PhnY and PhnZ comprise a new oxidative pathway for enzymatic cleavage of a carbon-phosphorus bond

Article abstract of DOI:10.1021/ja302072f

The sequential activities of PhnY, an α-ketoglutarate/Fe(II)- dependent dioxygenase, and PhnZ, a Fe(II)-dependent enzyme of the histidine-aspartate motif hydrolase family, cleave the carbon-phosphorus bond of the organophosphonate natural product 2-aminoethylphosphonic acid. PhnY adds a hydroxyl group to the α-carbon, yielding 2-amino-1-hydroxyethylphosphonic acid, which is oxidatively converted by PhnZ to inorganic phosphate and glycine. The PhnZ reaction represents a new enzyme mechanism for metabolic cleavage of a carbon-phosphorus bond.

Full text of DOI:10.1021/ja302072f

Communication
pubs.acs.org/JACS
PhnY and PhnZ Comprise a New Oxidative Pathway for Enzymatic
Cleavage of a Carbon−Phosphorus Bond
†
§
‡
‡
†
Fern R. McSorley, Peter B. Wyatt, Asuncion Martinez, Edward F. DeLong, Bjarne Hove-Jensen,
,
†
and David L. Zechel*
†
Department of Chemistry, Queen’s University, 90 Bader Lane, Kingston, Ontario, Canada K7L 3N6
§
School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, U.K.
‡
Division of Biological Engineering, Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 77
Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
*
S Supporting Information
5
NOP). This mechanism is unusual in that the organo-
phosphonate substrate must be incorporated into ribose as a
ABSTRACT: The sequential activities of PhnY, an α-
ketoglutarate/Fe(II)-dependent dioxygenase, and PhnZ, a
Fe(II)-dependent enzyme of the histidine-aspartate motif
hydrolase family, cleave the carbon−phosphorus bond of
the organophosphonate natural product 2-aminoethyl-
phosphonic acid. PhnY adds a hydroxyl group to the α-
carbon, yielding 2-amino-1-hydroxyethylphosphonic acid,
which is oxidatively converted by PhnZ to inorganic
phosphate and glycine. The PhnZ reaction represents a
new enzyme mechanism for metabolic cleavage of a
carbon−phosphorus bond.
5
-phospho-α-D-ribosyl-1-alkylphosphonate, whereupon CP
bond cleavage yields 5-phospho-α-D-ribosyl-1,2-cyclic phos-
phate and an alkane (i.e., methylphosphonic acid leads to
6
methane). A milestone study revealed that PhnJ cleaves the
CP bond of 5-phospho-α-D-ribosyl-1-alkylphosphonate through
a radical reaction that requires a [4Fe-4S] cluster and S-
7
adenosylmethionine. Although the precise mechanism of PhnJ
has yet to be defined, the formation of the 1,2-cyclic phosphate
suggests an essential role for the 2-hydroxyl of ribose in enzyme
turnover.
The global distribution of organophosphonates in what are
typically phosphate-poor environments leads one to consider
that other CP bond-cleaving mechanisms may have evolved to
mong many examples of naturally occurring organo-
8
A
phosphonates, 2-aminoethylphosphonic acid (1) is the
catabolize alternative forms of phosphorus. This was the
1
,2
most prevalent in the environment (Scheme 1a). Many
inspiration behind a recent study that screened marine-derived
metagenomic DNA for genes that restored the ability of a Δphn
mutant of Escherichia coli to grow in the presence of 1 as the
organisms, particularly phytoplankton, use 1 as a substitute for
3
phosphocholine for the synthesis of phosphonolipids.
9
Although incorporation of 1 into lipids can be a response to
phosphate deprivation, the carbon−phosphorus (CP) bond
may also afford protection against hydrolytic enzymes, alter
membrane bilayer structure, and contribute to intercellular
sole source of phosphate. In addition to finding genes
encoding the phosphonoacetaldehyde hydrolase noted above,
a new pair of genes was retrieved, phnY and phnZ, that
complemented the Δphn mutation. PhnY belongs to a large
and catalytically diverse class of non-heme Fe(II)/α-ketogluta-
rate-dependent dioxygenases (family code: pfam05721), which
include phytanoyl-CoA dioxygenases and halogenases. PhnY
also shows high sequence similarity to Penicillium decumbens
EpoA and Pseudomonas stutzeri WM88 HtxA, which epoxidize
cis-propenylphosphonic acid and oxidize hypophosphite to
2
communication. 1 also represents an important source of
inorganic phosphate (P ) for microorganisms living in
i
phosphate-limited environments. Studies have so far uncovered
two general enzyme mechanisms for cleaving a CP bond in
1,4
order to liberate P for metabolic use. The “electron-sink”
i
mechanism is applied to alkylphosphonates with a β-carbonyl
group, where the CP bond is cleaved by direct attack of a
nucleophile, either water or an enzyme side chain, on the
phosphorus center, with the enzyme using either a Schiff base
or interaction of a metal ion with the carbonyl group to assist
delocalization of the resulting carbanion. The phosphonoace-
taldehyde, phosphonopyruvate, and phosphonoacetate hydro-
10,11
phosphite, respectively.
PhnZ is predicted to be a metal
ion-dependent phosphohydrolase of the HD superfamily and
belongs to a large subclass of uncharacterized enzymes that are
frequently associated with the CP lyase encoding phn operon in
several species of bacteria (family codes: pfam01966 and
12
TIGR03276). The intriguing functions predicted for PhnY9
and PhnZ suggest a new strategy for cleavage of a CP bond,
which warranted further investigation.
4
lases typify this electron-sink mechanism. Phosphoenolpyr-
uvate mutase also falls into this category of CP bond cleavage
4
Synthetic phnY and phnZ genes were expressed separately in
E. coli and the encoded enzymes purified in a single step by
affinity chromatography (Supporting Information (SI) Figure
(
and is a homologue of phosphonopyruvate hydrolase),
although the primary function of this enzyme is to synthesize
CP bonds by catalyzing the equilibrium between phosphoe-
2
nolpyruvate and phosphonopyruvate. The second CP bond
cleavage mechanism is found in CP lyase, a multienzyme
complex encoded by the phn operon (phnCDEFGHIJKLM-
Received: March 1, 2012
Published: May 8, 2012
©
2012 American Chemical Society
8364
dx.doi.org/10.1021/ja302072f | J. Am. Chem. Soc. 2012, 134, 8364−8367

Journal of the American Chemical Society
Communication
a
Scheme 1. Proposed Reactions and Mechanisms for PhnY and PhnZ
a
(
a) Hydroxylation of 1 by the Fe(II)/α-ketoglutarate-dependent dioxygenase PhnY to give 2 followed by CP bond cleavage by PhnZ to yield
23
glycine 3 and inorganic phosphate (P ). (b) Hypothetical mechanism for PhnZ by analogy to the calculated mechanism for MIOX.
i
1
). We first examined the activity of PhnY, as the potent
resulted in conversion to P (SI Figure 2b). However, reaction
i
oxidizing activity of this class of enzyme seemed ideal for
of PhnZ directly with (±)-2 resulted in only 50% conversion
(Figure 1e), suggesting that PhnZ (and likewise PhnY) is
stereospecific. This indicates that 2 is the substrate for PhnZ,
and that PhnZ catalyzes the cleavage of the CP bond of 2 to
modifying 1 to render the CP bond susceptible to cleavage. The
31
reaction of PhnY with 1 was monitored by P NMR
spectroscopy. No change in the signal for 1 (δ 17.3 ppm)
was observed with PhnY alone or in the presence of α-
ketoglutarate (Figure 1a). However, a mixture of Fe(II), α-
ketoglutarate, ascorbate, and PhnY enabled conversion of 1 to a
new compound with a signal at δ 14.3 ppm (Figure 1b).
Omitting ascorbate markedly reduced conversion of 1 by PhnY
yield P . PhnZ activity is abolished by the addition of EDTA
i
(data not shown), indicating the essential role of a metal ion.
After the EDTA was removed by dialysis, PhnZ was incubated
with 2 generated by PhnY in the presence of various metal ions
2
+
3+
2+
2+
2+
2+
2+
2+
2+
(Fe , Fe , Co , Mn , Ni , Ca , Cu , Zn , or Mg , 0.1
(SI Figure 2a), reflecting the well-known ability of reducing
mM each). 31P NMR spectroscopy revealed that only Fe(II)
agents to enhance the activity of α-ketoglutarate/Fe(II)-
enabled PhnZ to completely convert 2 to P (SI Figure 3).
i
dependent dioxygenases by maintaining the iron cofactor in
Partial activity was observed with the other metal ions,
presumably due to the presence of residual Fe(II) from the
PhnY reaction, with the exception of Mg where no conversion
was observed, which suggests that this metal ion is inhibitory.
Repeating this analysis with synthetic (±)-2 confirmed the
requirement for Fe(II) for activity (SI Figure 4). ICP-MS
analysis of the metal ion content of PhnZ confirmed the
binding of 1.2 ± 0.1 mol of iron per mole of enzyme, as well as
0.5 ± 0.1 mol of calcium per mole of enzyme. (See SI for more
details.)
13
the catalytically active ferrous form. Given the precedent of α-
14
2+
hydroxylation of taurine by E. coli TauD and 2-hydrox-
15
yethylphosphonic acid by Streptomyces luridis DhpA, we
anticipated that hydroxylation at the α-carbon of 1 by PhnY
would yield the known natural product 2-amino-1-hydrox-
9
yethylphosphonic acid 2 (Scheme 1a). This was confirmed by
adding synthetic (±)-2 and observing an increase in the signal
at δ 14.3 ppm (Figure 1c). In contrast, methylphosphonic and
ethylphosphonic acids were not substrates for PhnY (data not
shown), which agrees with the previous observation that these
compounds do not sustain the growth of E. coli Δphn
The dependence on Fe(II) for activity suggested that PhnZ
might cleave the CP bond of 2 through an oxidative
mechanism. A precedent for such a reaction was reported for
the Fe(II)-dependent enzyme 2-hydroxyethylphosphonate
dioxygenase (HEPD). Although HEPD normally catalyzes
9
transformed with phnY and phnZ genes.
We next examined the role of PhnZ in the conversion of 1.
When PhnZ was included with the PhnY reaction, 31P NMR
16
spectroscopy revealed complete conversion of 1 to P (δ 2.9
oxidative cleavage of a carbon−carbon bond, elegant studies
i
ppm, Figure 1d). Incubation of PhnZ alone with 1 did not
afford conversion to a new product. Denaturation of PhnY (95
with substrate analogues revealed promiscuous CP bond
17,18
cleaving activity toward 1-hydroxyalkylphosphonic acids.
°C, 10 min) after forming 2 followed by addition of PhnZ also
This suggests that 1-hydroxyalkylphosphonic acids are
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Journal of the American Chemical Society
Communication
glycine 3 (Scheme 1a). Indeed, treatment of a PhnY/PhnZ
reaction mixture with phenyl isothiocyanate followed by HPLC
analysis revealed that glycine 3 is the alkyl product resulting
from CP bond cleavage of 2 (Figure 2).
Figure 2. HPLC detection of glycine as a product of the PhnY and
PhnZ reaction. All solutions were reacted with phenyl isothiocyanate
prior to HPLC analysis (see SI). Phenyl isothiocyanate adducts are
denoted with *. (a) Chromatogram of the phenyl isothiocyanate
adduct of 1. (b) Reaction of 1 with PhnY in the presence of Fe(II), α-
ketoglutarate, and ascorbate yielding 2. (c) Addition of PhnZ to the
PhnY reaction described in (b). (d) Chromatogram of the phenyl
isothiocyanate adduct of glycine 3.
Although PhnZ and HEPD catalyze similar reactions, the
mechanism of PhnZ may actually more closely resemble that
19
proposed for myo-inositol oxygenase (MIOX). Unlike HEPD,
a mononuclear enzyme that belongs to the cupin superfamily,
MIOX has significant structural homology to the HD hydrolase
20
family. Indeed, PhnZ and MIOX share the conserved “HD”
motif that distinguishes this family of enzymes, despite sharing
little overall sequence homology (10% identity, 19% similarity,
see SI). In the case of MIOX, the HD residues act as ligands for
a mixed-valence Fe(II)/Fe(III) cofactor to catalyze the
oxidative cleavage of a carbon−carbon bond. Thus, analogous
to MIOX, PhnZ may utilize the Fe(II) ion to reduce molecular
Figure 1. In vitro reconstitution of the degradation of 2-amino-
ethylphosphonate 1 to inorganic phosphate by PhnY and PhnZ
monitored by 31P NMR spectroscopy. (a) Spectrum of the reaction of
21
1
(δ 17.3 ppm) with PhnY and α-ketoglutarate. (b) Reaction of 1
17.3 ppm) with PhnY in the presence of Fe(II), α-ketoglutarate, and
ascorbate, yielding 2-amino-1-hydroxyethylphosphonic acid (2) (δ
(
oxygen to form a superoxo-Fe(III) species in proximity of the
22,23
substrate 2 (I in Scheme 1b).
MIOX employs the second
1
4.3 ppm). (c) Addition of synthetic (±)-2 to the PhnY reaction
Fe(III) ion to bind the vicinal hydroxyl groups of myo-inositol
described in (b). (d) Result of the addition of PhnZ to the PhnY
20
in a bidentate fashion. A similar Lewis acid interaction with
the phosphonate and α-hydroxyl oxygens of 2 can be
envisioned for PhnZ (Scheme 1b). Although PhnZ co-purifies
with approximately one equivalent of iron rather than two, it is
possible that the purified enzyme is not fully loaded with metal
ion. Also, spontaneous oxidation of excess Fe(II) in the PhnZ
reaction described in (b). Inorganic phosphate appears at δ 2.9 ppm.
(
e) Result of the reaction of PhnZ with synthetic (±)-2 (δ 14.3 ppm)
to yield P (δ 2.9 ppm). *Residual P from purification of PhnY.
i
i
inherently sensitive to oxidative CP bond cleavage, and that a
similar oxidative reaction by PhnZ with 2 would yield P and
i
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Journal of the American Chemical Society
Communication
reactions described above would provide Fe(III) to serve this
substrate binding role.
ACKNOWLEDGMENTS
■
This work was supported by the Gordon and Betty Moore
Foundation (AM, EFD), the Natural Sciences and Engineering
Research Council of Canada (NSERC), and Queen’s
University. D.L.Z. is additionally supported by an Ontario
Early Researcher Award. We thank Prof. D. Beauchemin and
Dr. A. Asfaw for assistance with ICP-MS analysis, as well as
Prof. R. S. Brown for assistance with HPLC analyses. We also
thank Profs. N. H. Williams, R. Kluger, and D. A. Pratt for
inspiring discussions on the mechanism of PhnZ.
The calculated reaction manifold for MIOX provides a
23
plausible template for CP bond cleavage by PhnZ. The
superoxo-Fe(III) species can be expected to abstract the α-
hydrogen of 2 (I to II, Scheme 1b). Hydroperoxylation or
hydroxylation of a substrate radical intermediate utilizing low
valence Fe(III) or high valence Fe(IV)-oxo species as electron
acceptors, respectively, have been proposed to follow hydrogen
19
abstraction by MIOX. However, calculations suggest that
rather than forming a carbon-centered radical, hydrogen
abstraction by MIOX is accompanied by single electron
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■
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5
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(
7
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26
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AUTHOR INFORMATION
■
Corresponding Author
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dlzechel@chem.queensu.ca
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Notes
The authors declare no competing financial interest.
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