Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl- l -methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration

Article abstract of DOI:10.1021/jacs.7b09890

Many cobalamin (Cbl)-dependent radical S-adenosyl-l-methionine (SAM) methyltransferases have been identified through sequence alignment and/or genetic analysis; however, few have been studied in vitro. GenK is one such enzyme that catalyzes methylation of the 6′-carbon of gentamicin X₂ (GenX₂) to produce G418 during the biosynthesis of gentamicins. Reported herein, several alternative substrates and fluorinated substrate analogs were prepared to investigate the mechanism of methyl transfer from Cbl to the substrate as well as the substrate specificity of GenK. Experiments with deuterated substrates are also shown here to demonstrate that the 6′-pro-R-hydrogen atom of GenX₂ is stereoselectively abstracted by the 5′-dAdo· radical and that methylation occurs with retention of configuration at C6′. Based on these observations, a model of GenK catalysis is proposed wherein free rotation of the radical-bearing carbon is prevented and the radical SAM machinery sits adjacent rather than opposite to the Me-Cbl cofactor with respect to the substrate in the enzyme active site.

Full text of DOI:10.1021/jacs.7b09890

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Communication
Reaction Catalyzed by GenK, a Cobalamin-Dependent
Radical SAM Methyltransferase in the Biosynthetic Pathway
of Gentamicin, Proceeds with Retention of Configuration
Hak Joong Kim, Yung-nan Liu, Reid M. McCarty, and Hung-wen Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09890 • Publication Date (Web): 01 Nov 2017
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Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical SAM
Methyltransferase in the Biosynthetic Pathway of Gentamicin, Pro-
ceeds with Retention of Configuration
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Hak Joong Kim, Yung-nan Liu, Reid M. McCarty, and Hung-wen Liu*
Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and Department of Chemistry, University of
Texas at Austin, Austin, Texas 78712, United States
Supporting Information Placeholder
4) at the C6′ position to yield G418 (5)5 during the biosyn-
ABSTRACT: Many cobalamin (Cbl)-dependent radical S-
adenosyl-L-methionine (SAM) methyltransferases have
been identified through sequence alignment and/or genetic
analysis; however, few have been studied in vitro. GenK is
thesis of gentamicin C1 (6) and C2 (Scheme 1), which are
the major and most potent gentamicins.5 The GenK-
catalyzed reaction is thus important for the production of
this class of clinically useful aminoglycoside antibiotics.6
one such enzyme that catalyzes methylation of the 6′-
H
H
OH
O
OH
O
NHMe
O
6'
OH
O
Me
HO
HO
Me
HO
HO
carbon of gentamicin X2 (GenX2) to produce G418 during
the biosynthesis of gentamicins. Reported herein, several
alternative substrates and fluorinated substrate analogs
were prepared to investigate the mechanism of methyl
transfer from Cbl to the substrate as well as the substrate
specificity of GenK. Experiments with deuterated sub-
strates are also shown here to demonstrate that the 6′-pro-
R-hydrogen atom of GenX2 is stereoselectively abstracted
by the 5′-dAdo• radical and that methylation occurs with
retention of configuration at C6′. Based on these observa-
tions, a model of GenK catalysis is proposed wherein free
rotation of the radical-bearing carbon is prevented and the
radical SAM machinery sits adjacent rather than opposite
to the Me-Cbl cofactor with respect to the substrate in the
enzyme active site.
6'
HO
HO
HO
HO
NH2
NH2
NH2
NH2
NH2
H2N
H2N
H2N
O
H2N
O
O
O
HO
NH2
HO
NH2
NH2
HO
HO
O
OH
O
OH
O
OH
O
OH
O
GenK
O
O
GenD1
O
4"
HO
HO
HO
NHMe
Me
NHMe
NHMe
NHMe
Me
HO
gentamicin A (3)
Me
gentamicin X2 (4)
G418 (5)
gentamicin C1 (6)
Scheme 1. C6′ methylation reaction catalyzed by GenK
in the gentamicin biosynthetic pathway.
To probe the ability of GenK to accept different sub-
strates, paromamine (7), neamine (8), and five pseudotri-
saccharides including gentamicin A (3), A2 (9) and B (10),
along with JI-20A (11) and 2′-deamino-2′-hydroxy-GenX2
(12) were synthesized as described in the Supporting In-
formation and tested for their competence as GenK sub-
strates (Figure 1). These compounds were separately incu-
bated with GenK in 50 mM Tris•HCl buffer (pH 8.0) in the
presence of 10 mM DTT, 4 mM SAM, 1 mM methyl vio-
logen, 4 mM NADPH, and 1 mM methylcobalamin for 18
h.5 Reactions were then characterized by mass spectrome-
try and HPLC with UV detection at 254 nm. The assay was
also performed with GenX2 (4) as a reference. HPLC anal-
ysis of the reactions mixtures showed 5′-dAdo (13) for-
mation when GenK was incubated with the pseudotrisac-
charides 3 and 9-12 but not the pseudodisaccharides 7 and
8. Likewise, mass spectroscopic analysis of the product
mixtures demonstrated that all pseudotrisaccharides tested
were converted to their methylated products (see Figure
S1) albeit with reduced efficiency compared to GenX2 (4)
as measured in terms of 5′-dAdo production (see Table S1).
Therefore, GenK can recognize and process alternative
substrates; however, the biologically relevant substrate
GenX2 appears to be preferred.
Methylation is a ubiquitous and important reaction in the
biosynthesis of natural products.1 A typical biological
methylation reaction proceeds via an SN2-like mechanism
that involves direct attack of a nucleophilic functional
group at the electrophilic methyl of the sulfonium moiety in
S-adenosyl-L-methionine (SAM, 1). During the reaction, S-
adenosyl-L-homocysteine (SAH, 2) serves as the leaving
group released with each turnover.2 However, a group of
methyltransferases has been recently discovered that be-
long to the radical SAM superfamily of enzymes. Distinct
from the classic paradigm for biochemical methylation,3
the mechanisms of these enzymes involve radical chemistry
with methyl transfer to unactivated (i.e., non-nucleophilic)
sp2- or sp3-hybridized carbons or phosphinate centers.4
Radical SAM methyltransferases (RSMTs) can be divid-
ed into four groups based on protein architecture, predicted
reaction mechanism, and cofactor dependence.4 The sub-
group comprising the cobalamin (Cbl)-dependent RSMTs
has the most known members and the greatest recognized
diversity of reactions catalyzed. One such enzyme is GenK,
which catalyzes the methylation of gentamicin X2 (GenX2,
With regards to the mechanism of GenK-catalyzed meth-
ylation, three hypotheses have been proposed (see Scheme
2).5 Similar to other radical SAM enzymes,7 the reaction is
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initiated by reductive homolysis of SAM via single electron trol. However, none of these analogs showed any evidence
transfer from the reduced [4Fe-4S]1+ cluster in the active
site to produce methionine (14) and a 5′-deoxyadenosyl
radical (5′-dAdo•, 15) that abstracts a hydrogen atom from
C6′ of GenX2 (4). In the first mechanism (see Scheme 2A),
the resulting substrate radical (16) then abstracts the methyl
group from Me-Cbl to give G418 (5) and Cbl(II). Single
electron reduction of Cbl(II) followed by methyl transfer
from SAM regenerates Me-Cbl after each turnover. Analo-
gous catalytic cycles have been proposed for most other
Cbl-dependent RSMTs.8–12 However, due to the decreased
pKa of the 5′ α-hydroxyalkyl radical in 16, this intermediate
may be deprotonated to form a ketyl radical anion (17/18)
as proposed in the reactions catalyzed by DesII13–20 and
(R)-2-hydroxyacetyl-CoA dehydratase.21–22 Subsequent
nucleophilic attack by the ketyl intermediate 18 at Me-Cbl
could result in an alkoxy radical (19) and a Cbl(I) interme-
diate. The alkoxy radical could then be reduced either by an
external electron followed by protonation (mechanism B in
Scheme 2) or via hydrogen atom transfer from 5′-dAdo
(13) (mechanism C in Scheme 2). In the latter scenario, the
SAM so produced could serve to regenerate Me-Cbl such
that SAH is the sole reaction byproduct and 5′-dAdo is not
produced. However, the 1:1:1 stoichiometry of G418 (5),
SAH (2), and 5′-dAdo (13) formation observed in early
experiments casts doubt on mechanism C.5
of reaction with GenK (Figure S2). Instead, [6′-F]-GenX2
(20) was found to be a weak inhibitor for GenK via end-
point assays conducted in the presence of 1 mM GenX2
with the concentration of [6'-F]-GenX2 varied from 0.01 to
20 mM (see Figure S3). These results imply that the 6′-F
analog does bind into the GenK active site and that the 6′-
OH group is likely important to properly orient the sub-
strate to facilitate initial hydrogen atom abstraction.
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Figure 1. Compounds tested as substrates for GenK. The
structural variations from GenX2 (4) are highlighted in red.
To distinguish between the mechanisms in Scheme 2,
three mechanistic probes, [6′-F]-GenX2 (20), [6′-OMe]-
GenX2 (21), and [5′-F]-GenX2 (22), were prepared (see
Figure 2) as described in the Supporting Information. If
GenK can accept [6′-F]-GenX2 (20) as a substrate, then
mechanisms B and C would be ruled out, because the α-
fluoroalkyl radical intermediate would be electron-deficient
at C6′ and is thus not expected to be a Lewis base. The
same mechanistic prediction also holds for [6′-OMe]-
GenX2 (21). As for [5′-F]-GenX2 (22), if mechanism A is
operative, the turnover product should still retain the fluoro
group at C5′. In contrast, elimination of the 5′-fluoride is
possible if the GenK reaction proceeds via mechanisms B
or C due to predicted carbanionic character of C6′ in the
ketyl intermediate.
Scheme 2. Possible mechanisms of catalysis by GenK.
(A) Methylation occurs at C6′ of 16 by radical-mediated
methyl transfer. (B) Methylation occurs at C6′ via SN2-type
chemistry involving a ketyl radical anion intermediate
17/18 as the nucleophile. (C) Same as mechanism B, except
the product radical 19 is quenched by reclaiming a hydro-
gen atom from 5′-dAdo (13) to regenerate SAM (1).
To probe the orienting role of the 6′-OH group in sub-
strate binding and to gain more insight into the catalytic
cycle of GenK, the stereochemical course of the conversion
of GenX2 (4) to G418 (5) was also investigated. This was
accomplished by synthesizing the deuterated substrate
isotopologs [6′S-2H]-GenX2 (23) and [6′R-2H]-GenX2 (24)
as detailed in the Supporting Information. These isotopo-
logs (23 and 24, 1 mM each) were separately incubated
These substrate analogs were, therefore, separately incu-
bated with GenK, with GenX2 (4) used as a positive con-
2
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with GenK as described above. The 5′-dAdo (13) produced
regenerated SAM is expected to be at least partially labeled
with deuterium when [6'R-2H]-GenX2 (24) serves as the
substrate. This provides additional evidence arguing against
the plausibility of mechanism C.
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was collected by HPLC and deuterium incorporation was
analyzed by mass spectroscopy (Figures S3 and S4). When
[6′S-2H]-GenX2 (23) was used in the GenK reaction, the
isotopic envelope of the isolated 5′-dAdo was not found to
differ significantly from that produced in a control reaction
with unlabeled GenX2 (4) indicating natural abundance
labeling (Figure 3a vs. 3b). However, when [6'R-2H]-
GenX2 (24) was employed, the peak corresponding to in-
corporation of a single deuterium was found to be dominant
(Figure 3c) indicating deuterium transfer from 24 to 5′-
dAdo• (15) to produce deuterated 5′-dAdo.
The stereochemical course of only a handful of reactions
catalyzed by Cbl-dependent RSMTs have been reported.24
For GenD1 (3 → 4)11 and MoeK5,25 this information can
be directly deduced from the structures of the substrate and
product as methylation occurs at a stereogenic center in
both cases (e.g., C4′′ of 3 for GenD1). In contrast, the ste-
reospecificity of the β-methylation reactions in the biosyn-
thesis of bottromycin26 and the β-methylation step of 2-
hydroxyethylphosphonate (HEP) catalyzed by Fom3 during
fosfomycin biosynthesis9,27 were determined experimental-
ly by feeding studies with labeled precursors. In all of these
cases, the reaction occurs with inversion of configuration at
the carbon center where methylation takes place.24 These
findings led to the suggestion that Me-Cbl and the radical
SAM machinery are situated on opposite sides of the center
to be methylated once bound in the active site of these
enzymes.9,11,24 This hypothesis is consistent with the only
known crystal structure of a Cbl-dependent radical SAM
enzyme, OxsB.28 Though not a RSMT, the substrate bind-
ing site of OxsB is believed to be interposed equidistant
between the C5' of SAM and the Co of the Cbl cofactor.
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Figure 2. Analogs used to probe the GenK mechanism.
The G418 product (5) formed during incubation of GenK
with the isotopologs 23 and 24 was also analyzed by mass
spectrometry. When [6'S-2H]-GenX2 (23) was utilized as
the substrate, the deuterium label was found to be retained
in the G418 consistent with the observation of natural
abundance labeling in the 5′-dAdo produced (see Figure
3e). In contrast, when [6′R-2H]-GenX2 (24) was used, the
majority of the Gen418 produced was unlabeled (Figure
3f). Nevertheless, the isotopic envelope in this reaction
deviated significantly from that expected for natural abun-
dance labeling (compare Figure 3d vs. 3f) implying partial
retention (i.e., ca. 20%) of the deuterium label in G418
when [6′R-2H]-GenX2 is the substrate. Similar observations
were made when the assay mixtures were treated with 1-
fluoro-2,4-dinitrobenzene at the end of the incubation and
the resulting dinitrophenyl (DNP)-derivatized G418 was
isolated by HPLC and analyzed by mass spectrometry (see
Figure S5).
These results reveal that H-atom abstraction from C6′ of
GenX2 is stereoselective for the pro-R C6′–H; however, the
primary deuterium kinetic isotope effect (KIE) on [2H]-
atom abstraction from [6′R-2H]-GenX2 can lead to in-
creased abstraction of the otherwise disfavored pro-S H-
atom. Consequently a mixture of labeled and unlabeled
Gen418 are observed with this isotopolog. Since G418 (5)
is known to have the R-configuration at C6′,23 these results
demonstrate that the majority (i.e., at least 95% assuming a
primary deuterium KIE greater than 5) of the reaction flux
proceeds with retention of configuration as the pro-R H-
atom is abstracted and the methyl group is transferred from
Me-Cbl to the GenX2 substrate radical.
Figure 3. Electrospray ionization (ESI) mass data of (a-
c) 5′-dAdo (13, [M + H]+ = 252 m/z) and (d-f) G418 (5, [M
+ H]+ = 497.28 m/z) generated during incubation of GenK
with (a and d), GenX2 (4, [M + H]+ = 483.28 m/z), (b and e)
[6′S-2H]-GenX2 (23), and (c and f) [6′R-2H]-GenX2 (24).
The generality of this hypothesis has, however, been
brought into question by recent studies on Fom3 where the
substrate was found to be cytidylyl-HEP and the methyla-
tion reaction was shown to be non-stereoselective.10 The
present results with GenK provide further evidence that
reactions catalyzed by this class of enzymes do not neces-
sarily proceed via a mechanism that is stereochemically
uniform over all cases. While rotation of the carbon center
bearing the unpaired electron may occur in the case of the
Fom3 substrate radical intermediate, such free rotation is
not applicable to the mechanism of GenK catalysis given
the stereoselectivity of the reaction. Thus, the C5′ hy-
droxymethyl functionality of GenX2 is predicted to be
constrained in a single orientation during the pro-R H-atom
In contrast to the 5′-dAdo and Gen418 reaction products,
no deuterium incorporation was detected in either the SAH
(2) produced during the reaction nor the residual SAM (1)
regardless of which isotopolog (i.e., 23 or 24) was included
as the substrate (Figure S6 and S7). These results are in-
consistent with mechanism C where either SAH or the
3
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abstraction event to generate a transient α-hydroxyalkyl or
1
2
3
4
5
6
7
8
ketyl radical intermediate. As the results with the 6′-fluoro
(20), 6′-OMe (21), and 6′-amino (11) derivative suggest,
this configuration may be crucial for H-atom abstraction
from the substrate during turnover. Finally, this constraint
is maintained during the subsequent radical or nucleophilic
attack on the Me-Cbl donor that likely sits adjacent rather
than opposite to the radical SAM machinery in the GenK
Michaelis complex, thereby leading to retention of configu-
ration during H-atom abstraction and methyl addition.
In summary, the Cbl-dependent RSMTs represent the
largest and most diverse collection of RSMTs presently
known. Based on prior in vitro investigations of eight of
these enzymes (TsrM,29-31 Fom3,8-10 GenD1,11 ThnK,12
PhpK,32-34 PoyC,35 CysS,36 and GenK5), a model of sub-
strate binding wherein the substrate is sandwiched between
the Cbl cofactor and the radical SAM machinery has been
suggested. However, the present results with GenK suggest
that this may not necessarily be a general property of these
enzymes. Instead, the GenK Michaelis complex is predict-
ed to involve a front-side juxtaposition of both cofactors
with respect to the substrate. Hence, from just this small
number of Cbl-dependent RSMTs studied to date, the
chemistry and structural features of these enzymes are
proving to be quite varied, and they are expected to be rich
source of new discoveries for future investigations.
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ASSOCIATED CONTENT
Supporting Information. Details regarding GenK assays
along with HPLC protocols, and chemical syntheses of
related compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
h.w.liu@mail.utexas.edu
Notes
The authors declare no competing financial interest.
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This work was supported by grants from the National Insti-
tutes of Health (GM035906) and the Welch Foundation (F-
1511).
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