Preparation, Assay, and Application of Chlorinase SalL for the Chemoenzymatic Synthesis of S-Adenosyl-L-Methionine and Analogs

Article abstract of DOI:10.1016/bs.mie.2018.02.012

S-adenosyl-L-methionine (SAM) is universal in biology, serving as the second most common cofactor in a variety of enzymatic reactions. One of the main roles of SAM is the methylation of nucleic acids, proteins, and metabolites. Methylation often imparts regulatory control to DNA and proteins, and leads to an increase in the activity of specialized metabolites such as those developed as pharmaceuticals. There has been increased interest in using SAM analogs in methyltransferase-catalyzed modification of biomolecules. However, SAM and its analogs are expensive and unstable, degrading rapidly under physiological conditions. Thus, the availability of methods to prepare SAM in situ is desirable. In addition, synthetic methods to generate SAM analogs suffer from low yields and poor diastereoselectivity. The chlorinase SalL from the marine bacterium Salinispora tropica catalyzes the reversible, nucleophilic attack of chloride at the C5′ ribosyl carbon of SAM leading to the formation of 5′-chloro-5′-deoxyadenosine (ClDA) with concomitant displacement of L-methionine. It has been demonstrated that the in vitro equilibrium of the SalL-catalyzed reaction favors the synthesis of SAM. In this chapter, we describe methods for the preparation of SalL, and the chemoenzymatic synthesis of SAM and SAM analogs from ClDA and L-methionine congeners using SalL. In addition, we describe procedures for the in situ chemoenzymatic synthesis of SAM coupled to DNA, peptide, and metabolite methylation, and to the incorporation of isotopes into alkylated products.

Full text of DOI:10.1016/bs.mie.2018.02.012

CHAPTER ELEVEN
Preparation, Assay, and
Application of Chlorinase SalL
for the Chemoenzymatic Synthesis
of S-Adenosyl-L-Methionine
and Analogs
Tony D. Davis*, Sylvia Kunakom†, Michael D. Burkart*,1,
Alessandra S. Eustaquio†,1
*University of California San Diego, San Diego, CA, United States
†University of Illinois at Chicago, College of Pharmacy, and Center for Biomolecular Sciences, Chicago, IL,
United States
1Corresponding authors: e-mail address: mburkart@ucsd.edu; ase@uic.edu
Contents
1. Introduction
368
371
371
374
376
376
378
2. Preparation and Assay of Chlorinase SalL
2.1 Purification of Recombinant SalL From Escherichia coli
2.2 SalL Activity Assay
3. Chemoenzymatic Synthesis of SAM Analogs
3.1 Overview of Chemoenzymatic Methods to Prepare SAM and SAM Analogs
3.2 SalL-Mediated Chemoenzymatic Synthesis of SAM and SAM Analogs
3.3 In Situ Chemoenzymatic Synthesis of SAM Coupled to DNA Methylation
by HhaI
380
381
383
3.4 In Situ Chemoenzymatic Synthesis of SAM Coupled to Teicoplanin
Methylation by MtfA
3.5 In Situ Chemoenzymatic Synthesis of 13C-Labeled SAM Coupled With
Isotope Incorporation Into Teicoplanin via Methylation by MtfA
3.6 In Situ Chemoenzymatic Synthesis of SAM Coupled to RGG Peptide
Methylation by rPRMT1
384
385
385
385
4. Summary and Conclusions
Acknowledgments
References
Abstract
S-adenosyl-L-methionine (SAM) is universal in biology, serving as the second most com-
mon cofactor in a variety of enzymatic reactions. One of the main roles of SAM is the
methylation of nucleic acids, proteins, and metabolites. Methylation often imparts
#
Methods in Enzymology, Volume 604
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2018.02.012
2018 Elsevier Inc.
All rights reserved.
367

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Tony D. Davis et al.
regulatory control to DNA and proteins, and leads to an increase in the activity of spe-
cialized metabolites such as those developed as pharmaceuticals. There has been
increased interest in using SAM analogs in methyltransferase-catalyzed modification
of biomolecules. However, SAM and its analogs are expensive and unstable, degrading
rapidly under physiological conditions. Thus, the availability of methods to prepare SAM
in situ is desirable. In addition, synthetic methods to generate SAM analogs suffer from
low yields and poor diastereoselectivity. The chlorinase SalL from the marine bacterium
Salinispora tropica catalyzes the reversible, nucleophilic attack of chloride at the C50
ribosyl carbon of SAM leading to the formation of 50-chloro-50-deoxyadenosine
(ClDA) with concomitant displacement of L-methionine. It has been demonstrated that
the in vitro equilibrium of the SalL-catalyzed reaction favors the synthesis of SAM. In this
chapter, we describe methods for the preparation of SalL, and the chemoenzymatic syn-
thesis of SAM and SAM analogs from ClDA and L-methionine congeners using SalL. In
addition, we describe procedures for the in situ chemoenzymatic synthesis of SAM
coupled to DNA, peptide, and metabolite methylation, and to the incorporation of iso-
topes into alkylated products.
1. INTRODUCTION
S-adenosyl-L-methionine (SAM or AdoMet) is ubiquitous in nature
and the second most utilized enzyme substrate after adenosine triphosphate
(ATP). SAM is not only a source of methyl groups to methylate a variety of
substrates, including nucleic acid, proteins, and small molecules (Cantoni,
1975; Thomsen, Vogensen, Buchardt, Burkart, & Clausen, 2013), but also
a source of methylene, amino, ribosyl, and aminopropyl groups, in addition
to 50-deoxyadenosyl radicals (Fontecave, Atta, & Mulliez, 2004). Remark-
ably, SAM has also recently been shown to serve as a cofactor in coordinat-
ing pericyclic reactions via electrostatic catalysis (Ohashi et al., 2017).
Another role for SAM is to serve as a substrate in reactions catalyzed by
SAM-dependent halogenases of the 50-halo-50-deoxyadenosine (XDA)
synthase class (Agarwal et al., 2017; Dong et al., 2004; Eusta´quio, Pojer,
Noel, & Moore, 2008).
Halogenated compounds are pervasive across all domains of life. Over
5000 organohalogens are known. Organochlorines and organobromines
are the most abundant with >2000 known examples of each, whereas
organoiodines are more scarce (<200) and organofluorines are very rare
with only five rigorously identified compounds (Chan & O’Hagan, 2012;
Gribble, 2012; Wang, Zhou, Fredimoses, Liao, & Liu, 2014). Halogenating
enzymes employ various mechanistic strategies depending on the properties

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
369
of the halogen atom and the reactivity of the organic substrate. Most
halogenases activate chloride, bromide, and iodide via oxidation by utilizing
cofactors such as heme-iron, vanadium, flavin, and Fe(II)/α-ketoglutarate
(Agarwal et al., 2017). For fluoride, on the other hand, which evades oxi-
dation due to its extreme electronegativity, an orthogonal strategy has been
described that uses SAM as a cosubstrate in an SN2-type, nucleophilic sub-
stitution reaction (Fig. 1). The only fluorinase described thus far (FlA) cat-
alyzes the first committed step in the biosynthesis of fluoroacetate and
fluorothreonine in the soil bacterium Streptomyces cattleya in which fluoride
attacks the C50 electrophilic carbon of SAM, displacing L-methionine and
generating 50-fluoro-50-deoxyadenosine (FDA) (Dong et al., 2004). FlA
was later identified in other bacteria in addition to S. cattleya (Deng et al.,
2014; Huang et al., 2014).
Four years after the structural characterization of the fluorinase, the
chlorinase SalL was reported from the marine bacterium Salinispora tropica
(Eusta´quio et al., 2008). SalL is analogous to fluorinase and the only other
structurally characterized SAM-dependent halogenase of the XDA synthase
class (Fig. 1). SalL catalyzes the first committed step in the biosynthesis of
´
proteasome inhibitor salinosporamide A (Eustaquio et al., 2009, 2008;
Feling et al., 2003). Both FlA and SalL can also catalyze the reverse
reaction, i.e., synthesis of SAM from XDA. For FlA, the in vitro equilibrium
Fig. 1 Reaction catalyzed by SAM-dependent halogenases. Fluorinase FlA from soil
bacterium Streptomyces cattleya (Dong et al., 2004) and chlorinase SalL from marine
bacterium Salinispora tropica (Eustáquio et al., 2008) catalyze reversible SN2-type, nucle-
ophilic substitution reactions in which the halide attacks the C50 ribosyl carbon of SAM,
generating 50-halo-50-deoxyadenosine (XDA) and releasing L-methionine. FlA and SalL
catalyze the first committed step in the biosynthesis of fluorometabolites and
salinosporamide A, respectively.

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Tony D. Davis et al.
lies in favor of FDA by a factor of three (Dong et al., 2004). That the forward
reaction is favored by FlA is unsurprising given that the C–F bond is the
strongest bond in organic chemistry. For chlorinase SalL, on the other
hand, synthesis of SAM from 50-chloro-50-deoxyadenosine (ClDA) and
L-methionine (L-met) is favored, with the relative enzyme efficiency
(kcat/Km) for the reverse reaction (SAM synthesis) being several orders of
magnitude higher than the forward halogenation reaction (Eusta´quio
et al., 2008). The fluorinase was also reported to favor the reverse reaction
in vitro when using chloride as the halide (Deng et al., 2006).
As a versatile methyl donor to biomolecules, there has been considerable
interest in exploring the synthesis of SAM analogs of chemical and biological
utility. For instance, nonnatural SAM analogs could be used to modify cel-
lular targets such as DNA, RNA, proteins, and metabolites. Moreover, SAM
itself is an expensive and unstable cofactor, degrading rapidly under physi-
ological or alkaline conditions. Thus, the availability of methods to prepare
SAM in situ is desirable.
Traditionally, SAM analogs have been chemically synthesized from
S-adenosyl-L-homocysteine. However, this synthesis suffers from low yields
and lack of diastereoselectivity (Dalhoff, Lukinavicius, Klimasauskas, &
Weinhold, 2006a, 2006b). Furthermore, SAM synthesis is challenging due
to its inherent lability and tendency to racemize (Hoffman, 1986). Chemo-
enzymatic synthesis using chlorinase SalL and ClDA offers an alternative to
chemical synthesis. In addition to being stable and commercially available,
ClDA can also be synthesized economically from adenosine and SOCl2
(Sinhababu, Bartel, Pochopin, & Borchardt, 1985). Chemoenzymatic syn-
thesis of SAM and analogs in situ could also be coupled with downstream
applications such as methylation or alkylation by methyltransferases. This
strategy can also be used for the facile incorporation of isotopes into
alkylated products.
We have demonstrated the use of SalL for the diastereoselective synthesis
of SAM and SAM congeners from ClDA and L-met analogs (see Section 3).
We have also coupled the SalL-catalyzed production of SAM with DNA and
with metabolite methylation (Lipson et al., 2013). Importantly, we have
demonstrated that in situ generated SAM analogs were recognized by rat
protein arginine methyltransferase 1 (rPRMT1) which alkylated its peptide
substrate (Thomsen et al., 2013).
In this chapter, we describe procedures for the preparation and assay of
SalL, the SalL-mediated chemoenzymatic synthesis of SAM analogs from

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
371
ClDA and L-met congeners, and the in situ chemoenzymatic synthesis of
SAM coupled to DNA, peptide, and metabolite methylation, and to the
incorporation of isotopes into alkylated products.
2. PREPARATION AND ASSAY OF CHLORINASE SalL
2.1 Purification of Recombinant SalL From Escherichia coli
Recombinant SalL containing a N-terminal His8 tag was obtained by gene
expression in and protein purification from E. coli BL21(DE3) (Eusta´quio
et al., 2008).
2.1.1 Cloning of the salL Gene Into an Expression Vector
The salL gene can be amplified by PCR using total genomic DNA isolated
from S. tropica CNB-440 as a template and appropriate primers. The follow-
ing are suggested primers that were used to clone salL into pHIS8 (Jez,
Ferrer, Bowman, Dixon, & Noel, 2000) by restriction/ligation, yielding
plasmid pAEM7 (Eusta´quio et al., 2008):
for: 50-CGTGGTTCCCATGGCATGCAGCACAATCTCATTGC-
30 (NcoI site underlined)
rev: 50-GCTCGAATTCAAGCTTGTCAGCTACCCGAGCACCG-
30 (HindIII site underlined)
The primers were designed to ensure that the ATG (Met start codon,
bold) was in-frame with the His8 tag. The priming sites are in italics. Bases
upstream of the restriction sites are random sequences to facilitate cutting of
the PCR product with the respective restriction enzyme. salL was amplified
by PCR using the Expand High Fidelity PCR system (Roche) following the
manufacturer’s instructions and using 5% DMSO (v/v) and annealing tem-
perature of 58°C. Alternative, proofreading DNA polymerases include Q5®
High Fidelity polymerase and Phusion® High Fidelity polymerase (New
England Biolabs). The expected size of the PCR product when using the
primers above is 888bp. Alternatively, synthetic DNA can be ordered from
GenScript. The accession code for the salL gene (Strop_1026) of S. tropica
CNB-440 is NC_009380.1 (position 1,162,700 to 1,163,551, comple-
ment). If using the primers above, digest the PCR product with NcoI and
HindIII and ligate it into the same sites of pHIS8 to yield the pHIS8-salL
expression vector (pAEM7). While the PCR product can be digested and
cloned directly into pHIS8, we subcloned the PCR product into the
pGEM-T Easy vector (Promega) by following the manufacturer’s

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instructions. The insert of the obtained plasmid (pAEM5) was sequenced
and confirmed to contain no mistakes. Following digestion of pAEM5 with
HindIII, NcoI, and DraI (DraI cuts only the vector backbone), and iso-
propanol precipitation, the HindIII–NcoI salL fragment (868bp) was cloned
into the same sites of pHIS8. pHIS8 contains an N-terminal, 8ꢀHis tag that
will facilitate protein purification. Note that the pAEM7 salL expression
plasmid used here contains native DNA from S. tropica rather than DNA
which is codon-optimized for expression in E. coli.
2.1.2 Expression of salL in E. coli BL21(DE3) and Purification of
Recombinant SalL Enzyme
2.1.2.1 Materials
•
•
•
•
•
•
•
Competent E. coli BL-21 (DE3) cells (Novagen)
salL expression vector (pAEM7)
Luria–Bertani (LB) agar (Becton Dickinson)
LB broth (Becton Dickinson)
Terrific broth (TB) medium (Becton Dickinson)
Ni2+-NTA resin (Qiagen)
Lysis buffer: 50mM sodium phosphate buffer (pH 8.0), 500mM NaCl,
20mM imidazole (pH 8.0), 20mM β-mercaptoethanol, 10% (v/v) glyc-
erol, and 1% (v/v) Tween-20, and 1mg/mL lysozyme
Wash buffer: 50mM sodium phosphate buffer (pH 8.0), 500mM NaCl,
20mM imidazole (pH 8.0), 20mM β-mercaptoethanol, and 10% (v/v)
glycerol
•
•
Elution buffer: 50mM sodium phosphate buffer (pH 8.0), 500mM
NaCl, 250mM imidazole (pH 8.0), 20mM β-mercaptoethanol, and
10% (v/v) glycerol
•
•
•
•
Storage buffer: 50mM sodium phosphate buffer (pH 7.9), 20% glycerol
Disposable polypropylene column (from Qiagen)
Disposable PD-10 desalting columns (Sephadex G-25, GE Healthcare)
SDS-PAGE (10% Bis-Tris NuPAGE gel with MOPS running buffer,
ThermoFisher)
2.1.2.2 Procedure
1. Competent E. coli BL-21 (DE3) cells are commercially available (e.g.,
Novagen, cat. No. 69450-3). Follow the manufacturer’s instructions to
introduce the salL-containing expression vector into E. coli BL-21
(DE3) by chemical transformation. Plate the transformation reaction

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
373
on LB agar containing 50mg/L kanamycin (pHIS8 contains a
kanamycin-resistance gene) and incubate the plates overnight at 37°C.
2. Inoculate a single colony onto 50mL LB containing 50mg/L
kanamycin (use a 250-mL flask) and incubate the flask overnight at
37°C, 200rpm.
3. Prepare cryo stocks for long-term storage by mixing the E. coli BL21
(DE3) culture containing the salL plasmid 1:1 with sterile glycerol
40%, giving a final glycerol concentration of 20%. Store cryo stocks
at ꢁ80°C.
4. Use the overnight LB culture to inoculate 1L of TB media containing
50mg/L kanamycin so that the starting A600 ¼0.1 (10–20mL of the
overnight E. coli seed culture are expected to be necessary). Incubate
the flask at 25°C, 225rpm until A600 ¼0.8. Once an A600 of 0.8 is
reached, induce cells with 0.25mM IPTG (final concentration) and
incubate overnight at 20°C, 225rpm. A 2.8-L Fernbach flask is
recommended for cultivation of 1L cultures.
5. Harvest cells by centrifugation (e.g., 15min at 5000ꢀg) and resuspend
the cell pellet in ꢂ20mL of cold lysis buffer. Incubate on ice for 30min.
6. Note: take care to keep the preparation cold by cooling centrifuges,
buffers, and keeping the sample on ice.
7. Lyse cells by sonication on ice (e.g., 2s on, 2s off for 3min total).
Alternatively, a French press can be used for cell lysis.
8. Centrifuge the lysate at high speed (>10,000ꢀg) for 30min at 4°C.
9. The clear lysate is then purified using Ni2+-NTA resin (Qiagen) and a
modified batch purification method as briefly described below (refer to
the manufacturer’s instructions “The QIAexpressionist” for more
details).
10. Transfer the clear lysate to a clean, autoclaved beaker on ice, and add
5mL Ni2+-NTA to the clear lysate. Mix gently using a magnetic stirrer
for 1h. Alternatively, use a rotary shaker for mixing the sample
contained in a conical tube.
11. If using a beaker for mixing, transfer the lysate/Ni-NTA suspension to
a conical tube and centrifuge at 3000ꢀg for 5min at 4°C to collect the
Ni-NTA resin.
12. Discard the supernatant and wash the resin twice with 25mL wash
buffer each as following. Add 25mL wash buffer to the Ni-NTA pellet,
resuspend gently, and centrifuge at 3000ꢀg for 5min at 4°C. Discard
supernatant and repeat.

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Tony D. Davis et al.
13. After the last wash, resuspend the Ni-NTA pellet in ꢂ5mL wash buffer
and transfer the resin to an empty polypropylene column with the bot-
tom outlet capped. Remove bottom cap and discard flow-through.
Wash once with 25mL cold wash buffer. Discard flow-through.
14. Elute the His8-tagged SalL with 2ꢀ 5mL elution buffer.
15. Assess the quality of the protein preparation using SDS-PAGE
(e.g., 10% Bis-Tris NuPAGE gel with MOPS running buffer, Thermo-
Fisher). The expected size of the His8-tagged protein is 32.4kDa
(Fig. 2).
16. The expected yield is >60mg recombinant, soluble SalL per liter of
culture. Protein yield can be calculated using the Bradford assay
(Bradford, 1976).
17. Desalting can be performed using PD-10 columns (Sephadex G-25):
cut the bottom tip off the column and let the storage liquid flow (dis-
card); equilibrate the column with 25mL storage buffer; load sample in
a total volume of 2.5mL per PD-10 column; discard the flow-through;
elute with 3.5mL storage buffer. Alternatively, dialysis can be used for
desalting.
2.2 SalL Activity Assay
The reaction catalyzed by chlorinase SalL is reversible. Chloride, bromide,
and iodide are accepted as the halide cosubstrates in the reaction with SAM
(forward reaction) to yield XDA. Kinetic studies demonstrated that the
Fig. 2 SDS-PAGE analysis of purified, recombinant SalL. 10% Bis-Tris NuPAGE gel with
MOPS running buffer (ThermoFisher). L, Fisherbrand EZ-Run protein marker; (1) soluble
protein before induction; (2) soluble protein after induction; and (3) His8-SalL purified by
Ni-NTA affinity chromatography.

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
375
reverse reaction (formation of SAM from XDA and L-met) is favored
in vitro, with the relative enzyme efficiency (kcat/Km) of the reverse reaction
being orders of magnitude higher than the forward reaction (Eusta´quio et al.,
2008). The activity of recombinant SalL can be investigated by a HPLC-
based method as described below.
2.2.1 Reagents
•
•
•
•
•
•
S-adenosyl-L-methionine (Sigma-Aldrich)
KCl (analytical grade)
L-Methionine (Sigma-Aldrich)
50-Chloro-50-deoxyadenosine (Sigma-Aldrich)
50mM sodium phosphate buffer pH 7.9
Recombinant SalL (Section 2.1)
2.2.2 Instrumentation and Solvents
•
•
•
•
•
Agilent HPLC 1200
C18 column (250ꢀ4.60mm, 5μm particle size)
Mobile phase A: 95% 50mM KH2PO4; 5% acetonitrile
Mobile phase B: 80% 50mM KH2PO4; 20% acetonitrile
Note: 50mM KH2PO4 gives pH ꢂ4.5; there is no need to adjust the pH.
2.2.3 Procedure
1. In order to test if your SalL preparation is active, set up two different
reactions by incubating SalL (400nM) with
(i) SAM (0.5mM) and KCl (400mM)
(ii) L-Methionine (10mM) and ClDA (0.5mM)
in 50mM sodium phosphate buffer (pH 7.9) at 37°C for 1h. In
addition, set up following controls: reactions with inactivated
enzyme (the enzyme is first boiled for 5min and then, the reagents
are added) and boiled enzyme only (the enzyme is boiled for 5min
in buffer).
2. Boil the reaction mixture for 2min at 95°C and centrifuge for 30min,
10,000ꢀg to remove precipitated protein. 10μL of clear supernatant
is then analyzed by HPLC using a C18 column (250ꢀ4.60mm, 5μm
particle size) and 1mL/min flow rate. After equilibrating the system
for at least 15min using 100% mobile phase A, run following gradient:
100% A for 1min, 0%–100% B for 29min, 100%–0% B over 5min,
100% A for 5min. Inject also SAM and ClDA standards for comparison.

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Tony D. Davis et al.
The retention times obtained under the conditions described were
16.4min (ClDA) and 18.1min (SAM).
3. Notes:
•
•
SAM is unstable and SAM solutions should be freshly prepared.
Recombinant SalL purified from E. coli copurifies with adenosine
and ClDA. Thus, the boiled enzyme negative control may have a
small peak corresponding to ClDA.
3. CHEMOENZYMATIC SYNTHESIS OF SAM ANALOGS
3.1 Overview of Chemoenzymatic Methods to Prepare
SAM and SAM Analogs
SAM is biosynthesized by methionine adenosyltransferases (MAT) from
methionine and ATP (Cantoni, 1975; Fig. 3). SAM is an essential cofactor
for SAM-dependent methyltransferases to methylate various substrates
including nucleic acids, proteins, lipids, and small molecules (Bauerle,
Schwalm, & Booker, 2015; Lin, 2011; Martin & McMillan, 2002;
Struck, Thompson, Wong, & Micklefield, 2012). While transmethylation
produces small and modest chemical changes, these drastically affect molec-
ular recognition of substrates by cellular targets to control processes such as
Fig. 3 Biosynthesis, utilization, and chemoenzymatic synthesis of S-adenosyl-L-
methionine (SAM) analogs. (A) Biosynthesis of SAM in vivo is catalyzed by
L-methionine-adenosyltransferases (MAT). Subsequently, nucleic acid, protein, or small
molecule substrates are methylated by SAM-dependent methyltransferases (MT). SAH,
S-adenosyl-L-homocysteine. (B) Chemoenzymatic synthesis of SAM analogs in vitro
using chlorinase SalL from Salinispora tropica. ClDA, 50-chloro-50-deoxyadenosine.

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
377
epigenetic regulation of protein expression, neurotransmission, and natural
products biosynthesis. The prevalence of SAM in cellular processes
has inspired the development of analogs as chemical reporters for nucleic
acid (Dalhoff et al., 2006a, 2006b; Lukinavicius et al., 2007; Motorin
et al., 2011; Vranken et al., 2016), protein (Guo et al., 2014; Islam et al.,
2012, 2013; Islam, Zheng, Yu, Deng, & Luo, 2011; Peters et al., 2010;
Wang et al., 2013; Wang & Luo, 2013; Wang, Zheng, Yu, Deng, &
Luo, 2011), small molecule (Lee et al., 2010; Singh et al., 2014; Stecher
et al., 2009; Thomsen et al., 2013; Zhang, Weller, Thorson, & Rajski,
2006), and natural products methyltransferases (Winter et al., 2013;
Zhang et al., 2006).
The preparation of SAM and its analogs is especially challenging due to
inherent lability, tendency to racemize under experimental conditions, and
rapid degradation at physiological pH (Hoffman, 1986). Chemical synthesis
of SAM and SAM derivatives involves nucleophilic substitution between
S-adenosyl-L-homocysteine (SAH or AdoCy) and an alkyl halide to pro-
duce a mixture of two sulfonium diastereomers, usually in variable yields
(Dalhoff et al., 2006a, 2006b; de la Haba, Jamieson, Mudd, & Richards,
1959; Stecher et al., 2009). The S,S diastereomer serves as the methyl donor,
while the S,R diastereomer inhibits the methyltransferase (Borchardt & Wu,
1976; Khani-Oskouee, Jones, & Woodard, 1984). Additionally, analogs of
SAM where sulfur is replaced with selenium are more reactive methyl
donors due to the longer, weaker Se–C bonds and do not racemize
(Iwig & Booker, 2004). Notably, select Se-adenosyl-L-Selenomethionines
(SeAM), i.e., propargyl SeAM, are more stable than their SAM analogs
(Bothwell et al., 2012; Bothwell & Luo, 2014). Overall, these early synthetic
efforts ignited investigations in which nonnatural SAM analogs could be
used by endogenous methyltransferases to modify and profile various
cellular targets.
To complement chemical synthesis of SAM analogs, we pioneered
chemoenzymatic methods to expand the range of SAM analogs available
for labeling methyltransferase substrates (Lipson et al., 2013; Thomsen
et al., 2013; Vranken et al., 2016). We employed two homologous
halogenases, SalL and FlA, to produce SAM and SAM analogs. As discussed
earlier, SalL catalyzes the reaction of SAM with chloride to produce ClDA
and L-methionine via nucleophilic attack in S. tropica (Fig. 1). Similarly,
S. cattleya uses fluorinase FlA to react fluoride with SAM to afford FDA
and L-methionine. Based on Le Chatelier’s Principle, we hypothesized that

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Tony D. Davis et al.
under low salt concentrations and excess L-methionine, these halogenases
could catalyze the biologically reversed reaction to produce SAM in situ
(Fig. 3) from easily prepared ClDA. Indeed, we observed SAM production
by both SalL and FlA in various buffers, including Tris, HEPES, and phos-
phate buffers at pH 7–8, although SalL produced SAM in substantially
higher yields than FlA. Furthermore, we generated a W190A point mutant
of SalL that could accommodate larger alkyl substrates (Thomsen et al.,
2013). Both wild type (WT) and mutant SalL generated alkyl, allylic, and
benzylic SAM analogs, although the WT was most effective at generating
small SAM analogs (methyl, ethyl), whereas both the WT and W190A
mutant were equally effective at producing larger SAM analogs (propyl,
butyl, allyl, and benzyl). Importantly, we demonstrated that our in situ-
generated SAM congeners were recognized by nucleic acid, peptide, and
small molecule methyltransferases and alkylated their respective substrates
(Thomsen et al., 2013).
3.2 SalL-Mediated Chemoenzymatic Synthesis of SAM
and SAM Analogs
3.2.1 Reagents
•
•
•
•
•
•
DMSO (Sigma-Aldrich)
HPLC grade acetonitrile (Fisher Scientific)
50-chloro-50-deoxyadenosine (Sigma-Aldrich)
L-methionine (Sigma-Aldrich)
S-ethyl-L-homocysteine (Sigma-Aldrich)
S-propyl-L-homocysteine (prepared as described by Thomsen et al.,
2013)
•
•
•
S-butyl-L-homocysteine (Toronto Research Chemicals, Inc.)
S-allyl-L-homocysteine (prepared as described by Thomsen et al., 2013)
S-propargyl-L-homocysteine (prepared as described by Thomsen et al.,
2013)
•
•
S-benzyl-L-homocysteine (prepared as described by Thomsen et al.,
2013)
S-phenylethyl-L-homocysteine (prepared as described by Thomsen
et al., 2013)
•
•
S-(2-amino-2-carboxylethyl)-L-homocysteine (Sigma-Aldrich)
S-(2-amino-2-oxoethyl)-L-homocysteine (prepared as described by
Thomsen et al., 2013)
•
S-adenosyl-L-methionine (New England Biolabs)

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
379
3.2.2 Enzyme
•
SalL (expressed and purified as described in Section 2.1.2)
3.2.3 Stock Solutions and Buffers
•
•
•
•
•
•
•
300mM ClDA in DMSO
150mM L-methionine in 250mM NaOH
15–150mM L-methionine analog in 250mM NaOH
32mM SAM in 5mM H2SO4 and 10% ethanol
50mM sodium phosphate buffer, pH 6.8
400mM sodium formate, pH 3.0
50mM HEPES, pH 7.3
3.2.4 Analytical Instrumentation
•
•
•
Agilent 6230 ESI-TOF LC-MS
Agilent HPLC 1200
Agilent Poroshell 300SB (5μm, 2.1ꢀ75mm) column
3.2.5 Procedure
1. Equilibrate 200μM ClDA and 3μM SalL in 50mM phosphate buffer, pH
6.8 at 37°C.
2. Add 1.5–15mM L-methionine or analog from stock. Due to the poor
solubility of the benzyl and phenylethyl analogs of L-methionine, these
were added at a final concentration of 1.5mM from a 15mM stock
solution in 250mM NaOH. L-Methionine and all other analogs were
added at a final concentration of 15mM from a 150mM stock solution
in 250mM NaOH. In all assays, the addition of L-methionine or analog
was 10% of the total reaction volume, resulting in a final pH of 7.4.
3. Quench reactions at various time points (0–5h) by the addition of equal
volumes of 400mM sodium formate. Store quenched aliquots at 4°C
until HPLC analysis.
4. Analyze aliquots using analytical reversed phase HPLC using a Synergi™
Hydro RP column (4μm, 4.6ꢀ150mm, Phenomenex) using the
following gradient:
Buffer A: 10mM NH4HCO2, pH 3.2
Buffer B: 90% CH3CN, 10mM NH4HCO2, pH 3.2
0min: 100% buffer A, 1mL/min
5min: 98% buffer A, 1.5mL/min
10min: 70% buffer A, 2mL/min
12min: 0% buffer A, 2mL/min

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Tony D. Davis et al.
Table 1 LC-MS Retention Times and Chromatographic Analyses of Select SAM Analogs
Analog
Retention Time
Observed Monoisotopic Mass
SAM
4min
399.1555
Ethyl-SAM
Propyl-SAM
Butyl-SAM
Allyl-SAM
Benzyl-SAM
aMixture of diastereomers.
5min
413.1671
8.5, 8.9mina
427.1784
9.5min
441.2366
7.2, 7.9mina
10.4min
425.1862
475.2242
Note: Complete conversion of ClDA (retention time¼11min) into
SAM (retention time¼4min) was observed within 30min.
5. Verify the identity of ClDA and SAM by collecting 0.1mL fractions
from the HPLC and analyze by LC-MS. Table 1 provides a summary
of LC-MS data for select SAM analogs. See Thomsen et al. (2013) for
representative LC-MS traces and chromatograms.
3.3 In Situ Chemoenzymatic Synthesis of SAM Coupled
to DNA Methylation by HhaI
HhaI is a DNA methyltransferase that methylates cytosine at C5 (Sankpal &
Rao, 2002). In this assay, HhaI is coupled with in situ SAM production with
methylation of lambda DNA.
3.3.1 Reagents
•
N6-methyladenine-free lambda DNA (New England Biolabs)
3.3.2 Enzymes
•
•
•
SalL (expressed and purified as described in Section 2.1.2)
HhaI methyltransferase (New England Biolabs)
0.2U/μL HinP1I restriction enzyme (New England Biolabs)
3.3.3 Stock Solutions and Buffers
•
•
•
300mM ClDA in DMSO
150mM L-methionine in 250mM NaOH
HhaI buffer (5mM 2-mercaptoethanol, 10mM EDTA, 50mM Tris–
HCl, pH 7.5)

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
381
•
NEB4 buffer (10mM KOAc, 4mM Tris–HOAc, 2mM MgOAc2,
0.2mM dithiothreitol (DTT), pH 7.9)
3.3.4 Procedure
1. Equilibrate 50ng/μL N6-methyladenine-free lambda DNA, 4U/μL
HhaI, 200μM ClDA, and 15mM L-methionine in HhaI buffer at
37°C.
Note: The buffers used for DNA methyltransferase HhaI and SalL
activity assays were incompatible. HhaI was inactive in 50mM sodium
phosphate buffer, pH 7.9; however, the activity of SalL was reduced
by 30% in HhaI buffer. Thus, coupled assays were conducted in HhaI
buffer.
2. Add SalL to a final concentration of 900nM.
3. After 1h, quench reaction by heating at 80°C for 5min.
4. Dilute reaction in an equal volume of HinP1I in 2ꢀ NEB4 buffer to
afford a final concentration of 0.1U/μL HinP1I.
5. Incubate 1h at 37°C.
6. Analyze lambda DNA cleavage by gel electrophoresis on 2% agarose gel.
3.4 In Situ Chemoenzymatic Synthesis of SAM Coupled
to Teicoplanin Methylation by MtfA
MtfA is a small molecule methyltransferase from the chloroeremomycin
biosynthetic pathway in Amycolatopsis orientalis that catalyzes N-methylation
of vancomycin-like glycopeptides (Shi et al., 2009). In this assay, MtfA is
used to couple in situ SAM production with methylation of teicoplanin.
3.4.1 Reagents
•
Teicoplanin (TargetMol)
3.4.2 Enzymes
•
•
SalL (expressed and purified as described in Section 2.1.2)
MtfA methyltransferase (expressed and purified as described below)
3.4.3 Stock Solutions and Buffers
•
300mM ClDA in DMSO
150mM L-methionine (or [methyl-13C]-L-methionine) in 250mM
•
NaOH
•
50mM HEPES, pH 7.3

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Tony D. Davis et al.
3.4.4 Expression and Purification of MtfA Methyltransferase
One liter of 2ꢀ YT medium (Sigma-Aldrich) supplemented with
50μg/mL kanamycin was inoculated with E. coli BL21 (DE3) cells trans-
formed with mtfA/pET28a plasmid (obtained from Prof. Gerard D. Wright,
McMaster University). The cells were cultured at 37°C to an OD600 of 0.6.
The cells were cooled to 0°C on ice, induced with 100μM isopropyl β-D-1-
thiogalactopyranoside (IPTG), and grown for 20h at 16°C. Cells were
harvested via centrifugation at 2000ꢀg, 30min, 4°C, and the pellet was
resuspended in lysis buffer (50mM HEPES, 500mM NaCl, 20mM
imidazole, and 1mM DTT, pH 7.5) and lysed by passage through a French
pressure cell. The lysate was clarified by centrifugation at 4150ꢀg, 1.5h,
4°C. His6-MtfA was purified using 2mL Ni-NTA resin (GE Healthcare)
that was equilibrated with lysis buffer. The column was washed with 40mL
of buffer A (50mM HEPES, 1M NaCl, 20mM imidazole, 1mM DTT, pH
7.5), followed by 40mL of buffer B (50mM HEPES, 400mM NaCl,
30mM imidazole, 1mM DTT, pH 7.5). The protein was eluted with
buffer C (50mM HEPES, 400mM NaCl, 200mM imidazole, 1mM
DTT, pH 7.5). We obtained around 50mg of MtfA/L of cell culture.
Eluted proteins were visualized with 12% SDS-PAGE with Coomassie
Brilliant Blue staining. Imidazole was removed using spin concentration
into 20mM HEPES, 20mM NaCl, and 5mM DTT, pH 7.5. Pure
protein was stored at ꢁ20°C in 10mM HEPES, 10mM NaCl, 2.5mM
DTT, pH 7.5 containing 50% glycerol. The protein is stable as a glycerol
stock at ꢁ20°C.
3.4.5 Procedure
1. Equilibrate a 50μL mixture of 20mM ClDA, 50mM L-methionine, and
1.4μM SalL in 32mM phosphate buffer, pH 7.9 at 37°C.
2. After 1–3h, add 250μL of a solution containing: 1mM teicoplanin,
1mg/mL BSA, and 54μM MtfA in 50mM HEPES, pH 7.3.
3. Monitor the methylation of teicoplanin by removing aliquots at various
times and analyzing by LC-MS using a C18 column (3μm,
2.1ꢀ150mm, Atlantis) and an isocratic mixture of 3:1 H2O/CH3CN
(v/v) with 0.1% formic acid. Under these conditions, the retention times
of teicoplanin and N-methyl-teicoplanin were 9 and 9.5min,
respectively.

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
383
3.5 In Situ Chemoenzymatic Synthesis of 13C-Labeled SAM
Coupled With Isotope Incorporation Into Teicoplanin via
Methylation by MtfA
We envisioned SalL-mediated in situ chemoenzymatic synthesis of SAM as a
compelling and facile way to incorporate isotopes into biomolecules. Here,
we describe the use of this technology to incorporate 13C into teicoplanin
for sensitive NMR identification (Lipson et al., 2013).
3.5.1 Reagents
•
d6-DMSO (Cambridge Isotope Laboratories, Item No. DLM-10-10)
[Methyl-13C]-L-methionine (Cambridge Isotope Laboratories, Item No.
CLM-206-PK)
•
•
Teicoplanin (TargetMol)
3.5.2 Enzymes
•
SalL (expressed and purified as described in Section 2.1.2)
MtfA methyltransferase (expressed and purified as described in
Section 3.4.4)
•
3.5.3 Stock Solutions and Buffers
•
•
•
300mM ClDA in d6-DMSO
300mM [methyl-13C]-L-methionine in d6-DMSO
10mM phosphate buffer, pH 7.5
3.5.4 Analytical Instrumentation
Varian VX500 NMR spectrometer equipped with an X-Sens cold probe
•
3.5.5 Procedure
1. Prepare 800μL of a mixture of 350nM SalL and 350nM MtfA in 10mM
phosphate buffer, pH 7.5.
2. Prepare 200μL of a mixture of 7mM teicoplanin, 142mM [methyl-13C]-
L-methionine, and 14.2mM ClDA in 10mM phosphate buffer, pH 7.5
containing 50% d6-DMSO.
3. Transfer the mixture of teicoplanin, [methyl-13C]-L-methionine, and
ClDA mixture to the mixture of SalL and MtfA.
4. Transfer the entire solution to a clean 5mm NMR tube.

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Tony D. Davis et al.
5. Collect 13C-NMR spectra on a Varian VX500 NMR spectrometer
at various time points. For each time point, we acquired data for ꢃ64
scanswith a delaytime (d1)of5s. In our hands, weobservedtheappearance
of [methyl-13C]-N-teicoplaninwithin3–9h, asevidenced bya 13C-NMR
signal at δ¼33.7ppm.
3.6 In Situ Chemoenzymatic Synthesis of SAM Coupled to RGG
Peptide Methylation by rPRMT1
Protein arginine methyltransferase 1 (PRMT1) is responsible for the major-
ity of arginine methylation in mammalian cells. In the assay described
below, rPRMT1, a rat homolog that dimethylates arginine residues, is used
to couple in situ SAM production with methylation of arginine residues
in the RGG peptide. Transfer of ethyl, allyl, and benzyl congeners of
L-methionine has also been observed (Thomsen et al., 2013). Overall, sterics
likely influences transfer since we observed RGG allylation, but did not
observe RGG propylation, although the corresponding SAM analogs have
similar sizes and are formed in situ at similar rates. RGG allylation most likely
occurs via an SN20 mechanism.
3.6.1 Reagents
•
RGG peptide (expressed and purified as described by Thomsen et al.,
2013)
3.6.2 Enzymes
•
SalL (expressed and purified as described in Section 2.1.2)
•
rPRMT1 (expressed and purified as described by Thomsen et al., 2013)
3.6.3 Stock Solutions and Buffers
•
•
•
•
300mM ClDA in DMSO
150mM L-methionine in 250mM NaOH
400mM sodium formate, pH 3.0
50mM phosphate buffer, pH 7.4
3.6.4 Procedure Peptide Methylation Using rPRMT1
1. Equilibrate a mixture containing 200μM ClDA, 15mM L-methionine,
1μM SalL, 10μM RGG peptide, and 2μM rPRMT1 in 50mM phos-
phate buffer, pH 7.4 at 37°C.
2. After 12h, quench reaction by the addition of an equal volume of
400mM sodium formate.
3. Assess the formation of methylated RGG peptide using LC-MS.

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
385
4. SUMMARY AND CONCLUSIONS
We described procedures for the chemoenzymatic synthesis of SAM
and analogs that employ chlorinase SalL discovered from a marine actino-
mycete bacterium. Recombinant SalL can be purified from E. coli in rela-
tively high yields, i.e., over 60mg soluble enzyme per liter culture. The
SalL-catalyzed synthesis of SAM analogs from L-methionine congeners
and ClDA is diastereoselective. Moreover, we described methods for in situ
synthesis of SAM coupled with DNA, protein, and metabolite methylation,
and with the incorporation of isotopes into biomolecules.
In addition to SAM-dependent halogenases, numerous groups have used
methionine adenosyltransferase wild type and mutants that accommodate
structural changes in L-methionine and ATP to generate stable SAM analogs
that alkylate a variety of acceptor substrates (Huber, Johnson, Zhang, &
Thorson, 2016; Huber et al., 2016; Singh et al., 2014; Wang et al.,
2013). Overall, the development of chemoenzymatic methods and protein
engineering efforts has expanded the structural scope of SAM analogs that
can be generated for biological studies.
ACKNOWLEDGMENTS
Financial support for this work was provided by the National Center for Advancing
Translational Sciences, National Institutes of Health (NIH), under Grant KL2TR002002
(to A.S.E.), NIH GM095970 (to M.D.B), and startup funds from the Department of
Medicinal Chemistry and Pharmacognosy and the Center for Biomolecular Sciences,
University of Illinois at Chicago (to A.S.E.). T.D.D. was supported by the NIH/NIGMS
IRACDA K12 GM068524 award. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the NIH. Plasmid pAEM7 for the
expression of salL was constructed in Prof. Bradley S. Moore’s laboratory (Scripps
Institution of Oceanography). The authors also graciously thank Prof. Joseph P. Noel
(Salk Institute for Biological Studies) for vector pHIS8, Prof. Gerard D. Wright
(McMaster University) for the mtfA gene construct, Dr. Yongxuan Su (UC San Diego)
for mass spectral analyses, and Drs. Anthony Mrse and Xuemei Huang (UC San Diego)
for assistance with acquiring NMR spectral data.
REFERENCES
Agarwal, V., Miles, Z. D., Winter, J. M., Eusta´quio, A. S., El Gamal, A. A., & Moore, B. S.
(2017). Enzymatic halogenation and dehalogenation reactions: Pervasive and mechanis-
tically diverse. Chemical Reviews, 117, 5619–5674.
Bauerle, M. R., Schwalm, E. L., & Booker, S. J. (2015). Mechanistic diversity of radical
S-adenosylmethionine (SAM)-dependent methylation. Journal of Biological Chemistry,
290, 3995–4002.
Borchardt, R. T.,
S-adenosylmethionine-dependent methyltransferases V: Role of the asymmetric
&
Wu, Y. S. (1976). Potential inhibitors of

386
Tony D. Davis et al.
sulfonium pole in the enzymatic binding of S-adenosyl-L-methionine. Journal of Medicinal
Chemistry, 19, 1099–1103.
Bothwell, I. R., Islam, K., Chen, Y., Zheng, W., Blum, G., Deng, H., et al. (2012).
Se-adeonsyl-L-selenomethionine cofactor analogue as a reporter of protein methylation.
Journal of the American Chemical Society, 134, 14905–14912.
Bothwell, I. R., & Luo, M. (2014). Large-scale, protection free synthesis of Se-adenosyl-L-
selenomethionine analogues and their application as cofactor surrogates of met-
hyltransferases. Organic Letters, 16, 3056–3059.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemis-
try, 72, 248–254.
Cantoni, G. L. (1975). Biological methylation: Selected aspects. Annual Review of Biochemis-
try, 44, 435–451.
Chan, K. K., & O’Hagan, D. (2012). The rare fluorinated natural products and biotechno-
logical prospects for fluorine enzymology. Methods in Enzymology, 516, 219–235.
Dalhoff, C., Lukinavicius, G., Klimasauskas, S., & Weinhold, E. (2006a). Direct transfer of
extended groups from synthetic cofactors by DNA methyltransferases. Nature Chemical
Biology, 2, 31–32.
Dalhoff, C., Lukinavicius, G., Klimasauskas, S., & Weinhold, E. (2006b). Synthesis of
S-adenosyl-L-methionine analogs and their use for sequence-specific transalkylation of
DNA by methyltransferases. Nature Protocols, 1, 1879–1886.
de la Haba, G., Jamieson, G. A., Mudd, S. H., & Richards, H. H. (1959).
S-adenosylmethionine: The relation of configuration at the sulfonium center to enzy-
matic reactivity. Journal of the American Chemical Society, 81, 3975–3980.
Deng, H., Cobb, S. L., McEwan, A. R., McGlinchey, R. P., Naismith, J. H., O’Hagan, D.,
et al. (2006). The fluorinase from Streptomyces cattleya is also a chlorinase. Angewandte
Chemie, 45, 759–762.
Deng, H., Ma, L., Bandaranayaka, N., Qin, Z., Mann, G., Kyeremeh, K., et al. (2014).
Identification of fluorinases from Streptomyces sp. MA37, Norcardia brasiliensis, and
Actinoplanes sp. N902-109 by genome mining. Chembiochem, 15, 364–368.
Dong, C., Huang, F., Deng, H., Schaffrath, C., Spencer, J. B., O’Hagan, D., et al. (2004).
Crystal structure and mechanism of a bacterial fluorinating enzyme. Nature, 427,
561–565.
Eusta´quio, A. S., McGlinchey, R. P., Liu, Y., Hazzard, C., Beer, L. L., Florova, G., et al.
(2009). Biosynthesis of the salinosporamide
A polyketide synthase substrate
chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine. Proceedings of the
National Academy of Sciences of the United States of America, 106, 12295–12300.
Eusta´quio, A. S., Pojer, F., Noel, J. P., & Moore, B. S. (2008). Discovery and characteri-
zation of a marine bacterial SAM-dependent chlorinase. Nature Chemical Biology, 4,
66–74.
Feling, R. H., Buchanan, G. O., Mincer, T. J., Kauffman, C. A., Jensen, P. R., & Fenical, W.
(2003). Salinosporamide A: A highly cytotoxic proteasome inhibitor from a novel
microbial source, a marine bacterium of the new genus Salinospora. Angewandte Chemie,
42, 355–357.
Fontecave, M., Atta, M., & Mulliez, E. (2004). S-adenosylmethionine: Nothing goes to
waste. Trends in Biochemical Sciences, 29, 243–249.
Gribble, G. W. (2012). Recently discovered naturally occurring heterocyclic organohalogen
compounds. Heterocycles, 84, 157–207.
Guo, H., Wang, R., Zheng, W., Chen, Y., Blum, G., Deng, H., et al. (2014). Profiling
substrates of protein arginine N-methyltransferase 3 with S-adenosyl-L-methionine
analogues. ACS Chemical Biology, 9, 476–484.
Hoffman, J. L. (1986). Chromatographic analysis of the chiral and covalent instability of
S-adenosyl-L-methionine. Biochemistry, 25, 4444–4449.

Chemoenzymatic Synthesis of SAM and Analogs Using SalL
387
Huang, S., Ma, L., Tong, M. H., Yu, Y., O’Hagan, D., & Deng, H. (2014). Fluoroacetate
biosynthesis from the marine-derived bacterium Streptomyces xinghaiensis NRRL
B-24674. Organic and Biomolecular Chemistry, 12, 4828–4831.
Huber, T. D., Johnson, B. R., Zhang, J., & Thorson, J. S. (2016). AdoMet analog
synthesis and utilization: Current state of the art. Current Opinion in Biotechnology, 42,
189–197.
Huber, T. D., Wang, F., Singh, S., Johnson, B. R., Zhang, J., Sunkara, M., et al. (2016).
Functional AdoMet isosteres resistant to classical AdoMet degradation pathways. ACS
Chemical Biology, 11, 2484–2491.
Islam, K., Bothwell, I., Chen, Y., Sengelaub, C., Wang, R., Deng, H., et al. (2012).
Bioorthogonal profiling of protein methylation using azido derivative of S-adenosyl-
L-methionine. Journal of the American Chemical Society, 134, 5909–5915.
Islam, K., Chen, Y., Wu, H., Bothwell, I. R., Blum, G. J., Zeng, H., et al. (2013). Defining
efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation.
Proceedings of the National Academy of Sciences of the United States of America, 110,
16778–16783.
Islam, K., Zheng, W., Yu, H., Deng, H., & Luo, M. (2011). Expanding cofactor repertoire of
protein lysine methyltransferase for substrate labeling. ACS Chemical Biololgy, 6,
679–684.
Iwig, D. F., & Booker, S. J. (2004). Insight into the polar ractivity of the onium chalcogen
analogues of S-adenosyl-L-methionine. Biochemistry, 43, 13496–13509.
Jez, J. M., Ferrer, J. L., Bowman, M. E., Dixon, R. A., & Noel, J. P. (2000). Dissection of
malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mech-
anism of a plate polyketide synthase. Biochemistry, 39, 890–902.
Khani-Oskouee, S., Jones, J. P., & Woodard, R. W. (1984). Stereochemical course of the
biosynthesis of L-aminocyclopropane-1-carboxylic acid. I. Role of the asymmetric
sulfonium pole and the α-amino acid center. Biochemical and Biophysical Research
Communications, 121, 181–187.
Lee, B. W., Sun, H. G., Zang, T., Kim, B. J., Alfaro, J. F., & Zhou, Z. S. (2010). Enzyme-
catalyzed transfer of a ketone group from an S-adenosylmethionine analogue: A tool
for the functional analysis of methyltransferases. Journal of the American Chemical Society,
132, 3642–3643.
Lin, H. (2011). S-adenosylmethionine-dependent alkylation reactions: When are radical
reactions used? Bioorganic Chemistry, 39, 161–170.
Lipson, J. M., Thomsen, M., Moore, B. S., Clausen, R. P., La Clair, J. J., & Burkart, M. D.
(2013).
A tandem chemoenzymatic methylation of S-adenosyl-L-methionine.
Chembiochem, 14, 950–953.
Lukinavicius, G., Lapiene, V., Stasevskij, Z., Dalhoff, C., Weinhold, E., & Klimasauskas, S.
(2007). Targeted labeling of DNA by methyltransferase-directed transfer of activated
groups (mTAG). Journal of the American Chemical Society, 129, 2758–2759.
Martin, J. L.,
& McMillan, F. M. (2002). SAM (dependent) I AM: The
S-adenosylmethionine-dependent methyltransferase fold. Current Opinion in Structural
Biology, 12, 783–793.
Motorin, Y., Burhenne, J., Teimer, R., Koynov, K., Willnow, S., Weinhold, E., et al.
(2011). Expanding the chemical scope of RNA: Methyltransferases to site-specific
alkynylation of RNA for click labeling. Nucleic Acids Research, 39, 1943–1952.
Ohashi, M., Liu, F., Hai, Y., Chen, M., Tang, M. C., Yang, Z., et al. (2017). SAM-
dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis.
Nature, 549, 502–506.
Peters, W., Willnow, S., Duisken, M., Kleine, H., Macherey, T., Duncan, K. E., et al.
(2010). Enzymatic site-specific functionalization of protein methyltransferase substrates
with alkynes for click labeling. Angewandte Chemie International Edition England, 49,
5170–5173.

388
Tony D. Davis et al.
Sankpal, U. T., & Rao, D. N. (2002). Structure, function, and mechanism of HhaI DNA
methyltransferases. Critical Reviews in Biochemistry and Molecular Biology, 37, 167–197.
Shi, R., Lamb, S. S., Zakeri, B., Proteau, A., Cui, Q., Sulean, T., et al. (2009). Structure and
function of the glycopeptide N-methyltransferase MtfA, a tool for the biosynthesis of
modified glycopeptide antibiotics. Chemistry & Biology, 16, 401–410.
Singh, S., Zhang, J., Huber, T. D., Sunkara, M., Hurley, K., Goff, R. D., et al. (2014). Facile
chemoenzymatic strategies for the synthesis and utilization of S-adenosyl-L-methionine
analogues. Angewandte Chemie International Edition, 53, 3965–3969.
Sinhababu, A. K., Bartel, R. L., Pochopin, N., & Borchardt, R. T. (1985). Mechanism of
action of S-adenosyl-L-homocysteine hydrolase. Measurement of kinetic isotope effects
using adenosine-30-d and S-adenosyl-L-homocysteine-30-d as substrates. Journal of
American Chemical Society, 107, 7628–7632.
Stecher, H., Tengg, M., Ueberbacher, B. J., Remler, P., Schwab, H., Griengl, H., et al.
(2009). Biocatalytic Friedel-Crafts alkylation using non-natural cofactors. Angewandte
Chemie International Edition England, 48, 9546–9548.
Struck, A. W., Thompson, M. L., Wong, L. S.,
& Micklefield, J. (2012).
S-adenosyl-methionine-dependent methyltransferases: Highly versatile enzymes in
biocatalysis, biosynthesis and other biotechnological applications. Chembiochem, 13,
2642–2655.
Thomsen, M., Vogensen, S. B., Buchardt, J., Burkart, M. D., & Clausen, R. P. (2013).
Chemoenzymatic synthesis and in situ application of S-adenosyl-L-methionine analogs.
Organic and Biomolecular Chemistry, 11, 7606–7610.
Vranken, C., Fin, A., Tufar, P., Hofkens, J., Burkart, M. D., & Tor, Y. (2016).
Chemoenzymatic synthesis and utilization of a SAM analog with an isomorphic
nucleobase. Organic and Biomolecular Chemistry, 14, 6189–6192.
Wang, R., Islam, K., Liu, Y., Zheng, W., Tang, H., Lailler, N., et al. (2013). Profiling
genome-wide chromatin methylation with engineered posttranslation apparatus within
living cells. Journal of the American Chemical Society, 135, 1048–1056.
Wang, R., & Luo, M. (2013). A journey toward bioorthogonal profiling of protein meth-
ylation inside living cells. Current Opinion in Chemical Biology, 17, 729–737.
Wang, R., Zheng, W., Yu, H., Deng, H., & Luo, M. (2011). Labeling substrates of protein
arginine methyltransferase with engineered enzymes and matched S-adenosyl-L-
methionine analogues. Journal of the American Chemical Society, 133, 7648–7651.
Wang, L., Zhou, X., Fredimoses, M., Liao, S., & Liu, Y. (2014). Naturally occurring
organoiodines. RSC Advances, 4, 57350–57376.
Winter, J. M., Chiou, G., Bothwell, I. R., Xu, W., Garg, N. K., Luo, M., et al. (2013).
Expanding the structural diversity of polyketides by exploring the cofactor tolerance
of an inline methyltransferase domain. Organic Letters, 15, 3774–3777.
Zhang, C., Weller, R. L., Thorson, J. S., & Rajski, S. R. (2006). Natural product diversi-
fication using a non-natural cofactor analogue of S-adenosyl-L-methionine. Journal of the
American Chemical Society, 128, 2760–2761.