An unusual carbon-carbon bond cleavage reaction during phosphinothricin biosynthesis

Article abstract of DOI:10.1038/nature07972

Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture. One such compound, phosphinothricin tripeptide, contains the unusual amino acid phosphinothricin attached to two alanine residues. Synthetic phosphi

Full text of DOI:10.1038/nature07972

Vol 459
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11 June 2009
|
doi:10.1038/nature07972
LETTERS
An unusual carbon
–carbon bond cleavage reaction
during phosphinothricin biosynthesis
Robert M. Cicchillo1,2,3*, Houjin Zhang2,4*, Joshua A. V. Blodgett5, John T. Whitteck1,2, Gongyong Li1,
Satish K. Nair2,4, Wilfred A. van der Donk1,2,3,4 & William W. Metcalf2,5
Natural products containing phosphorus–carbon bonds have
found widespread use in medicine and agriculture1. One such com-
pound, phosphinothricin tripeptide, contains the unusual amino
acid phosphinothricin attached to two alanine residues. Synthetic
phosphinothricin (glufosinate) is a component of two top-selling
herbicides (Basta and Liberty), and is widely used with resistant
transgenic crops including corn, cotton and canola. Recent genetic
and biochemical studies showed that during phosphinothricin
tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is
converted to hydroxymethylphosphonate (HMP)2. Here we report
the in vitro reconstitution of this unprecedented C(sp3)–C(sp3)
bond cleavage reaction and X-ray crystal structures of the enzyme.
The protein is a mononuclear non-haem iron(II)-dependent dioxy-
genase that converts HEP to HMP and formate. In contrast to most
other members of this family, the oxidative consumption of HEP
does not require additional cofactors or the input of exogenous
electrons. The current study expands the scope of reactions
catalysed by the 2-His–1-carboxylate mononuclear non-haem iron
family of enzymes.
Hydroxyethylphosphonate dioxygenase (HEPD) was overexpressed
in Escherichia coli with an amino-terminal hexa-histidine tag and puri-
fied by Ni21-affinity chromatography. As isolated the protein was
inactive, but in the presence of Fe(II) and O2 conversion of HEP to
HMP (Fig. 1) was observed by 31P NMR spectroscopy and high-
performance liquid chromatography (HPLC). One molar equivalent
ofFe(II)wassufficientforfullreconstitutionofactivity.Externalreduc-
tants or cofactors were not required for catalysis, in contrast to most
other non-haem Fe(II) oxygenases3,4. Thus, all four electrons required
for reduction of O2 are provided by HEP.
The fate of the excised carbon was determined using synthetic HEP
labelled with 13C at carbon 2 (2-13C-HEP). Stoichiometric amounts
of HMP and a new product with a resonance of 171 parts per million
(p.p.m.) in the 13C NMR spectrum were observed, corresponding to
13C-formate (Supplementary Fig. 1). Additionally, HMP formed
with 1-13C-HEP and 2-13C-HEP was analysed by 31P NMR spectro-
scopy, resulting in a doublet (from 13C-HMP, Fig. 2a) and a singlet
(from 12C-HMP) signal, respectively. The hydrogens at C1 are not
removed in this process because the HEPD-catalysed oxidation of
1-2H2-HEP produced 1-2H2-HMP (Fig. 2a, inset).
The enzyme reaction was also performed in the presence of 18O2 (99
atom per cent), with 2-13C-HEP as substrate to circumvent complica-
tions from spurious formate5. After derivatization of formate to its
tert-butyldimethylsilyl ester, the products were monitored by gas chro-
matography–mass spectrometry (GC–MS), displaying ions at m/z 106
and 104 corresponding to loss of the tert-butyl group (M-57) from
derivatized 18O,13C-formate and 16O,13C-formate, respectively. The
18O in formate exchanges with solvent in a time-dependent fashion in
the protocol used (Supplementary Fig. 2), explaining the presence of the
two products. The shortest exposure to the work-up and derivatization
conditions resulted in .85% 18O-formate (Fig. 2b). Incorporation of
18O into HMP was assessed using liquid chromatography–MS (LC–
MS). Approximately 60% of HMP contained 18O (m/z 115), with
40% containing 16O (m/z 113; Fig. 2c). This result was unexpected
because the primary alcohol of HMP did not exchange under the
reaction conditions. In an effort to understand the lower than expected
18O content in HMP, the reaction was also performed in the presence of
80% (v/v) H218O (95 atom per cent)/H216O. LC–MS analysis revealed
69% 2-16O-HMP and 31% 2-18O-HMP (Supplementary Fig. 3). When
corrected for the 18O content of the labelled water, the two comple-
mentary experiments (18O2 and H218O) are in good agreement and
indicate the intermediacy of a species in which oxygen derived from
O2 exchanges with water. These results also demonstrate that HEPD is a
dioxygenase. The results of all labelling studies are summarized in
Fig. 2d.
HEPD does not have sequence homology with other proteins in
the databases except for enzymes that probably catalyse the same
transformation judging from their operon context. To gain three-
dimensional information, the structure of HEPD was determined by
2–
CO2
O
P
O
OPO3
CO2H
O
P
O
H
HO
–O
HO
–O
H
CO2H
H H
H
–O
Me
O
HCO2H O2
O
P
O
P
P
OH
HO
HO
–O
OH
–O
HEPD
Fe(II)
HMP
HEP
Ala–Ala
H2N
O
O
P
PTT
O
P
HO
H
Me
H
HO
–O
HO
Me
–O
HppE
H
O
Fosfomycin
HPP
Figure 1
|
Structure of phosphinothricin tripeptide and the reaction
catalysed by HEPD. The biosyntheses of the commercial herbicide
phosphinothricin (boxed in the phosphinothricin tripeptide (PTT)
structure) and the clinically used antibiotic fosfomycin share several early
steps starting with phosphoenolpyruvate before the pathways diverge after
formation of HEP2,20. HEPD catalyses the unprecedented conversion of HEP
to HMP (for the steps from HMP to PTT, see Supplementary Fig. 8). The
structurally related enzyme HppE converts HPP to fosfomycin21.
1Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. 2Institute for Genomic Biology, 3Howard Hughes Medical Institute, 4Department of
Biochemistry, and 5Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
*These authors contributed equally to this work.
871
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ligands is similar to that of HppE, but the disposition of these ligands
within the b-barrel is not conserved because Glu 176 of HEPD is
situated on a different b-strand to the equivalent Glu 142 of
HppE7. Furthermore, the spacing between the first two metal ligands
in HEPD (HX46E, where X represents any amino acid) is unique as
these residues are closely spaced (HX1–4E/D) in other facial triad
enzymes7. In the second repeat in HEPD, the canonical 2-His–1-
Asp/Glu is replaced by an arrangement of 2-His–1-Asn, and steric
occlusion by residues Tyr 358 and Lys 404 precludes formation of a
competent metal-binding pocket. Combined with the observed
requirement for one equivalent of iron for full enzyme activity, the
second repeat is probably vestigial and does not participate in cata-
lysis. Given the structural similarity to the epoxidase HppE, HEPD
was incubated with 2-HPP, the substrate for HppE. 2-HPP was con-
verted to 2-oxopropylphosphonate (2-OPP), rendering the enzyme
inactive in the process (Supplementary Fig. 5). No evidence was
found for hydrogen peroxide formation in this transformation.
Attempts to produce crystals of Fe(II)–HEPD have failed owing to
the high concentrations of Cd(II) required for crystallization. Crystals
of SeMet-labelled Cd(II)–HEPD were grown in the presence of HEP
a
b 30
25
20
15
10
5
100
80
60
40
20
0
115
106
*
80 100 120 140 160
m/z
HEP HMP
104
0
90
95 100 105 110 115 120
25.0 22.5 20.0 17.5 15.0 12.5
p.p.m.
m/z
c
100
80
60
40
20
0
115
30
20
15
10
5
d
O
113
*
P
HO
–O
OH
D
D
80 100 120 140 160 180
60
HEPD
18O2
m/z
˚
and solved to a resolution of 1.92 A, revealing electron density con-
sistent with bidentate coordination of substrate to Cd21 (Fig. 3d),
which is similar to that observed for binding of 2-HPP to Co(II)–
HppE7. In the HppE co-crystal structures, substrate binding induces
substantial reorganization of the active site. In contrast, binding of
HEP to Cd(II)–HEPD results only in the torsional movement of
Tyr 98 about x1 (the torsion angle between the a- and b-carbons),
O
P
18O
18OH
D
+
HO
H
OH
*
–O
D
4
5
6
7
8
9
10 11
Time (min)
˚
resulting in a hydrogen bond interaction (2.3 A) with one of the
phosphonate oxygen atoms of the substrate. An additional hydrogen
Figure 2
|
NMR and mass spectral data from in vitro labellingstudies. a, 31
P
NMR spectrum showing production of 13C-HMP (d (chemical shift)
17.2 p.p.m.) from 1-13C-HEP (d 19.9 p.p.m.) after 50% conversion. Inset:
˚
bond interaction occurs between N2 of Asn 126 (2.8 A) and a differ-
ent phosphonate oxygen.
mass spectrum of HMP derived from a reaction using 1,1-2H2-HEP. b, Mass
spectrum of 18O,13C-formate (m/z 106) produced by HEPD from a reaction
using 18O2 and 2-13C-HEP. Spurious formate is denoted by an asterisk (m/z
103). c, Analysis of HMP produced from the HEPD reaction performed with
18O2. Extracted ion chromatograms demonstrate that ,60% of the HMP
contains 18O (black line) whereas 40% contains 16O (red line). Inset:
summed mass spectrum from the total ion chromatographic peak.
d, Summary of labelling studies from the HEPD reaction.
Although oxidative scission of carbon–carbon bonds is well docu-
mented, previously characterized proteins typically act on substrates
that contain aromatic3, alkene8 or 1,2-dihydroxy9 functionalities. No
such activating groups are present in HEP, nor does HEPD need
multiple successive oxidations such as in P450-mediated cleavage
of the C20–C22 side chain of sterols10. Figure 4 presents a working
model for the HEPD reaction. The substrate is proposed to bind to
Fe(II) in a bidentate manner followed by reaction with O2, resulting
in a Fe(III)–[O22] intermediate. This species is proposed to abstract a
hydrogen atom from C2 of HEP to generate intermediate II, akin to
similar steps proposed for isopenicillin N synthase and myo-inositol
oxygenase9,11. One important difference between isopenicillin N
synthase and HEPD is that the former contains a sulphur ligand from
the substrate that has been suggested on the basis of computational
studies to stabilize the Fe(III)–[O22] intermediate and overcome the
unfavourable energetics of O2 binding and activation12. Although a
thiolate ligand is lacking in HEPD, the observation that external
electrons are not required leaves no alternate pathways to convert
intermediate I into a more reactive species found in other non-haem
iron oxygenases4. Furthermore, HEP does not have low-energy elec-
trons that can be provided by the substrate, as in the extradiol diox-
ygenases that use highly electron-rich catechol substrates. HEPD also
does not contain other nearby metals that are important for activity,
in contrast to the binuclear iron enzyme myo-inositol oxygenase13.
Hence, the only option for HEPD seems to be hydrogen atom
abstraction from C2 by intermediate I to produce the hydroperoxo
complex II and a substrate radical.
single-wavelength anomalous diffraction data collected from crystals
of selenomethionine-incorporated protein, and refined to a Bragg
˚
˚
¨
limit of 1.8 angstrom (A). Crystallization was contingent on the
presence of 50 mM CdCl2 in the precipitant. The overall structure
consists of imperfect tandem repeats of a bi-domain architecture
(Fig. 3a). Each of the repeats is composed of an all-a-helical domain
linked to a b-barrel fold characteristic of the cupin superfamily6.
Despite the lack of appreciable sequence similarity, each of the two
repeats is structurally homologous to the monomer of HppE7, a non-
haem iron-dependent enzyme that converts (S)-2-hydroxypropyl-
phosphonic acid (2-HPP) to fosfomycin (Fig. 1). However, the
repeats of HEPD are not entirely discretely folded domains as the
b-barrel domain of the first repeat is composed of interlacing
b-strands from different parts of the molecule.
The disposition of the tandem domains of HEPD recapitulates
many of the elements of the quarternary structure of HppE7, with
the tandem arrangement found in HEPD structurally analogous to
one-half of the HppE tetramer. This tetramer is stabilized through
crossover interactions between the a-helical domains of the indi-
vidual monomers. In contrast, the tandem arrangement in HEPD
is held in place by two short orthogonal a-helices located at the
junction between each helical and b-barrel domain (Fig. 3b). The
b-barrel fold of the first repeat contains all of the canonical active site
elements of non-haem iron enzymes, including a 2-His–1-Glu facial
triad3. A Cd(II) ion is situated at the base of the active site and is
coordinated by His 129, Glu 176 and His 182 (Fig. 3c). Well-defined
spherical electron densities have been modelled as three water ligands
forming a six-coordinate metal centre. The constellation of metal
Two different pathways can be envisioned to account for the label-
ling studies. The substrate radical could attack the hydroperoxide
generating a ferryl species (Fe(IV)5O) and a hemiacetal (Fig. 4).
The latter can undergo a retro-Claisen type-C–C bond scission with
the incipient negative charge on C1 attacking the electrophilic ferryl,
potentially by means of a Horner–Wadsworth–Emmons-like inter-
mediate stabilized by metal coordination. A ferryl would account for
the required intermediacy of a species in which oxygen derived from
O2 can exchange with solvent14–19 (Supplementary Fig. 6). A second
872
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LETTERS
Figure 3
Cd(II)
|
HEPD
Structures of Cd(II)
–HEPD and
b
a
–
–
HEP. a, b, Orthogonal views of
Cd(II)–HEPD showing the tandem arrangement
of the HppE fold including cupin domains (blue
and purple), a-helical domains (red and cyan),
cadmium (white sphere) and metal ligands
(green). c, Stereoview of electron density maps
using model phases (Fo – Fc). The first map
(contoured at 3s in blue) was calculated by
omitting metal-bound solvents (red sphere)
before one round of refinement. The second map,
contoured at 5s (purple mesh) and 14s (yellow
mesh), was calculated by omitting the cadmium
(gold sphere) before one round of refinement.
d, Stereoview of electron density maps (Fo – Fc)
calculated using HEPD–HEP model phases. The
map was calculated by omitting non-protein
metal-bound ligands before one round of
90º
c
Glu 176
Cd(II)
Glu 176
Cd(II)
refinement (contoured at 3s in blue mesh and 6s
in red). HEP carbons are shown in green.
His 129
His 129
His 182
His 182
d
Glu 176
Cd(II)
Glu 176
Cd(II)
HEP
HEP
His 129
His 129
Tyr 98
Tyr 98
His 182
His 182
Asn 126
Asn 126
Figure 4
|
Working models for the mechanism of
HEP
O2
H
catalysis by HEPD. The ferrous ion reacts with
O2 to generate the formal ferric–superoxide
complex I. Hydrogen atom abstraction from C2
of HEP generates a substrate radical and
H
OH2
OH
Fe(II)
–O
–O
Glu 176
His 129
Glu 176
Glu 176
His 129
Fe(III)
Fe(III)
His 129
P
P
OH2
O
O
ferric–hydroperoxide (II). From intermediate II
two possible pathways can be envisaged. Radical
attack on the ferric–hydroperoxide may cleave
the O–O bond and produce a ferryl species and a
hemiacetal (hydroxylation). C–C bond cleavage
and attack of the resulting resonance-stabilized
anion at the ferryl species generates formate and
HMP. The weak hemiacetal ligand is posited to
first dissociate from the iron to account for
solvent exchange (Supplementary Fig. 6).
Alternatively, intermediate II could be converted
to the alkyl hydroperoxide III
His 182
His 182
His 182
HO
HO
I
II
Hydroperoxylation
Hydroxylation
–
H
+ H2O
H
H
L
–O
OH2
H
–O
Glu 176
Glu 176
His 129
L
–
O
L
Fe(II)
Fe(IV)
H + H2
Glu 176
His 129
P
O
P
His 129
Fe(II)
O
HO
His 182
P
HO
O
His 182
HO
His 182
III
(hydroperoxylation), which can undergo a
Criegee-type rearrangement to generate formyl-
HMP. Hydrolysis must then occur at the
methylene carbon to account for the oxygen
labelling studies. Filled circles represent oxygen
derived from O2 and half-filled circles represent
oxygens originally derived from O2 that have
exchanged with water. L, undefined metal ligand.
H
H
H
H
O
H
H
O
O
–
–
L
H
CH2
Glu 176
His 129
Glu 176
OH
OH
L
Glu 176
His 129
2 P
O
Fe(IV)
Fe(II)
Fe(II)
P
O
OH
P
His 129
O
HO
His 182
His 182
His 182
HO
873
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7. Higgins, L. J., Yan, F., Liu, P., Liu, H. W. & Drennan, C. L. Structural insight into
antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme. Nature 437,
mechanism that can account for the observed data involves conver-
sion of intermediate II to the hydroperoxide III, which can undergo a
Criegee-type rearrangement to provide the formate ester of HMP
(Fig. 4). Hydrolysis of this ester is expected to occur at the carbonyl
carbon, but such a mechanism would not account for the incorpora-
tion of oxygen derived from solvent into HMP. This observation can
be explained if hydrolysis took place via attack at C1 by solvent-
exchangeable hydroxide released in the Criegee rearrangement.
Both models can also explain the observed conversion of 2-HPP into
2-OPP, with the additional methyl group in the substrate inducing
the cleavage of a C–O bond instead of a C–C bond to form a ketone
(Supplementary Fig. 7). However, the lack of hydrogen peroxide
production in this latter transformation favours the hydroxylation
mechanism over the hydroperoxylation model. Further studies will
be required to provide insights into the factors that result in epoxide
formation by HppE and C–C bond cleavage by HEPD.
838
8. Grogan, G. Emergent mechanistic diversity of enzyme-catalysed beta-diketone
cleavage. Biochem. J. 388, 721 730 (2005).
9. Xing, G. et al. Evidence for C H cleavage by an iron-superoxide complex in the
glycol cleavage reaction catalyzed by myo-inositol oxygenase. Proc. Natl Acad. Sci.
USA 103, 6130 6135 (2006).
10. Lieberman, S. & Lin, Y. Y. Reflections on sterol sidechain cleavage process
catalyzed by cytochrome P450(scc). J. Steroid Biochem. Mol. Biol. 78, 1 14 (2001).
11. Burzlaff, N. I. et al. The reaction cycle of isopenicillin N synthase observed by X-ray
diffraction. Nature 401, 721 724 (1999).
–844 (2005).
–
–
–
–
–
12. Brown, C. D., Neidig, M. L., Neibergall, M. B., Lipscomb, J. D. & Solomon, E. I.
VTVH-MCD and DFT studies of thiolate bonding to [FeNO]7/[FeO2]8 complexes
of isopenicillin N synthase: substrate determination of oxidase versus oxygenase
activity in nonheme Fe enzymes. J. Am. Chem. Soc. 129, 7427–7438 (2007).
13. Xing, G. et al. Oxygen activation by a mixed-valent, diiron(II/III) cluster in the
glycol cleavage reaction catalyzed by myo-inositol oxygenase. Biochemistry 45,
5402
14. Kikuchi, Y., Suzuki, Y. & Tamiya, N. The source of oxygen in the reaction catalysed
by collagen lysyl hydroxylase. Biochem. J. 213, 507 512 (1983).
–5412 (2006).
–
METHODS SUMMARY
15. Baldwin, J. E., Adlington, R. M., Crouch, N. P. & Pereira, I. A. C. Incorporation of
18O-labelled water into oxygenated products produced by the enzyme
deacteoxy/deacetylcephalosporin C synthase. Tetrahedron 49, 7499–7518
(1993).
16. Sabourin, P. J. & Bieber, L. L. The mechanism of alpha-ketoisocaproate oxygenase.
HEPD was purified by immobilized metal affinity chromatography. Iron ana-
lysis22 on the as-isolated protein revealed iron content below the detection limits
(0.05 equivalents) whereas fully active, Fe(II)-reconstituted protein contained
0.99 60.05 equivalent of Fe per polypeptide. Typical aerobic assays contained,
in a final volume of 1,000 ml: 50 mM K1-HEPES, pH 7.5, 2–10 mM HEPD,
5–10 mM HEP or isotopically labelled HEP. Formate produced was derivatized
at 20 uC using 0.5 ml N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide
(MTBSTFA) plus 0.1% tert-butyldimethylsilyl chloride (TBDMSCl) for 20 min
and monitored by GC–MS. HMP produced was analysed by LC–MS using atmo-
spheric pressure chemical ionization (APCI). The stoichiometry of formate to
HMP produced during the HEPD reaction was determined by 13C NMR spec-
troscopy. Thepotential for 18O incorporation from H2O into HMP was evaluated
by running the reaction in H218O. HEPD was anaerobically activated as described
in the Methods. The mixture (150 ml) was removed from the glove box and added
to an aerobic mixture of H218O (832.6ml, 95 atom per cent) and HEP (17.4 ml).
ThefinalconcentrationsofHEPD andHEPwere10.5 mMand3 mM, respectively.
H218O was diluted to 79% in the 1 ml reaction. Intermittent bubbling of O2
enabled thecompleteconsumptionofHEPover 1 h. HMP producedwasanalysed
and a representative isotopic distribution of 18O/16O is shown in Supplementary
Fig. 3. The reported percentage of 18O-HMP is corrected based on the amount of
H218O in the assay.
Formation of beta-hydroxyisovalerate from alpha-ketoisocaproate. J. Biol. Chem.
257, 7468–7471 (1982).
17. Lindblad, B., Lindstedt, G. & Lindstedt, S. The mechanism of enzymic formation of
homogentisate from p-hydroxyphenylpyruvate. J. Am. Chem. Soc. 92, 7446–7449
(1970).
18. Wackett, L. P., Kwart, L. D. & Gibson, D. T. Benzylic monooxygenation catalyzed
by toluene dioxygenase from Pseudomonas putida. Biochemistry 27, 1360–1367
(1988).
19. Pestovsky, O. & Bakac, A. Aqueous ferryl(IV) ion: kinetics of oxygen atom transfer
to substrates and oxo exchange with solvent water. Inorg. Chem. 45, 814–820
(2006).
20. Woodyer, R. D., Li, G., Zhao, H. & van der Donk, W. A. New insight into the
biosynthesis of fosfomycin: discovery of the missing link illuminates the
mechanism of methyl transfer. Chem. Commun. 359
21. Liu, P. et al. Protein purification and function assignment of the epoxidase
catalyzing the formation of fosfomycin. J. Am. Chem. Soc. 123, 4619 4620 (2001).
22. Beinert, H. Micro methods for the quantitative determination of iron and copper in
biological material. Methods Enzymol. 54, 435 445 (1978).
–361 (2007).
–
–
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Acknowledgements We thank B. Griffin, J. M. Bollinger, S. E. Denmark and T. Begley
for discussions. This work was supported by grants from the National Institutes of
Health (PO1 GM077596 to W.W.M., W.A.v.d.D. and S.K.N., and NIH RO1
GM59334 to W.W.M.) and by the University of Illinois.
Received 20 November 2008; accepted 4 March 2009.
1. Seto, H. & Kuzuyama, T. Bioactive natural products with carbon
bonds and their biosynthesis. Nat. Prod. Rep. 16, 589 596 (1999).
2. Blodgett, J. A. et al. Unusual transformations in the biosynthesis of the antibiotic
phosphinothricin tripeptide. Nature Chem. Biol. 3, 480 485 (2007).
3. Costas, M., Mehn, M. P., Jensen, M. P. & Que, L. Jr. Dioxygen activation at
mononuclear nonheme iron active sites: enzymes, models, and intermediates.
–phosphorus
–
Authors Contributions R.M.C. performed all biochemical assays shown, which
were designed and analysed by R.M.C. and W.A.v.d.D. All structural studies were
performed and interpreted by H.Z. and S.K.N. W.W.M. designed and J.A.V.B.
performed initial biochemical reactions and identified the products. J.T.W. and G.L.
synthesized all substrates. R.M.C., S.K.N. and W.A.v.d.D. wrote the manuscript.
–
Chem. Rev. 104, 939
4. Kovaleva, E. G. & Lipscomb, J. D. Versatility of biological non-heme Fe(II) centers
in oxygen activation reactions. Nature Chem. Biol. 4, 186 193 (2008).
–986 (2004).
Author Information Atomic coordinates and structure factors have been
deposited in the Protein Data Bank (PDB) under accession codes 3G7D (for the apo
structure) and 3GBF (for the liganded structure). Reprints and permissions
information is available at www.nature.com/reprints. Correspondence and
requests for materials should be addressed to S.K.N. (s-nair@life.uiuc.edu),
W.A.v.d.D. (vddonk@illinois.edu) or W.W.M. (metcalf@illinois.edu).
–
5. Shyadehi, A. Z. et al. The mechanism of the acyl-carbon bond cleavage reaction
catalyzed by recombinant sterol 14 alpha-demethylase of Candida albicans. J. Biol.
Chem. 271, 12445
6. Dunwell, J. M., Purvis, A. & Khuri, S. Cupins: the most functionally diverse protein
superfamily? Phytochemistry 65, 7 17 (2004).
–12450 (1996).
–
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0.5 ml MTBSTFA plus 0.1% TBDMSCl (Pierce). Typical derivatizations were
performed for 20 min at 25 uC. The solution (1–2 ml) was then directly injected
into the GC–MS. Under the given GC conditions derivatized formate had a
retention time of 7.4 min.
METHODS
Purification of recombinant HEPD. Recombinant HEPD was overexpressed as
previously described2. HEPD was purified by immobilized metal affinity chro-
matography using a Ni-NTA matrix (Qiagen). In a typical purification, 10 g of
cells was thawed in 30 ml of buffer A (50 mM HEPES, pH 7.5, 300 mM NaCl,
20 mM imidazole, 10% glycerol) and allowed to incubate with lysozyme
(1 mg ml21) for 30 min at 4 uC. Cells were lysed by two passages through a
French press at a setting of 20,000 psi, and the lysate was centrifuged at
35,000g for 1 h at 4 uC. The supernatant was loaded onto a Ni-NTA column
(1.5 3 3 cm) equilibrated in buffer A, and the column was washed with 10
column volumes of buffer A. The column was subsequently washed with buffer
A containing first 50 mM and then 100 mM imidazole. The remaining bound
protein was eluted with buffer A containing 250 mM imidazole. HEPD typically
elutes between 100 mM and 250 mM imidazole and was pooled based on SDS–
PAGE analysis. The protein was concentrated using an Amicon ultracentrifuga-
tion device with a 10 kDa membrane cutoff, and the buffer was exchanged to
50 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol by gel filtration. Protein
concentrations were determined by Bradford analysis using BSA as a standard.
The protein was frozen in 100-ml aliquots and stored at 280 uC until ready for
use.
HEPD activity assay. Anaerobic activation ofHEPD was achievedinananaerobic
chamber obtained from CoyLaboratory Products, Inc. under anatmosphere ofN2
and H2 (95%/5%). HEPD (30–70 mM) was treated with one molar equivalent of
Fe(II)(NH4)2(SO4)2 for 20 min in a final volume of 200 ml. The solution was sub-
sequently brought outside the glove box and combined with the remaining assay
components. The reaction was initiated by addition of HEP (see Methods
Summary for assay ingredients). For the quantification of formate, 150-ml aliquots
of the assay mixture were removed at designated times and added to 2 ml of 9.2 M
H2SO4 to quench the reaction. Precipitated protein was pelleted by centrifugation,
and 50ml of the supernatant was analysed by HPLC (Agilent Technologies 1200
series HPLC). Formate was chromatographed using an AMINEX HPX-87H
(BioRad) column (3003 7.8 mm) in an isocratic mobile phase (25 mM H2SO4).
The flow rate and temperature were 0.6 mlmin21 and 60uC, respectively. Formate
eluted at 14.1min.
HMP analysis by LC–MS. Analysis and quantification of HMP was carried out
by LC–MS using an Agilent 1200 series LC–MS equipped with a multimode
electrospray ionization/APCI spray chamber. For HMP, APCI ionization was
performed using positive ionization and chromatographic separations were
achieved using a 150 3 4.6 mm Synergi C18 Fusion-RP column with a 4 mm
particle size (Phenomenex). HMP has a retention time of approximately 6 min
at a flow rate of 0.5 ml min21 using a 0.1% formic acid isocratic mobile phase.
Injection volumes ranged from 5 ml to 15 ml. The nebulizer gas was N2 at
8 l min21 with a nebulizer pressure of 40 psi. The drying gas temperature was
300 uC and the vaporization temperature was 250 uC. The capillary and charging
voltages were both set to 2,000 V and the corona discharge current was 4 mA. The
gain was set to 1.0 and the fragmentor voltage was 70 V.
Analysis of HEPD products by NMR spectroscopy. 1H-coupled 31P-NMR and
1H-decoupled 31P-NMR analyses were performed on a Varian Inova 600 spectro-
meter equipped with a 5-mm Varian 600DB AutoX probe tuned for phosphorus at
242.79 MHz. 13C spectra were recorded on a Varian Unity 500 spectrometer.
Carbonchemicalshiftswerereferencedtoanexternalstandardof0.1%tetramethyl-
silane in CDCl3. Phosphorus chemical shifts are reported relative to an external
standard of 85% phosphoric acid (d50). HEPD assays were performed as
described above except that the reaction was terminated by removal of the protein
by an ultrafiltration device (Millipore Amicon) equipped with a 10 kDa molecular
weightcutoffmembrane.D2Owasthenaddedtothesampletoafinalconcentration
of 20% before data acquisition. The identification of the second product as formate
was established by 13C NMR and GC–MS as seen in Supplementary Fig. 1a, b.
18O2 incorporation assay. Assays using 18O2 were carried out as described. All
assay components were prepared and mixed anaerobically. 2-13C-HEP was sub-
stituted for HEP so that formate derived from substrate could be differentiated
from spurious formate during GC–MS analysis. The glass vial was fitted with a
tight rubber septum and brought outside of the glove box. The reaction was
initiated by the introduction of 1 ml of 18O2 (99 atom per cent, Isotec) via a gas-
tight syringe. The solution was allowed to stir slowly at room temperature (25 uC)
for 90min at which time sulphuric acid was added to a final concentration of
92mM to quench the reaction. After centrifugation to remove precipitated
protein, HMP was directly analysed by LC–MS. Formate was extracted and deri-
vatized as described above before GC–MS analysis. Time-dependent washout of
the 18O label in formate, under acidic conditions, was observed by GC–MS
(Supplementary Fig. 2) and has been noted elsewhere23.
Formate analysis by GC–MS. Formate production was monitored on an Agilent
6890N (Agilent Technologies) gas chromatograph with helium carrier gas.
Sample introduction was via split injection onto a HP-5 (5%-phenyl-methyl-
polylsiloxane) column (30 m, 0.32 mm inner diameter, 0.25 mm film thickness).
The injection temperature was 250 uC. The initial column temperature was
40 uC, and was held for 5 min after injection before increasing to 230 uC at
15 uC min21. The temperature was held at 230 uC for the remainder of the
27 min program. The HEPD reaction was performed as described above.
Sulphuric acid was added to a final concentration of 0.12 M to quench the
reaction. The acidified solutions were subjected to three separate 0.5 ml diethyl
ether extractions. The extracts were combined and formate was derivatized using
23. Shyadehi, A. Z. et al. The mechanism of the acyl
catalyzed by recombinant sterol 14 alpha-demethylase of Candida albicans. J. Biol.
Chem. 271, 12445 12450 (1996).
–carbon bond cleavage reaction
–
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