Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217

Article abstract of DOI:10.1016/j.bbapap.2014.09.009

Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. l-Phosphinothricin contains the only known naturally-occurring carbon-phosphorus-carbon bond linkage. In actinomycetes, the cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase PhpK catalyzes the formation of the second C-P bond to generate the complete C-P-C linkage in phosphinothricin. Here we use electron paramagnetic resonance and nuclear magnetic resonance spectroscopies to characterize and demonstrate the activity of a cobalamin-dependent radical SAM methyltransferase denoted SD-1168 from Shewanella denitrificans OS217, a marine bacterium that has not been reported to synthesize phosphinothricin. Recombinant, refolded, and reconstituted SD-1168 binds a four-iron, four-sulfur cluster that interacts with SAM and cobalamin. In the presence of SAM, a reductant, and methylcobalamin, SD-1168 surprisingly catalyzes the P-methylation of N-acetyl-demethylphosphinothricin and demethylphosphinothricin to produce N-acetyl-phosphinothricin and phosphinothricin, respectively. In addition, this enzyme is active in the absence of methylcobalamin if the strong reductant titanium (III) citrate and hydroxocobalamin are provided. When incubated with [methyl-¹³C] cobalamin and titanium citrate, both [methyl-¹³C] and unlabeled N-acetylphosphinothricin are produced. Our results suggest that SD-1168 catalyzes P-methylation using radical SAM-dependent chemistry with cobalamin as a coenzyme. In light of recent genomic information, the discovery of this P-methyltransferase suggests that S. denitrificans produces a phosphinate natural product.

Full text of DOI:10.1016/j.bbapap.2014.09.009

Biochimica et Biophysica Acta 1844 (2014) 2135–2144
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbapap
Spectroscopic characterization and mechanistic investigation of
P-methyl transfer by a radical SAM enzyme from the marine bacterium
Shewanella denitrificans OS217
1
⁎
Kylie D. Allen , Susan C. Wang
School of Molecular Biosciences, College of Veterinary Medicine, PO Box 647520, Washington State University, Pullman, WA 99164-7520, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2014
Received in revised form 5 September 2014
Accepted 7 September 2014
Available online 16 September 2014
Natural products containing carbon–phosphorus bonds elicit important bioactivity in many organisms. L-
Phosphinothricin contains the only known naturally-occurring carbon–phosphorus–carbon bond linkage. In ac-
tinomycetes, the cobalamin-dependent radical S-adenosyl-L-methionine (SAM) methyltransferase PhpK cata-
lyzes the formation of the second C–P bond to generate the complete C–P–C linkage in phosphinothricin. Here
we use electron paramagnetic resonance and nuclear magnetic resonance spectroscopies to characterize and
demonstrate the activity of a cobalamin-dependent radical SAM methyltransferase denoted SD_1168 from
Shewanella denitrificans OS217, a marine bacterium that has not been reported to synthesize phosphinothricin.
Recombinant, refolded, and reconstituted SD_1168 binds a four-iron, four-sulfur cluster that interacts with
SAM and cobalamin. In the presence of SAM, a reductant, and methylcobalamin, SD_1168 surprisingly catalyzes
the P-methylation of N-acetyl-demethylphosphinothricin and demethylphosphinothricin to produce N-acetyl-
phosphinothricin and phosphinothricin, respectively. In addition, this enzyme is active in the absence of
methylcobalamin if the strong reductant titanium (III) citrate and hydroxocobalamin are provided. When
incubated with [methyl-13C] cobalamin and titanium citrate, both [methyl-13C] and unlabeled N-
acetylphosphinothricin are produced. Our results suggest that SD_1168 catalyzes P-methylation using radical
SAM-dependent chemistry with cobalamin as a coenzyme. In light of recent genomic information, the discovery
of this P-methyltransferase suggests that S. denitrificans produces a phosphinate natural product.
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Radical S-adenosyl-L-methionine
Methyltransferase
Cobalamin
Phosphinate
1. Introduction
reactions thought to be involved in the biosynthesis of C–P compounds,
have inspired a wealth of research. The majority of C–P compound bio-
Phosphonate and phosphinate natural products contain carbon–
phosphorus (C–P) bonds and have noteworthy bioactivities due to
their structural similarities to phosphate esters, carboxylic acids, and
various tetrahedral intermediates in enzymatic reactions [1]. These
compounds are widely used as antimicrobial, antifungal, and herbicidal
agents [2–4]. Such applications, as well as the extraordinary biochemical
synthetic pathways begin with the reaction catalyzed by phosphoenol-
pyruvate mutase (PepM), which forms the initial C–P bond via
isomerization of phosphoenolpyruvate to generate phosphonopyruvate.
This energetically unfavorable reaction is usually driven by decarboxyl-
ation catalyzed by phosphonopyruvate decarboxylase (Ppd) to generate
phosphonoacetaldehyde [1].
L-Phosphinothricin (PT; L-2-amino-4-hydroxymethylphosphi-
nylbutanoate) is an unusual amino acid that contains the only known
naturally occurring C–P–C bond sequence (Fig. 1A). PT is a L-glutamate
analog and has herbicidal and antimicrobial activities through inhibiting
plant and bacterial glutamine synthetases [5–7]. PT is produced as
part of a tripeptide by Streptomyces hygroscopicus, Streptomyces
viridochromogenes, and Kitasatospora phosalacinea [8–10]. In the two
Streptomyces species, at least 24 genes are required for biosynthesis of
the PT tripeptide, L-PT-Ala–L-Ala (PTT) [11,12]. Although the biosyn-
thetic pathway for phosalacine (L-PT–L-Ala–L-Leu) in K. phosalacinea
has not been investigated, it is likely to be similar. These tripeptides
are readily absorbed by target cells, where intracellular peptidases re-
lease the active PT antibiotic. In the latter stages of PT biosynthesis, the
P-methyltransferase PhpK is thought to append a methyl group to the
Abbreviations: 2-HEP, 2-hydroxyethylphosphonate; 2-HPP, 2-hydroxypropyl-
phosphonate; [4Fe-4S], four-iron, four-sulfur; Ado-CH2•, 5′-deoxyadenosyl radical; Ado-
CH3, 5′-deoxyadenosine; Cbl, cobalamin; CH3Cbl, methylcobalamin; DMPT, L-2-amino-4-
hydroxyphosphinylbutanoate or demethylphosphinothricin; NAcDMPTT, NAcDMPT
tripeptide; NAcPT, N-acetylphosphinothricin; NAcPTT, NAcPT tripeptide; NMR, nuclear
magnetic resonance; PT, L-2-amino-4-hydroxymethylphosphinylbutanoate or L-
phosphinothricin; PTT, phosphinothricin tripeptide; SAH, S-adenosylhomocysteine; SAM,
S-adenosyl-L-methionine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis; UV–vis, ultraviolet–visible
⁎
Corresponding author at: Washington State University, School of Molecular
Biosciences, BLS 202, PO Box 647520, Pullman, WA 99164-7520, USA. Tel.: +1 509 335
7714.
E-mail addresses: kdallen@vt.edu (K.D. Allen), susan_wang@wsu.edu (S.C. Wang).
Present address: Department of Biochemistry, Virginia Polytechnic Institute and State
1
University, 111 Engel Hall, Mail Code 0308, Blacksburg, VA 24061, USA.
http://dx.doi.org/10.1016/j.bbapap.2014.09.009
1570-9639/© 2014 Elsevier B.V. All rights reserved.

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Here, we describe the characterization of a Cbl-dependent radical
SAM methyltransferase encoded by the sden_1168 gene from the
denitrifying marine bacterium Shewanella denitrificans OS217 [22]. We
will refer to the resulting protein as SD_1168. S. denitrificans OS217
has not been reported to biosynthesize known C–P compounds.
However, its genome encodes phosphoenolpyruvate mutase (PepM;
sden_1161) and phosphonopyruvate decarboxylase (Ppd; sden_1162,
annotated as a thiamine pyrophosphate-dependent enzyme) (Fig. 3)
near the sden_1168 gene, suggesting that this organism has the capacity
to produce C–P compounds [23].
Although the biological function of SD_1168 is currently un-
known, the protein shares significant identity and similarity with
both Fom3 and PhpK (34% and 53%, and 13% and 31%, respectively),
methyltransferases involved in C–P compound biosynthesis [15,
21]. Fom3 is required for the penultimate step of fosfomycin biosyn-
thesis, where it adds a methyl group to the sp3-hybridized C-2
carbon of 2-hydroxyethylphosphonate (2-HEP) to generate S-2-
hydroxypropylphosphonate (S-2-HPP) (Fig. 1C) [21,24,25]. The sig-
nificant sequence similarity between SD_1168 and Fom3 led us to
hypothesize that SD_1168 might catalyze a similar, or even the
same, C-methylation as Fom3. To date, we have not observed
SD_1168 C-methylation activity upon 2-HEP. Instead, we found
that SD_1168 unexpectedly catalyzes P-methylation upon both
NAcDMPT and DMPT to generate the final C–P–C bond sequence
found in PT (Fig. 1A and B). We show that SD_1168 is likely to per-
form radical SAM chemistry using a Cbl coenzyme for this difficult
reaction. Since PT and its derivatives are the only naturally-
occurring phosphinates discovered to date, our results suggest that
S. denitrificans synthesizes an as-yet undiscovered phosphinate nat-
ural product.
Fig. 1. P-methylation and C-methylation reactions of interest.
phosphinate precursor 2-acetylamino-4-hydroxyphosphinylbutanoate
(N-acetyldemethylphosphinothricin or NAcDMPT) to produce 2-
acetylamino-4-hydroxymethylphosphinylbutanoate (N-acetylphos-
phinothricin or NAcPT), which contains the final C–P–C bond sequence
(Fig. 1B) [11,13–15]. In a randomly-generated S. hygroscopicus mutant
that could not catalyze P-methylation, NAcDMPT and its tripeptide, N-
acetyldemethylphosphinothricin tripeptide (NAcDMPTT) accumulated,
suggesting that these two N-acetylated metabolites were substrates
for PhpK (Fig. 1B) [14]. Furthermore, only the N-acetylated precursors
were methylated by S. hygroscopicus cell lysates while the corresponding
non-acetylated precursors, demethylphosphinothricin (DMPT; 2-amino-
4-hydroxyphosphinylbutanoate) and DMPT tripeptide (DMPTT), were
not methylated. Isotopic labeling studies demonstrated that
methylcobalamin (CH3Cbl) was the methyl group donor for the P-
methylation reaction [5,13].
In 2001, PhpK was identified as a radical S-adenosyl-L-methionine
(SAM) superfamily member based on the presence of a highly con-
served CxxxCxxC motif whose cysteine residues coordinate a four-
iron, four-sulfur ([4Fe–4S]) cluster [16]. The reduced [4Fe–4S]+1 cluster
and SAM are used to generate a 5′-deoxyadenosyl radical (Ado-CH2•)
that abstracts a hydrogen atom from a substrate as the first step of catal-
ysis (Fig. 2) [17]. PhpK belongs to a subset of radical SAM enzymes that
contain a cobalamin (Cbl)-binding domain [16,18]. In vitro studies in
our laboratory demonstrated that PhpK from K. phosalacinea catalyzes
the P-methylation of NAcDMPT to produce NAcPT in a SAM-, sodium
dithionite-, and CH3Cbl-dependent manner (Fig. 1B) [15]. Three related
Cbl-dependent radical SAM methyltransferases, TsrM, GenK, and Fom3,
have been reported upon since the initial PhpK work [19–21]. These en-
zymes are found in bacterial biosynthetic pathways for the antibiotics
thiostrepton, gentamicin, and fosfomycin, respectively. Although some
similarities exist between known members of the Cbl-dependent radi-
cal SAM family, a variety of differences have been reported, and many
mechanistic details remain unresolved.
2. Materials and methods
2.1. Materials
Reagents were obtained from typical suppliers unless otherwise in-
dicated. Titanium(III) (Ti) citrate and [methyl-13C]Cbl were synthesized
as described elsewhere [26,27]. SAM was acquired from Safeway and
was purified as described elsewhere [15]. 2-HEP was synthesized as de-
scribed elsewhere [21].
2.2. Cloning of sden_1168 from S. denitrificans OS217 (GenBank accession
ABE54454.1)
S. denitrificans OS217 was obtained from the American Type Culture
Collection (ATCC) (ATCC-BAA 1090), was reconstituted in liquid media
according to ATCC recommendations, and was streaked onto marine
broth 2216 (Difco, Sparks, MD) agar plates to obtain isolated colonies.
A single colony was used to inoculate 5 mL of marine broth, and the cul-
ture was incubated with shaking at 30 °C overnight. Genomic DNA was
isolated from the overnight culture using the Wizard Genomic DNA pu-
rification kit (Promega, Madison, WI). The sden_1168 gene was ampli-
fied from S. denitrificans genomic DNA using PCR. The forward primer
was 5′-TATATACATATGCGACCAAATTTTTA-3′ and the reverse primer
Fig. 2. Radical SAM cleavage.

K.D. Allen, S.C. Wang / Biochimica et Biophysica Acta 1844 (2014) 2135–2144
2137
Fig. 3. Comparison of putative S. denitrificans OS217 and Kitasatospora NRRL F-6133 C–P homologous genes [23]. Genes in color are homologous. Red indicates Cbl-dependent radical SAM
methyltransferases. Genes in gray are not homologous. Not all genes from each putative pathway are shown. PepM: phosphoenolpyruvate mutase, Ppd: phosphonopyruvate
decarboxylase.
was 5′-TTTTGCGGCCGCTCATTCTTCATAAGCTT-3′. Restriction sites for
NdeI and NotI, respectively, are underlined in each primer. Digestion
and ligation of sden_1168 into pET-30a(+) (EMD Millipore/Merck
KGaA) were performed using standard molecular biology techniques.
Ligation mixtures were transformed into NovaBlue GigaSingles compe-
tent cells (EMD Millipore/Merck KGaA). Plasmids were isolated with the
Spin Miniprep Kit (Qiagen, Valencia, CA) and sequenced to confirm the
expected nucleotide sequence for sden_1168. The resulting construct
was denoted sden_1168-pET-30a.
magnesium sulfate (Buffer A) to a final concentration of ~30 μM, and
300 μL was transferred to an anaerobic cuvette for measurement.
2.5. DMPT synthesis
DMPT was synthesized according to a procedure developed by
AsisChem Inc. (Watertown, MA) with some modifications [15]. To
14.8 g (0.04 mol) Z-L-glutamic acid α-benzyl ester (Chem-Impex,
Wood Dale, IL) 50 mL benzene, 0.4 g copper acetate, and 224 μL pyridine
were added. The mixture was stirred at 20 °C for 1 h, then 50 mL ben-
zene and 8.8 g (0.027 mol) lead acetate were added. The reaction was
refluxed under argon for 24 h, filtered through a celite pad, and rinsed
with ethyl acetate (3 × 20 mL). The bright turquoise filtrate was washed
with water (3 × 100 mL) and brine (3 × 100 mL), and the resulting
yellow/brown organic layer was dried over 4 g anhydrous sodium sul-
fate. The product was purified by column chromatography over silica
gel (ethyl acetate:hexanes, 1:9). Fractions containing the desired
product were combined and concentrated by rotary evaporation.
To the resulting yellow syrup 2 mL methanol, 8 mL 50% aqueous
hypophosphorus acid, and 0.16 g 2,2′-azobis(2-methylpropionitrile)
were added. The reaction was stirred at 75 °C for 9 h. 30 mL
water was added and the pH was adjusted to ~8 with saturated
sodium carbonate. This mixture was washed with methyl tert-butyl
ether (2 × 40 mL). The aqueous layer was adjusted to pH ~2 with 6 M
hydrochloric acid. The desired product was extracted with dichloro-
methane (2 × 30 mL) and dried over anhydrous sodium sulfate. After
concentration via rotary evaporation, 15 mL 6 M hydrochloric acid
was added and the reaction was refluxed under argon for 3 h. The
resulting DMPT product was concentrated using rotary evaporation,
and the yellow gel was resuspended in 3 mL water. DMPT was purified
using cation exchange resin (Dowex AG-50, Acros Organics). The resin
was washed stepwise with water followed by 10 mM sodium carbonate
at pH 8, 9, and 10. DMPT was eluted with 10 mM sodium carbonate,
pH 11. The eluant was concentrated via rotary evaporation, and the
resulting yellow oil was transferred into the anaerobic chamber to
2.3. Overproduction, purification, and iron–sulfur cluster reconstitution of
SD_1168
Overproduction and purification of SD_1168 were conducted as de-
scribed elsewhere with minor modifications [15,21,28]. All purification
and protein manipulations took place in an anaerobic chamber (Coy
Laboratory Products, Grass Lake, MI). SD_1168 was allowed to refold
overnight by itself, in the presence of SAM, or in the presence of
hydroxocobalamin. Protein concentrations were estimated using the
method of Waddell [29]. Aliquots of SD_1168 were frozen anaerobically
and stored in liquid nitrogen.
2.4. Iron and sulfide content and spectrophotometric analysis
The amount of iron and sulfide bound by SD_1168 was determined
with an Agilent 8453 ultraviolet–visible (UV–vis) spectrophotometer
(Agilent Technologies, Santa Clara, CA) using methods described previ-
ously [21,30–32]. To determine iron content, samples contained 0.55–
3.27 nmol of SD_1168, and a standard curve was developed using
1–25 nmol FeCl3. For sulfide determination, samples contained 0.22–
0.87 nmol of SD_1168 and a standard curve was generated with
1–5 nmol Na2S. For spectrophotometric measurements, purified and
reconstituted SD_1168 was first thawed in the anaerobic chamber.
The protein was diluted with 20 mM potassium (K+
hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS) and 1 mM
) 4-(2-

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deaerate. The concentration of DMPT was determined by 31P nuclear
magnetic resonance (NMR) spectroscopy by comparison with a phos-
phonate standard of known concentration. DMPT was resuspended in
1 M K+ EPPS, pH 8 at a concentration of 80–100 mM and stored in ali-
quots at −80 °C.
phosphonates eluted during the initial wash and were collected
and concentrated via rotary evaporation. The concentrated, partially
purified reaction mixtures were resuspended in 550 μL deuterium
oxide (Cambridge Isotope Labs, Tewksbury, MA) for NMR analysis.
One-dimensional (1-D) 1H or 31P and two-dimensional (2-D) H–P
gradient heteronuclear single quantum correlation (gHSQC) NMR
spectra were collected at the Washington State University NMR Cen-
ter using a Varian 600 MHz spectrometer at 22 °C [15,22]. gHSQC
data were collected for 12–16 h for each enzymatic reaction sample.
Peak positions varied between samples (up to 2 ppm along 1H and/or
10 ppm along 31P) due to differences in final pH/pD of the partially
purified reactions.
2.6. NAcDMPT synthesis
Crude DMPT was N-acetylated by adding 20 mL acetic acid, 28 μL
triethylamine, and 4 mL acetic anhydride (in 500 μL aliquots over
~5 min). Acetylation was complete after ~24 h. The resulting NAcDMPT
was concentrated and purified over silica gel in 90% methanol followed
by cation exchange (Dowex AG-50, Acros Organics) eluted with water.
Fractions containing NAcDMPT were combined and concentrated by ro-
tary evaporation to leave a yellow oil. The concentration of NAcDMPT
was determined by NMR spectroscopy by comparison with a phospho-
nate standard of known concentration. NAcDMPT was resuspended in
1 M K+ EPPS, pH 8 at a concentration of ~100 mM and stored at −80 °C.
2.9. Partial unfolding of SD_1168 to produce SAM-free protein (“SD_1168
(−SAM)”)
SD_1168 (~100 μM) that had originally been refolded by itself with-
out added SAM and without HOCbl was thawed in the anaerobic cham-
ber at room temperature (20 °C). Next, the enzyme (3 mL) was dialyzed
stepwise into increasing concentrations of urea (1–3 M) in Buffer A (1 L)
using a Slide-A-Lyzer cassette (Thermo Fisher Scientific, Rockford, IL).
Three rounds of dialysis for 3 h each were performed at 20 °C in the an-
aerobic chamber. After the final round of dialysis (3 M urea), SD_1168
was light yellow in color, and the UV–vis spectrum indicated that the
protein no longer contained a [4Fe–4S] cluster. These results suggested
that the enzyme had partially unfolded and had released any bound
SAM. The enzyme was then transferred to a new container, 6-fold
molar excess ferrous ammonium sulfate and sodium sulfide and 5 mM
dithiothreitol (DTT) were added, and the mixture was diluted tenfold
in Buffer A. This mixture was incubated overnight in the anaerobic
chamber at 20 °C. The protein was then concentrated using centrifugal
filter units (Amicon Ultra-15, 30 kDa cut-off, EMD Millipore/Merck
KGaA) spun at 5000 rpm and 4 °C to ~100 μM. The final protein, denot-
ed “SD_1168 (−SAM)” was dialyzed into Buffer A and stored in liquid
nitrogen.
2.7. Electron paramagnetic resonance (EPR) sample preparation and spec-
tral collection
SD_1168 was thawed in the anaerobic chamber and diluted to a final
concentration of 100 μM in Buffer A. The protein was mixed with vari-
ous combinations of the following components: sodium dithionite
(4 mM), SAM (1 mM), NAcDMPT (1 mM), and CH3Cbl (0.5–1 mM).
After ~1 h, the samples were transferred to 4 mm EPR tubes (Norell,
Landisville, NJ), frozen in isopentane, and stored in liquid nitrogen.
Low temperature X-band EPR spectra were obtained on a Bruker
EMXplus spectrometer equipped with a Bruker EMXpremium micro-
wave bridge, an Oxford Instruments ESR 900 continuous flow cryostat,
and an Oxford Instruments ITC503 temperature controller. Bruker
Xenon software (version 1.1b50) was used to acquire and manipulate
spectra. Spectra were recorded under the following conditions:
9.379 GHz, 16 G modulation amplitude, 3400 G center field, 1000 G
sweep width, 1 mW power, 0.3 s time constant, 10 K, and 4 × 2 min
scans.
3. Results and discussion
2.8. Evaluation of enzymatic activities
3.1. Overexpression and refolding
SD_1168 was thawed in the anaerobic chamber and diluted to
100 μM with Buffer A. Ti(III) citrate (13.6 mM) or sodium dithionite
(4 mM) was added to the protein and the mixture was incubated for
30–60 min. When dithionite was used as a reductant, the protein was
exchanged into Buffer A to remove excess dithionite using a PD-10
desalting column (Sephadex G-25 M, GE Healthcare, Fairfield, CT).
Then SAM (1 mM), NAcDMPT (1 mM) or DMPT (800 μM) or 2-HEP
(1 mM), and CH3Cbl (1 mM), [methyl-13C]Cbl (1 mM), and/or
HOCbl (1 mM) were added. Control reactions were performed in
which one or more of the reaction components were omitted. Reac-
tions were set up in triplicates (3 × 1 mL) except for the [methyl-13C]
Cbl experiment, which was set up as 6 × 1 mL reactions. Reaction
mixtures were incubated anaerobically for ~18 h at ~20 °C before re-
moval from the anaerobic chamber. Centrifugation (10,000 rpm,
10 min) removed precipitated protein; any remaining protein was
removed using polyethersulfone centrifugal filters (VWR, Radnor,
PA). The resulting filtrate from reactions containing NAcDMPT was
partially purified with water elution from cation exchange resin
(AG-50, Acros Organics, Geel, Belgium) and concentrated via rotary
evaporation. The resulting filtrate from reactions containing DMPT
or 2-HEP was partially purified using a reverse-phase C4 column
(Grace Vydac, Deerfield, IL, 10 mm × 250 mm) connected to a high
performance liquid chromatography (HPLC) instrument (Beckman
Coulter, System Gold, Brea, CA). The elution included a 20 min
wash (0.1% formic acid in water, 1 mL/min) followed by a linear
gradient from 0 to 100% acetonitrile. The phosphinates and
The gene encoding SD_1168 from S. denitrificans was cloned into
pET-30a(+) for protein overexpression. SD_1168 was found exclusively
in inclusion bodies when overexpressed in either Escherichia coli or
Streptomyces lividans. All efforts to express soluble recombinant protein
were unsuccessful, as observed in our laboratory for related methyl-
transferases [15,21]. Therefore, we refolded SD_1168 using an
established refolding procedure based upon a literature precedent [15,
21,28]. In brief, the inclusion bodies were solubilized in urea. The
resulting soluble protein was subsequently refolded by dilution in
urea-free buffer in the presence of iron, sulfide, DTT, and SAM. The pro-
tein was then concentrated, the [4Fe–4S] clusters were reconstituted,
and the protein was dialyzed to remove excess iron, sulfide, and reduc-
tant. The final typical yield was ~12 mg of SD_1168 protein per g of
E. coli cell paste. SD_1168 was ~75% pure as assessed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Fig. S1), and it
migrated at its expected molecular mass of 64.8 kDa.
3.2. [4Fe–4S] cluster characterization
Reconstituted SD_1168 was dark brown in color and displayed a
UV–vis spectrum with a broad shoulder having an absorbance maxi-
mum at 420 nm (Fig. 4, black line), characteristic of proteins containing
[Fe–4S]+2 clusters [33,34]. This absorbance was quenched upon the ad-
dition of sodium dithionite or Ti citrate (Fig. 4, orange and blue lines, re-
spectively). Analysis of iron and sulfide content showed that SD_1168
bound 6.1 0.5 mol of iron per mol protein and 4.0 0.8 mol of sulfide

K.D. Allen, S.C. Wang / Biochimica et Biophysica Acta 1844 (2014) 2135–2144
2139
SD_1168. Upon incubation with sodium dithionite, an EPR signal cen-
tered at g = 1.93 was observed (Fig. 5B). This feature is characteristic
of the [4Fe–4S]+1 cluster in radical SAM enzymes.
When the protein was refolded in the presence of HOCbl instead
of SAM and then reduced with dithionite, a more intense signal for
the +1 cluster was observed (Fig. 5C) compared to the sample contain-
ing reduced SD_1168 refolded in the presence of added SAM (Fig. 5B).
This result suggests that Cbl binds near the [4Fe–4S] cluster and
may raise its reduction potential, thus facilitating generation of the +1
cluster [42]. Addition of SAM and dithionite to SD_1168 refolded in
the presence of HOCbl produced new features in the EPR signal at
g ~ 2.04 (Fig. 5D), providing evidence that SAM is a ligand for the cluster
as observed for other radical SAM enzymes [43,44]. A concomitant de-
crease in the intensity of the EPR signal for the +1 cluster (compare
Fig. 5C and D) was also observed. This result may be due to uncoupled
cleavage of SAM that yields the EPR-silent +2 form of the cluster
together with methionine and AdoCH3. When SAM is reductively
cleaved to generate Ado-CH2• in the first step of radical SAM catalysis,
the [4Fe–4S] cluster is oxidized to the EPR-silent +2 state (Fig. 2).
Many radical SAM enzymes catalyze uncoupled cleavage of SAM in
the absence of substrate, especially when dithionite is used as a reduc-
ing agent [37,45–47]. However, we have not detected these cleavage
products using other means (e.g., HPLC).
Fig. 4. UV–vis spectra of ~30 μM SD_1168. Black: reconstituted SD_1168; orange:
SD_1168 + 4 mM sodium dithionite; blue: SD_1168 + 10 mM Ti citrate.
per mol protein. The slightly higher than expected iron content (6.1
0.5 mol iron per mol protein) is comparable to our data for the related
Fom3 methyltransferase from Streptomyces wedmorensis [21] and sug-
gests that adventitious iron may be bound elsewhere in the protein.
To better characterize the [4Fe–4S] cluster, low temperature X-band
EPR spectroscopy was employed since it is a valuable method for char-
acterizing the oxidation state and chemical environment of paramag-
netic centers in proteins [35]. The g-value and shape of the observed
signal assist in the characterization of different types of unpaired elec-
tron spin in different chemical environments [36]. Refolded and
reconstituted SD_1168 was EPR-silent (Fig. 5A), which was consistent
with the resting [4Fe–4S]+2 clusters we observed with UV–vis spectro-
photometry. Signals for an incomplete [3Fe–4S]+1 and/or oxidized
[4Fe–4S]+3 cluster were not observed. Such catalytically-inactive
forms of the cluster have been found in other radical SAM enzymes
after purification and reconstitution [37–41], but our refolding and re-
constitution procedure produced the complete [4Fe–4S]+2 cluster in
3.3. Enzymatic activities
After determining that SD_1168 demonstrated some of the expected
characteristics of a radical SAM enzyme, we set out to determine its
in vitro activity. Reaction mixtures were analyzed using 2-D gHSQC
NMR spectroscopy [15]. Approximate levels of turnover were deter-
mined by integrating the resulting 2-D cross-peak volumes and com-
paring with the original amount of starting organophosphorus
substrate. Activity results for SD_1168 are summarized in Table 1.
Given the sequence similarities between SD_1168, Fom3, and PhpK,
we examined SD_1168 for both C-methyltransferase and P-
methyltransferase activities. Due to the high sequence similarity be-
tween SD_1168 and Fom3, we initially hypothesized that SD_1168
would catalyze C-methylation. The Fom3 substrate, 2-HEP, and product,
2-HPP (Fig. 1C), have distinguishable 1-D 31P and 2-D 1H–31P gHSQC
NMR spectra [21]. Depending on the pH/pD of the final sample, the
peaks for 2-HEP and 2-HPP are approximately 0.5–1 ppm apart along
the phosphorus axis. The 2-D gHSQC experiment, which is two-to-
fourfold more sensitive than 1-D 31P detection, was used to examine
SD_1168 for C-methylation activity upon 2-HEP. However, when
SD_1168 was incubated with dithionite or Ti citrate, SAM, 2-HEP, and
CH3Cbl, C-methylation activity was not observed (Table 1; Fig. S2).
This result suggests that SD_1168 may not be a C-methyltransferase, al-
though we have previously found that Fom3 is also quite difficult to
assay for reproducible turnover [21].
Knowing that SD_1168 shared lower but still significant similarity
with PhpK, the P-methyltransferase found in K. phosalacinea,
S. hygroscopicus, and S. viridochromogenes Tü494, we decided to test
SD_1168 for P-methylation activity. The potential substrates for
SD_1168, NAcDMPT and DMPT, and the products of P-methylation,
NAcPT and PT, respectively (Fig. 1A and B), have easily distinguishable
1-D 31P and 2-D 1H–31P gHSQC NMR spectra [15]. We assessed SD_1168
for P-methylation activity using the more sensitive 2-D gHSQC method
[15]. In these experiments, the signal positions depend upon final pH/
pD and therefore vary as much as 10 ppm along the phosphorus axis
and 2 ppm along the proton axis between samples. Standards at varying
pH/pD and enzymatic reaction samples spiked with authentic product
were used to confirm the identities of observed products.
Under our partial purification conditions, the phosphorus-coupled
C-4 methylene protons of NAcDMPT typically produce a strong cross-
peak centered at ~1.7 ppm (1H) and ~35 ppm (31P) (Fig. 6A). When
SD_1168 was incubated with dithionite, SAM, NAcDMPT, and CH3Cbl,
Fig. 5. EPR spectra of SD_1168. Spectrum A: Refolded and reconstituted SD_1168, spec-
trum B: SD_1168 + dithionite, spectrum C: SD_1168 refolded with HOCbl + dithionite,
spectrum D: SD_1168 refolded with HOCbl + dithionite + SAM, spectrum E: SD_1168
(−SAM) + dithionite.

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K.D. Allen, S.C. Wang / Biochimica et Biophysica Acta 1844 (2014) 2135–2144
Table 1
Methylation activity of SD_1168 upon various substrates. Unless otherwise noted, reactions were incubated anaerobically for ~18 h prior to NMR analysis. NR = no reaction.
Enzyme preparation
Reductant
Organophosphorus
substrate
Cobalamin
substrate
SAM present (+)/
absent (−)
% Turnover
[Product]
(μM)
Figure
–
Ti(III) citrate
Na2SO4
Ti(III) citrate
Ti(III) citrate
Na2SO4
Ti(III) citrate
Ti(III) citrate
Ti(III) citrate
–
2-HEP
2-HEP
2-HEP
CH3Cbl
CH3Cbl
CH3Cbl
CH3Cbl
CH3Cbl
CH3Cbl
CH3Cbl
CH3Cbl
CH3Cbl
–
+
+
+
+
+
+
+
+
+
+
−
–
NR
NR
NR
NR
NR
NR
NR
NR
–
SD_1168
SD_1168
S2
–
–
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
NAcDMPT
DMPT
S4A
S3
Not shown
Not shown
5A
S4B
S4C
S4D
S6
5C
5D
SD_1168
SD_1168
SD_1168
SD_1168
SD_1168
SD_1168
0.3%
3.7% (5 min)
4.8% (2 h)
7.4%
NR
NR
8.4%
NR
18
220
290
400
NR
NR
460
NR
460
1100
190
Ti(III) citrate
Ti(III) citrate
Ti(III) citrate
Ti(III) citrate
Ti(III) citrate
Ti(III) citrate
SD_1168 (refolded without exogenous SAM)
SD_1168 (−SAM)
SD_1168
SD_1168
SD_1168
CH3Cbl
CH3Cbl
HOCbl
+
+
+
8.5%
11.0%
3.5%
[Methyl-13C]Cbl
CH3Cbl
5B
a new cross-peak corresponding to the methyl phosphinate protons of
NAcPT was observed (Table 1, Fig. S3). This was an unexpected result
since the genome of S. denitrificans does not encode most of the
known enzymes required for PT biosynthesis. Turnover was poor
under these conditions and resulted in only 0.3% product formation
(Table 1). This is a significantly lower amount of turnover than PhpK
from K. phosalacinea is capable of under similar conditions [15]. This re-
sult suggested that the reaction conditions used were suboptimal, per-
haps due to differences in reduction potentials of the [4Fe–4S]+2/+1
couple or to as-yet unknown differences in the active sites of the two
enzymes. The reduction potential of similar [4Fe–4S]+2/+1 interconver-
sions in biotin synthase and clostridial lysine-2,3-aminomutase has
been estimated to be ~−450 mV [42,48]. In comparison, the reduction
potential of sodium dithionite has been measured and or estimated to
fall between −327 and −511 mV, depending upon concentration, pH,
and purity [49–52].
Our previous work with multiple Cbl-dependent radical SAM
methyltransferases including PhpK and Fom3 has revealed that
dithionite can inadvertently react with CH3Cbl to produce the presum-
ably catalytically-incompetent Cbl(II). We do not observe dithionite-
reduced SD_1168 P-methylation unless the enzyme is desalted prior
to adding the other reaction components. In addition, dithionite binds
to Cbl(II) [53], which may affect the ability of SD_1168 and related en-
zymes to perform more than one turnover. Finally, sodium dithionite
is largely impure, contains a variety of breakdown products, and is un-
stable even after purification efforts [54]. Therefore, we sought an alter-
native cluster reductant.
Ti citrate is a one-electron reductant with a redox potential of
−500 mV that has been successfully used to reduce [4Fe–4S] cofactors
found in carbon monoxide dehydrogenase/acetyl-CoA synthase and
the corrinoid iron–sulfur protein [55–57]. When Ti citrate was used in
SD_1168 reactions instead of dithionite, significantly greater P-
methylation activity (7.4% vs. 0.3%, Table 1) was observed as evidenced
by the cross-peak at 1.35 ppm (1H) and 57.0 ppm (31P) (Fig. 6A). In our
hands, PhpK also appears to be more active in the presence of Ti citrate
compared with dithionite [58]. For these reasons, Ti citrate was used for
subsequent experiments with SD_1168.
Since titanium(III) is paramagnetic and interferes with the EPR
signal for the +1 cluster, we did not obtain EPR evidence for Ti citrate
reduction of the [4Fe–4S] cluster. Instead, we used UV–vis spectropho-
tometry to monitor the cluster's oxidation state. In the presence of Ti cit-
rate, the broad shoulder characteristic of the [4Fe–4S]+2 cluster
disappeared (Fig. 4, blue line). This is evidence for successful reduction
of the +2 cluster to the +1 state and is similar to our observations
using sodium dithionite to reduce the cluster (Fig. 4, orange line). To
our knowledge, the use of Ti citrate to reduce [4Fe–4S] clusters of radical
SAM enzymes has not been reported elsewhere. Thus, this compound
represents a promising, low-potential reducing agent for other Cbl-
dependent radical SAM methyltransferases. In vivo, the cluster reduc-
tant for SD_1168 is most likely a ferredoxin or flavodoxin; multiple
forms of these electron carriers can be found in the genome of
S. denitrificans OS217. Although others have reported that flavodoxin
is a suitable reductant [20], we have tested recombinant E. coli
flavodoxin and have not successfully observed activity for any Cbl-
dependent radical SAM methyltransferase in our laboratory [58].
We also tested SD_1168 for methylation activity upon DMPT, anoth-
er proposed intermediate in the PT biosynthetic pathway [1]. This activ-
ity is not catalyzed by PhpK, the “original” P-methyltransferase, which
appears to require the N-acetyl modification for substrate recognition
and binding. This modification, added by the Bar or Pat acetyltransferase
depending on the microbe, confers resistance and is removed by a
deacetylase immediately before export of the antibiotic [59]. There is
currently no genomic evidence for the existence of either the acetyl-
transferase or the deacetylase in S. denitrificans OS217.
By gHSQC, the phosphorus-coupled C-4 methylene protons of DMPT
give rise to a cross-peak centered at 1.45 ppm (1H) and 26 ppm (31P)
(Fig. 6B). Due to long-range coupling, the C-3 methylene protons are
also observed as a cross-peak at 1.90 ppm (1H) and 26 ppm (31P).
When SD_1168 was incubated with Ti citrate, SAM, DMPT, and
CH3Cbl, approximately 3.5% turnover was observed as evidenced by
the new cross-peak corresponding to the methyl group protons of PT
at 1.12 ppm (1H) and 40.5 ppm (31P) (Table 1, Fig. 6B). Unlike PhpK,
SD_1168 does not require the N-acetyl modification for substrate recog-
nition. This highlights an important difference in the active sites of these
two enzymes and may provide a clue as to the structure of the physio-
logical substrate for SD_1168. Although approximately twice as much
product was formed from methylation of NAcDMPT compared to
DMPT (Table 1), it currently remains unclear which phosphinate sub-
strate is preferred by SD_1168.
3.4. Cofactor and substrate requirements and potential catalytic
mechanism
Control experiments were performed in which one of each hypoth-
esized required component was omitted. In the absence of SD_1168, re-
ductant, or CH3Cbl, NAcPT formation was not observed (Table 1,
Fig. S4A–C). However, when SAM was omitted from reactions contain-
ing SD_1168 purposely refolded without exogenous SAM, a cross-peak
corresponding to ~8.4% NAcPT formation was observed (Table 1,
Fig. S4D). We hypothesized that this result was due to SD_1168 binding
of SAM from cellular extracts during refolding and purification. There-
fore, we partially unfolded purified SD_1168, which had originally
been refolded without SAM and without HOCbl, with urea to release
any small molecules bound by the protein. The resulting preparation

K.D. Allen, S.C. Wang / Biochimica et Biophysica Acta 1844 (2014) 2135–2144
2141
Fig. 6. H–P gHSQC NMR spectra of partially purified, Ti citrate-reduced SD_1168-catalyzed reactions of interest. A. NAcDMPT + CH3Cbl reaction. The cross-peaks at (1.66, 35) ppm and
(1.95, 35) ppm correspond to the C-4 methylene protons and the C-3 methylene protons, respectively, of NAcDMPT. The cross-peak at (1.35, 56) ppm corresponds to the methyl protons
of NAcPT. B. DMPT + CH3Cbl reaction. The cross-peaks at (1.45, 26) ppm and (1.90, 26) ppm correspond to the C-4 methylene protons and the C-3 methylene protons, respectively, of
DMPT. The cross-peak at (1.12, 41) ppm corresponds to the methyl protons of PT. C. NAcDMPT + [methyl-13C]Cbl reaction. The two cross-peaks indicated by the double daggers (‡) at
(1.30, 56) ppm and (1.09, 56) ppm correspond to the methyl protons of [methyl-13C]NAcPT, which are split by the NMR-active 13C and 31P nuclei. The cross-peak indicated by an asterisk
(*) at (1.20, 56) ppm corresponds to the methyl protons of unlabeled NAcPT. D. NAcDMPT + HOCbl reaction. The major cross-peak at (1.68, 35) ppm corresponds to the C-4 methylene
protons of NAcDMPT, and the cross-peak at (1.29, 56.5) ppm corresponds to the methyl protons of NAcPT.
was refolded and reconstituted and was denoted SD_1168 (−SAM).
UV–vis spectrophotometry and EPR spectroscopy showed that
SD_1168 (−SAM) remained able to bind a reducible [4Fe–4S] cluster
(Figs. S5 and 5E). After incubation of SD_1168 (−SAM) with all hypoth-
esized reaction components except for SAM, no methylation activity
was observed (Table 1, Fig. S6). Thus, as expected for a radical SAM en-
zyme, SAM is required for SD_1168-catalyzed P-methylation.
Previous in vivo labeling experiments, knockout studies, and in vitro
enzymatic studies have highlighted the role of CH3Cbl as the methyl
group donor for other Cbl-dependent radical SAM methyltransferases
[13,15,60–62]. To investigate the methyl donor for the SD_1168-cata-
lyzed P-methylation of NAcDMPT, we performed the reaction in the
presence of [methyl-13C]Cbl. Under these conditions, the previously ob-
served cross-peak (indicated by an asterisk, *) corresponding to the
methyl protons of unlabeled NAcPT was observed at 1.20 ppm (1H)
and 56.4 ppm (31P, Fig. 6C). Two new cross-peaks (indicated by the dou-
ble daggers ‡) centered at 1.30 ppm (1H) and 56.5 ppm (31P) and at
1.09 ppm (1H) and 56.3 ppm (31P) were also observed. The splitting of
these cross-peaks results from passive couplings of 1H and 31P with
the newly added 13CH3 group from [methyl-13C]Cbl to produce
[methyl-13C]NAcPT [15]. This result suggests that an alternative methyl
group donor beyond CH3Cbl is used by the enzyme. We suspect that, as
with other characterized radical SAM methyltransferases, this donor is a
second molecule of SAM [19,20]. The approximate ratio of 13C to 12C
product was 1:1, indicating that SD_1168 was able to remethylate once
prior to NMR analysis. Since Ti citrate is not capable of reducing
13CH3Cbl to allow for methylation of Cbl(I) with unlabeled SAM
(Fig. S7), the 12C product must arise from a second turnover and not
from non-enzymatic side reactions. This observed result differs from our
previous work with PhpK which suggested that the K. phosalacinea P-
methyltransferase was only capable of a single turnover [15, K. Hu, W.J.
Werner, K.D. Allen, and S.C. Wang, submitted for publication]. However,
our previous PhpK studies used dithionite as the reducing agent [15]. As
described earlier, dithionite is likely not the ideal reductant to use with
SD_1168 and, perhaps, other related methyltransferases in vitro.
A hypothetical radical SAM P-methylation mechanism supported by
our data is depicted in Fig. 7 [1,18]. First, the [4Fe–4S]+1 cluster donates
an electron to the required molecule of SAM to produce Ado-CH2•. Next,
Ado-CH2• abstracts the phosphinyl hydrogen of NAcDMPT to generate a
phosphonyl radical that reacts with CH3Cbl to produce the methylated
product and Cbl(II), a dead end that cannot support further turnover.
Therefore, the enzyme must either bind a new molecule of CH3Cbl or
it must regenerate CH3Cbl to proceed with further catalysis. To date,
the use of CH3Cbl strictly as a substrate is unprecedented [63,64].
Thus, it is far more likely that CH3Cbl is a regenerated cofactor.
Remethylation of Cbl in Cbl-dependent radical SAM methyltransferases
may occur in a manner similar to the reactivation cycle of Cbl-
dependent methionine synthase [18]. In this cycle, flavodoxin reduces

2142
K.D. Allen, S.C. Wang / Biochimica et Biophysica Acta 1844 (2014) 2135–2144
Fig. 7. Proposed radical SAM P-methylation mechanism.
Cbl(II) to Cbl(I), which is then remethylated after nucleophilic attack on
SAM to form CH3Cbl [64]. Our hypothetical remethylation mechanism
for SD_1168 is supported by data from experiments with the Cbl-
dependent radical SAM methyltransferase GenK in which both
[methyl-13C]product and [methyl-13C]Cbl were observed after incuba-
tion with [methyl-13C]SAM, indicating that the methyl group from
SAM was transferred to both Cbl and the final GenK product [20].
To further test the hypothesis that CH3Cbl is not truly a substrate for
SD_1168 and instead is generated as an intermediate, we examined
SD_1168 for activity in the presence of Ti citrate, SAM, NAcDMPT, and
HOCbl. Under these conditions, the new cross-peak was equivalent to
approximately 8.5% NAcPT formation (Table 1, Fig. 6D), indicating that
CH3Cbl is not directly required. Together with our [methyl-13C]Cbl re-
sults, these data provide the first evidence for methylation of Cbl within
a P-methyltransferase enzyme. SD_1168 is apparently able to form
CH3Cbl using a methyl group from another source, then transfer this
methyl group to the final NAcPT product. Ti citrate can reduce any
Cbl(II) formed to Cbl(I), which can then attack a methyl group donor
such as SAM to generate CH3Cbl. This function of Ti citrate has been
well established in Cbl- and corrinoid-dependent enzymes [65–67].
Ti citrate can also reduce HOCbl to Cbl(I) (Fig. S8), so we cannot
yet rule out the possibility that the observed remethylation of Cbl
may be due to a non-enzymatic process. Additionally, further studies
using [methyl-13C]SAM are required to confirm whether SD_1168
remethylates Cbl using SAM as part of its catalytic cycle.
characterization is presumably due to the inherent complexity of these
systems as well as limitations in separation and detection technologies.
Since the genome of S. denitrificans encodes the first two genes
(sden_1161/pepM and sden_1162/ppd) required for most known C–P
compound biosynthetic pathways [23], it is likely that this bacterium
produces a C–P natural product. A comprehensive study recently
investigated the variety and prevalence of C–P biosynthetic genes
among microbes [23], illustrating a variety of pathways spanning
phosphonolipid and phosphonoglycan biosynthesis as well as produc-
tion of biologically-active C–P compounds [1,23,68–71]. The genes in
the immediate neighborhood of SD_1168 show striking similarity to a
group of genes identified in 2013 in an actinomycete, Kitasatospora sp.
NRRL F-6133 (compare Fig. 3, top and bottom) [23]. In fact, SD_1168
shares higher sequence identity and similarity (61% and 77%, respec-
tively) with the putative Cbl-dependent radical SAM methyltransferase
encoded by orf11 from F-6133 than with either Fom3 or PhpK. To date,
Orf11 has not been investigated. However, the high similarity suggests
that the biological roles of SD_1168 and Orf11 may be very similar,
whether for the production of bioactive C–P compounds or for
another biosynthetic purpose. In addition, a preliminary analysis of
S. denitrificans cells using 2-D high-resolution magic angle spinning
(HR-MAS) NMR spectroscopy suggests that this microbe may produce
a phosphinate as a cellular component [G. Helms and S.C. Wang, unpub-
lished data]. The relatively inert nature of the C–P bond to hydrolysis
may be an important way for S. denitrificans to retain phosphorus
from its marine environment. Thus, we currently speculate that the
true substrate for SD_1168 is an alkyl phosphinate. Knockout of the
sden_1168 gene in S. denitrificans OS217, synthesis of potential sub-
strates and, ultimately, the development of a kinetic assay for
SD_1168 and other Cbl-dependent radical SAM methyltransferases
will shed light on these questions and ultimately reveal the purpose of
this intriguing enzyme.
3.5. Potential biological role for SD_1168, conclusions, and future directions
Our work with SD_1168 has provided further insight into the unusu-
al reactions catalyzed by and the diversity of Cbl-dependent radical SAM
methyltransferases. SD_1168 requires a strong reductant, SAM, and Cbl
for P-methytransferase activity. This is consistent with the previously
hypothesized radical SAM mechanism for P-methylation (Fig. 7), our
laboratory's previous work on PhpK, and the recent study by Liu and co-
workers on the distantly-related GenK methyltransferase [15,18,20].
Our results also support the hypothesis that Cbl functions as a coen-
zyme, acting as an intermediate methyl group carrier that must be re-
generated for subsequent turnover.
The observed P-methylation activity of SD_1168 raises the obvious
question: what is the biological role of this protein? Though the P-
methyltransferase activity of SD_1168 upon both DMPT and NAcDMPT
is reasonable and, for the latter compound, comparable to that of
PhpK, it is unlikely that either phosphinate is the true physiological sub-
strate for this enzyme. We currently do not know whether SD_1168 or
PhpK is a “better” P-methyltransferase as evidenced by kcat and/or Km
since sensitivity and separation difficulties have prevented us from
performing kinetic analyses of SD_1168 or PhpK. To date, the only puta-
tive Cbl-dependent radical SAM methyltransferase for which any kinetic
information has been reported is TsrM, which apparently does not per-
form traditional radical SAM-dependent chemistry since Ado-CH2•
is reportedly not required for activity [19]. This lack of kinetic
Acknowledgements
We thank Dr. William H. Johnson Jr., for assistance with organic syn-
thesis; Dr. Gregory Helms, Dr. William Hiscox, and Dr. Kaifeng Hu for as-
sistance with NMR; Drs. Eva Schinko and Wolfgang Wohlleben for
assistance with expression in S. lividans; and Williard Werner and Drs.
Catherine Drennan, Perry Frey, and Christian Whitman for insightful
discussions. The Washington State University NMR Center equipment
was supported by the NIH (RR0631401 and RR12948), the NSF (CHE-
9115282 and DBI-9604689), and the MJ Murdock Charitable Trust.
K.D.A. was supported by NIH training grant T32GM008336 and a Bank
of America Poncin Trust Fellowship. This research was funded by
Washington State University and a Faculty Early Career Development
Award (CAREER) from the NSF to S.C.W. (CHE-0953721).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbapap.2014.09.009.

K.D. Allen, S.C. Wang / Biochimica et Biophysica Acta 1844 (2014) 2135–2144
2143
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