogues were designed, synthesized, and used as mechanistic probes to study this novel enzymatic transformation. The resulting data, together with insights obtained from density functional theory calculations, are consistent with a mechanism of HppE-catalyzed phosphono group migration that involves the formation of a carbocation intermediate. As such, this reaction represents a new paradigm for biological C-P bond cleavage.

Full text of DOI:10.1021/ja403441x

Communication
pubs.acs.org/JACS
Reaction of HppE with Substrate Analogues: Evidence for Carbon−
Phosphorus Bond Cleavage by a Carbocation Rearrangement
Wei-chen Chang, Steven O. Mansoorabadi, and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at
Austin, Austin, Texas 78712, United States
S
* Supporting Information
Scheme 1. Representative Enzyme-Catalyzed C−P Bond
Cleavage Reactions
ABSTRACT: (S)-2-Hydroxypropylphosphonic acid ((S)-
2-HPP) epoxidase (HppE) is an unusual mononuclear
non-heme iron enzyme that catalyzes the oxidative
epoxidation of (S)-2-HPP in the biosynthesis of the
antibiotic fosfomycin. Recently, HppE has been shown to
accept (R)-1-hydroxypropylphosphonic acid as a substrate
and convert it to an aldehyde product in a reaction
involving a biologically unprecedented 1,2-phosphono
migration. In this study, a series of substrate analogues
were designed, synthesized, and used as mechanistic
probes to study this novel enzymatic transformation.
The resulting data, together with insights obtained from
density functional theory calculations, are consistent with a
mechanism of HppE-catalyzed phosphono group migra-
tion that involves the formation of a carbocation
intermediate. As such, this reaction represents a new
paradigm for biological C−P bond cleavage.
hosphorus is an essential element for life.1 In biological
Psystems, P is typically present in the form of inorganic
phosphate or derivatives, such as organophosphate esters and
anhydrides.1 In recent years it has become increasingly apparent
that more highly reduced P compounds also play prominent
roles in biology.1,2 Many of these compounds, e.g., phosphonic
and phosphinic acids, contain stable C−P bonds in place of the
labile O−P bonds of the corresponding phosphate esters.2
Several such “C−P compounds” are bioactive natural products of
agricultural (e.g., phosphinothricin tripeptide, phosphonothrix-
in) and medical (e.g., fosfomycin, fosmidomycin) importance.1,2
Still other C−P compounds are metabolized as a source of
inorganic phosphate by microorganisms living in phosphate-
poor environments.1
Several distinct mechanisms of biological C−P bond cleavage
have been identified (Scheme 1).1,3−5 For example, C−P bond
cleavage in β-keto-phosphonates, such as phosphonoacetalde-
hyde (1), phosphonoacetate (2), and phosphonopyruvate (3),
catalyzed by the corresponding hydrolases, proceeds via
nucleophilic attack at the P center by a water molecule or an
active-site nucleophile. The negative charge developed during
turnover is stabilized by metal ions or a Schiff base.2,4,5 Another
example is the bacterial C−P lyase pathway, which utilizes a
noncanonical radical S-adenosyl-L-methionine (SAM) enzyme,
PhnJ, to cleave the C−P bond of 5-phospho-α-D-ribosyl-1-
alkylphosphonate (4).3 The detailed reaction mechanism of
PhnJ is not fully understood, although it has been proposed to
proceed via attack at the P center by a thiyl radical (5), leading to
homolytic cleavage of the C−P bond through a radical-mediated
process (6→7).3 Recently, the genes responsible for an
alternative organophosphonate catabolic pathway were identi-
fied in marine-derived metagenomic DNA. In this pathway, 2-
aminoethylphosphonate (8) is converted to glycine (13) and
inorganic phosphate (14) by the combined action of PhnY and
PhnZ.6 Specifically, PhnZ, a member of the histidine-aspartate
hydrolase superfamily, is proposed to catalyze the oxidative
cleavage of the C−P bond of 2-amino-1-hydroxyethyl-
phosphonate (9) through a mechanism reminiscent of that
used by the diiron enzyme myo-inositol oxygenase.6−8 In this
mechanism, the high-valent FeIII-FeIV-oxo intermediate 10
induces fragmentation of the C−P bond of the substrate, leading
to a metaphosphate intermediate (11) that recombines with the
carboxylate anion of glycine to give 12. Subsequent hydrolysis
yields 13 and 14.
More recently, an oxidative 1,2-phosphono group migration
was observed in the reaction of the non-heme iron enzyme (S)-2-
Received: April 6, 2013
© XXXX American Chemical Society
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dx.doi.org/10.1021/ja403441x | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Scheme 2. (A) Reaction Catalyzed by HppE, and Mechanisms
Proposed to Account for the Reactions of HppE with (B) (R)-
1-HPP (17) and (C) (1R,2R)- or (1R,2S)-2-Amino-1-HPP
Scheme 3. Possible Outcomes of Reaction of HppE with
Substrate Analogues Bearing (A) Electron-Withdrawing and
(B) Leaving Groups at C3, and (C) Mechanistic Probes Used
in This Study
a
(23 or 24)
phosphono migration (29→30, Scheme 3A) that reflects the
electron-withdrawing property of the C3 substituent of the
substrate analogues (R in 28), if the reaction proceeds via the
carbocation rearrangement mechanism (Scheme 2B, route a). In
contrast, if the radical-induced fragmentation/recombination
mechanism is operative (Scheme 2B, route b), analogues
containing a leaving group at C3 (e.g., 31) will partition between
formation of the migration product 33 (Scheme 3B, route b1)
and elimination of the leaving group from the enolate
intermediate 32, with concomitant formation of acrylaldehyde
34 and inorganic phosphate, respectively (Scheme 3B, route b2).
With these scenarios in mind, we prepared probes bearing
methoxy (36), monofluoro (37), or trifluoro (38) groups
(Scheme 3C).14 An analogue with a terminal methyl group (35)
was also synthesized.14 Compound 38 was synthesized as a
racemic mixture due to poor reactivity with porcine esterase and
the various lipases used to resolve the C1-OH chirality. The
remaining analogues were obtained in enantiomerically pure
form, as determined by measuring the enantiomeric excess by 31P
NMR using quinine as a chiral shift reagent15 (SI).
a
Route a, carbocation rearrangement. Route b, radical-induced
fragmentation/recombination.
hydroxypropylphosphonate (HPP, 15) epoxidase (HppE),
which catalyzes the conversion of 15 to fosfomycin (16)
(Scheme 2A).9 When incubated with the alternative substrate
(R)-1-HPP (17), HppE catalyzes the migration of the
phosphono group of 17 from C1 to C2 to give aldehyde 20 as
the product.10 From results obtained by X-ray crystallography,
model chemistry, and reactions with mechanistic probes (e.g., 23
and 24), it was proposed that cleavage of the C−P bond of 17 is
induced by the formation of a C2-centered carbocation
intermediate (19, Scheme 2B, route a).10 If correct, this would
represent a new paradigm for the enzymatic cleavage of a C−P
bond and would indicate that such reactions can occur via cation-
(Scheme 2B, route a) as well as anion- (Scheme 1A) and radical-
mediated (Scheme 1B,C) mechanisms. However, by analogy
with the proposed mechanism of PhnZ,6 the HppE-catalyzed C−
P bond migration observed with 17 can also be explained by a
radical-induced fragmentation to generate metaphosphate (21)
and an enolate intermediate (22), followed by recombination to
give 20 (Scheme 2B, route b). Similar mechanisms can also
account for the formation of 27 from the corresponding 2-amino
analogues 23 and 24 (Scheme 2C). To distinguish between these
two fundamentally different mechanisms, additional analogues of
17 bearing electron-withdrawing/leaving groups at C3 were
designed, synthesized, and analyzed as mechanistic probes of the
HppE-catalyzed 1,2-phosphono migration reaction.
Following the previously reported procedure,10 the reaction of
HppE with 35 was monitored using 1H NMR spectroscopy over
a 30 min period, and the extent of substrate consumption/
product formation at various time points was estimated by peak
integration and comparison with an internal standard (DMSO-
d6, δ 2.49). A new signal with a chemical shift consistent with an
aldehyde proton (δ 9.32) was observed. Its appearance is
accompanied by a shift of the methyl resonance from δ 0.75 (of
35) to 0.68 (of 39), indicating that HppE can accept 35 as a
substrate and convert it nearly quantitatively to the correspond-
ing migration product 39 (Figure 1a and Scheme 4).
When the experiments were repeated with 36, the distinctive
aldehyde proton peak of the migration product 40 (δ 9.42) was
detected, although the extent of conversion was only 16% during
the time monitored (Figure 1b). Neither methoxy group
elimination nor acrylaldehyde formation was noted. When 37
was incubated with HppE under the same conditions, only a trace
amount of the migration product 41 was detected by 1H NMR
The presence of one or more electron-withdrawing groups on
the C adjacent to the carbocation center will dramatically affect
the stability of the cation.11,12 For example, in studies of
prenyltransferase, which catalyzes a reaction generally accepted
to proceed a carbocation intermediate, the reaction rate was
decreased ∼3×107-fold when one of the methyl groups of the
substrate was replaced with a trifluoromethyl group.13 Thus, one
would expect a dramatic rate reduction of HppE-catalyzed
B
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Journal of the American Chemical Society
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To further explore the effects of electron-withdrawing
substitution with the (R)-1-HPP (17) analogues on the
energetics of the HppE-catalyzed reactions, density functional
theory (DFT) calculations were performed (SI). The calculated
C2−H bond dissociation energies (BDEs) for the methoxy-
containing substrate analogue 36 (93.9 kcal/mol) and the
monofluoro analogue 37 (93.5 kcal/mol) are very similar to that
of 17 (93.8 kcal/mol),10 whereas the BDE for the trifluoro
analogue 38 is significantly higher (98.4 kcal/mol). The
increased strength of the C−H bond of 38 may explain the
inability of HppE to accept the R-isomer as a substrate, but BDEs
alone cannot explain the observed differences in yield with 36
and 37 (16% and <5% versus 17, respectively). This prompted an
examination of the relative ionization energies (IEs) of each of
the corresponding C2-centered radicals of 36−38.17 The IEs of
the substrate analogue radicals were found to increase as a
function of the electron-withdrawing ability of the C3
substituent, from 4.5 to 15.2 to 26.3 kcal/mol (relative to the
IE of the (R)-1-HPP radical) for analogues 36−38, respectively.
This increase in IE closely follows the observed trend in reactivity
of HppE with the substrate analogues, supporting carbocation
formation in the 1,2-phosphono migration reaction (Scheme 2B,
route a).
The C2 radicals/cations formed in the reactions of HppE with
17 and analogues 36−38 are centered at a 2° C. It is well known
that the stability of both radicals and cations increases in the
order 1° < 2° < 3°.11,12 Moreover, the stabilization effects are
more dramatic for cations than the corresponding radicals.12 As a
result, an analogue of 17 proceeding through a 3° carbocation
intermediate is expected to be readily converted by HppE to the
subsequent migration product, while an analogue that requires
the formation of a 1° carbocation is likely to be a poor substrate.
Two compounds, 43 and 44, were therefore prepared to test this
hypothesis and gain further evidence for the proposed
carbocation mechanism (Scheme 5).14
1
Figure 1. Selected H and 19F NMR assays of HppE with substrate
analogues (a) 35, (b) 36, (c) 37, and (d) 38. The bottom trace of each
panel is the spectrum taken 3 min after mixing HppE with the substrate
analogue, and the top trace is recorded 27 min after initiation of the
reaction. The NMR signals and the corresponding protons in the
structures shown in Scheme 4 are color-coded.
Scheme 4. Reactions of HppE with 35−38
Compound 43 was synthesized as a racemic mixture. Upon
incubation with HppE, 43 was completely consumed to produce
acylphosphonate 45 (CH3, δ 0.86, d, JH−H = 7.2 Hz) and the
migration product 46 (CH3, δ 1.05, d, JP−H = 13.8 Hz; aldehyde-
H, δ 9.41) in a 1:1 ratio (as determined by integration of the
methyl signals of the products, Figure 2a). Unlike 43, the R- and
S-isomers of 44 were prepared separately.14 When (S)-44 was
incubated with HppE, quantitative turnover to the acyl-
phosphonate product was observed (Figure S2). However,
under the same conditions, <15% of (R)-44 was converted to the
migration product 47 (methylene protons, δ 2.72, dd, JP−H = 19.8
Hz, JH−H = 4.2 Hz; aldehyde-H, δ 9.40, t, JH−H = 4.2 Hz, Figure
(Scheme 4 and Figure S1). The conversion was <5% based on
integration of the 19F NMR peaks (Figure 1c). Interestingly, in
addition to the fluoro resonances of the substrate and product (δ
−218 and −225, respectively), a fluoride anion signal at δ −121
was detected. However, this F− peak remains visible in the
absence of enzyme, and no inorganic phosphate formation was
discernible using 31P NMR. Thus, the appearance of F− during
incubation is unlikely a catalytically relevant event.
When the substrate analogue 38 was incubated with HppE,
∼50% of it was converted to acylphosphonate 42 (estimated by
integration of the 19F NMR resonances at δ −63.2 of 38 and δ
−61.6 of 42, Figure 1d). A quintet (JH−F = 10.8 Hz) at δ 3.60 in
Scheme 5. Reactions of HppE with 43 and 44
1
the H NMR spectrum (Figure 1d) is characteristic for the C2
methylene protons of 42. No obvious formation of the migration
product was discernible. The fact that only half of 38 was
consumed during the course of the reaction is not surprising
since 38 used in the incubation is a racemic mixture. It appears
that only the S-epimer is accepted as a substrate by HppE and is
converted to the expected acylphosphonate product 42, whereas
the R-epimer, which should give rise to the migration product,
does not react (Scheme 4).10,16 Given the inverse correlation
between the extent of conversion of the analogues to the
corresponding migration products and the electron-withdrawing
ability of the substituents, these results are more consistent with a
mechanism involving the formation of a carbocation inter-
mediate along the reaction coordinate.
C
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Journal of the American Chemical Society
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AUTHOR INFORMATION
Corresponding Author
h.w.liu@mail.utexas.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is supported in part by grants from the National
Institutes of Health (GM040541) and the Welch Foundation (F-
1511). Dedicated to Prof. Koji Nakanishi on the occasion of his
88th birthday. We thank Steve Sorey for help in acquiring the
NMR spectra.
REFERENCES
■
(1) (a) Seto, H.; Kuzuyama, T. Nat. Prod. Rep. 1999, 16, 589.
(b) White, A. K.; Metcalf, W. W. Annu. Rev. Microbiol. 2007, 61, 379.
(2) Metcalf, W. W.; van der Donk, W. A. Annu. Rev. Biochem. 2009, 78,
65.
Figure 2. Selected 1H NMR assays of HppE with substrate analogues (a)
43 and (b) (R)-44, (c) preincubation with (R)-44 followed by addition
of 15, and (d) preincubation with 17 followed by addition of 15. The
bottom trace in panels (a) and (b) is the spectrum taken 3 min after
mixing HppE with the substrate analogue, and the top trace is recorded
27 min after initiation of the reaction. The NMR signals and the
corresponding protons in the structures shown in Scheme 5 are color-
coded.
(3) Kamat, S. S.; Williams, H. J.; Raushel, F. M. Nature 2011, 480, 570.
(4) Quinn, J. P.; Kulakova, A. N.; Cooley, N. A.; McGrath, J. W.
Environ. Microbiol. 2007, 9, 2392.
(5) Kim, J.; Dunaway-Mariano, D. Biochemistry 1996, 35, 4628.
(6) McSorley, F. R.; Wyatt, P. B.; Martinez, A.; Delong, E. F.; Hove-
Jensen, B.; Zechel, D. L. J. Am. Chem. Soc. 2012, 134, 8364.
(7) Xing, G.; Barr, E. W.; Diao, Y.; Hoffart, L. M.; Prabhu, K. S.; Arner,
R. J.; Reddy, C. C.; Krebs, C.; Bollinger, J. M., Jr. Biochemistry 2006, 45,
5402.
2b). Further experiments showed that preincubation of HppE
with (R)-44 led to enzyme inactivation, as the resulting enzyme
failed to convert 15 to 16 (Figure 2c). In control reactions where
HppE was preincubated with 17, the enzyme remained active
and capable of catalyzing fosfomycin production from 15 (Figure
2d). These data suggest that HppE is able to abstract a H atom
from C2 of (R)-44 to initiate the reaction, but only a fraction of
the radicals generated are oxidized to the corresponding cation
and converted to the migration product 47; the remaining
radicals react with the enzyme, rendering it inactive. This
interpretation is consistent with the results of DFT calculations
on 43 and 44, which show that the C2−H BDE and IE of the 3°
analogue 43 are 3.3 and 7.3 kcal/mol lower, respectively, than for
17, while those for the 1° analogue 44 are 3.7 and 10.5 kcal/mol
greater than for 17.
(8) Xing, G.; Diao, Y.; Hoffart, L. M.; Barr, E. W.; Prabhu, K. S.; Arner,
R. J.; Reddy, C. C.; Krebs, C.; Bollinger, J. M., Jr. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 6130.
(9) (a) Liu, P.; Murakami, K.; Seki, T.; He, X.; Yeung, S. M.; Kuzuyama,
T.; Seto, H.; Liu, H.-w. J. Am. Chem. Soc. 2001, 123, 4619. (b) Zhao, Z.;
Liu, P.; Murakami, K.; Kuzuyama, T.; Seto, H.; Liu, H.-w. Angew. Chem.,
Int. Ed. 2002, 41, 4529. (c) Liu, P.; Liu, A.; Yan, F.; Wolfe, M. D.;
Lipscomb, J. D.; Liu, H.-w. Biochemistry 2003, 42, 11577. (d) Higgins, L.
J.; Yan, F.; Liu, P.; Liu, H.-w.; Drennan, C. L. Nature 2005, 437, 838.
(10) Chang, W.-c.; Dey, M.; Liu, P.; Mansoorabadi, S. O.; Moon, S.-J.;
Zhao, K. Z.; Drennan, C. L.; Liu, H.-w. Nature 2013, 496, 114.
(11) Blanksby, S. J.; Ellison, G. B. Acc. Chem. Res. 2003, 36, 255.
(12) Kim, C. K.; Lee, K. A.; Bae, S. Y.; Han, I. S.; Kim, C. K. Bull. Kor.
Chem. Soc. 2004, 25, 311.
(13) (a) Poulter, C. D.; Rilling, H. C. Biochemistry 1976, 15, 1079.
(b) Poulter, C. D.; Satterwhite, D. M. Biochemistry 1977, 16, 5470.
(14) See SI for experimental details.
In summary, these results, coupled with information obtained
from previous mechanistic studies,10,18 are consistent with the
following mechanism of HppE-catalyzed 1,2-phosphono migra-
tion (Scheme 2B and Figure S3). First, (R)-1-HPP (17) binds to
the ferrous iron of HppE in a bidentate fashion through its
hydroxyl and phosphonate moieties and organizes the iron
center to coordinate molecular oxygen. O2 then binds, generating
a ferric-superoxo species that abstracts the pro-R H atom from
C2. Proton-coupled electron transfer to the resulting ferric-
hydroperoxo species generates a highly reactive ferryl
intermediate (18) that oxidizes the C2-centered substrate radical
to the corresponding 2° carbocation (19). The carbocation thus
formed induces a 1,2-shift of the phosphono moiety to generate
the aldehyde product (20). Finally, product release and
reduction of the ferric iron back to the ferrous state completes
the catalytic cycle. To our knowledge, this is the first example of
enzymatic cleavage of a C−P bond induced by the formation of a
carbocation. As such, it represents a new paradigm for biological
C−P bond cleavage.
̇
(15) Zyman
Tetrahedron: Asymmetry 1996, 7, 1277.
́ ́
czyk-Duda, E.; Skwarczynski, M.; Lejczak, B.; Kafarski, P.
(16) The configuration of the substrate governs its enzymatic fate.9b
The S-epimer of 1-HPP is converted to an acylphosphonate, whereas the
phosphonate migration reaction is observed with the R-epimer.10
(17) The calculated IEs were obtained as the difference in the
electronic energies of the radical and cationic species using the
optimized structure of the corresponding radical. This was done because
the phosphonate group was found to migrate to the 2 position during
geometry optimization of the cationic species. The reported values can
thus be considered an upper limit for the IE differences.
(18) (a) Yan, F.; Moon, S. J.; Liu, P.; Zhao, Z.; Lipscomb, J. D.; Liu, A.;
Liu, H.-w. Biochemistry 2007, 46, 12628. (b) Mirica, L. M.; McCusker, K.
P.; Munos, J. W.; Liu, H.-w.; Klinman, J. P. J. Am. Chem. Soc. 2008, 130,
8122. (c) Yun, D.; Dey, M.; Higgins, L. J.; Yan, F.; Liu, H.-w.; Drennan,
C. L. J. Am. Chem. Soc. 2011, 133, 11262. (d) Huang, H.; Chang, W.-c.;
Pai, P.-J.; Romo, A.; Mansoorabadi, S. O.; Russell, D. H.; Liu, H.-w. J.
Am. Chem. Soc. 2012, 134, 16171.
ASSOCIATED CONTENT
* Supporting Information
Experimental details and computational methods. This material
is available free of charge via the Internet at http://pubs.acs.org.
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