Mechanistic Investigation of 1,2-Diol Dehydration of Paromamine Catalyzed by the Radical S-Adenosyl- l -methionine Enzyme AprD4

Article abstract of DOI:10.1021/jacs.1c00076

AprD4 is a radical S-adenosyl-l-methionine (SAM) enzyme catalyzing C3′-deoxygenation of paromamine to form 4′-oxo-lividamine. It is the only 1,2-diol dehydratase in the radical SAM enzyme superfamily that has been identified and characterized in vitro. The AprD4 catalyzed 1,2-diol dehydration is a key step in the biosynthesis of several C3′-deoxy-aminoglycosides. While the regiochemistry of the hydrogen atom abstraction catalyzed by AprD4 has been established, the mechanism of the subsequent chemical transformation remains not fully understood. To investigate the mechanism, several substrate analogues were synthesized and their fates upon incubation with AprD4 were analyzed. The results support a mechanism involving formation of a ketyl radical intermediate followed by direct elimination of the C3′-hydroxyl group rather than that of a gem-diol intermediate generated via 1,2-migration of the C3′-hydroxyl group to C4′. The stereochemistry of hydrogen atom incorporation after radical-mediated dehydration was also established.

Full text of DOI:10.1021/jacs.1c00076

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Mechanistic Investigation of 1,2-Diol Dehydration of Paromamine
Catalyzed by the Radical S‑Adenosyl‑L‑methionine Enzyme AprD4
Yu-Cheng Yeh,$ Hak Joong Kim,$ and Hung-wen Liu*
Cite This: J. Am. Chem. Soc. 2021, 143, 5038−5043
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ABSTRACT: AprD4 is a radical S-adenosyl-L-methionine (SAM) enzyme catalyzing C3′-deoxygenation of paromamine to form 4′-
oxo-lividamine. It is the only 1,2-diol dehydratase in the radical SAM enzyme superfamily that has been identified and characterized
in vitro. The AprD4 catalyzed 1,2-diol dehydration is a key step in the biosynthesis of several C3′-deoxy-aminoglycosides. While the
regiochemistry of the hydrogen atom abstraction catalyzed by AprD4 has been established, the mechanism of the subsequent
chemical transformation remains not fully understood. To investigate the mechanism, several substrate analogues were synthesized
and their fates upon incubation with AprD4 were analyzed. The results support a mechanism involving formation of a ketyl radical
intermediate followed by direct elimination of the C3′-hydroxyl group rather than that of a gem-diol intermediate generated via 1,2-
migration of the C3′-hydroxyl group to C4′. The stereochemistry of hydrogen atom incorporation after radical-mediated
dehydration was also established.
resulting product radical is subsequently reduced back to an
even-electron state and, if necessary, reduced further in a
subsequent step to complete the net deoxygenation.
INTRODUCTION
■
The reductive deoxygenation of biological metabolites is a
process found in countless biosynthetic pathways and
accomplished by a mechanistically diverse array of enzyme
catalysts and pathways.1−14 Arguably one of the best known
motifs involves a two-step process where the C−O bond is first
cleaved via Lewis acid−base chemistry to form a desaturated
compound that is subsequently reduced in the second step,
which typically involves hydride transfer from a biological
reductant such as NADPH.8−10 The lyase activity in this
process requires the presence of an acidic carbon suitably
located relative to the oxy-leaving group so as to effect its
elimination upon deprotonation. Consequently, such an acidic
carbon must be introduced or otherwise activated, often by a
neighboring electron withdrawing group or coupling with an
organic cofactor.8−14 However, a number of dehydrations are
also known to proceed despite the absence of an activated
carbon. These reactions instead rely on oxidation of the
substrate to form a radical intermediate that can only be
generated via a limited number of specialized active site
features, such as a B12 coenzyme in glycerol dehydratase15−19
or an amino acid radical species in ribonucleotide reduc-
tase20,21 among others.15−19,22−25 Following dehydration, the
The C3′ deoxygenation of paromamine (1) also involves the
formation of radical intermediates, because it too lacks an
activating functional group on the glucosamine subunit.
However, in contrast to previous examples, the initial,
radical-mediated dehydration reaction involves a radical S-
adenosyl-L-methionine (SAM) enzyme, AprD4.26−28 The
resulting product, 4′-oxo-lividamine (3), retains the same
oxidation state as the paromamine substrate and must
therefore be reduced by its NADPH-dependent reductase
partner AprD3 in order to generate the deoxygenated 3′-
deoxy-pseudo-disaccharide lividamine (4; see Scheme 1).26−28
Although radical SAM enzymes have been shown to function
Received: January 4, 2021
Published: March 30, 2021
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© 2021 American Chemical Society
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Scheme 1. C3′-Deoxygenation of Paromamine (1) to
Scheme 2. Proposed Mechanism for AprD4-Catalyzed 1,2-
Diol Dehydration of Paromamine (1)
a
Lividamine (4) Catalyzed by AprD4 and AprD3
as amino-lyases (e.g., TDP-4-amino-4,6-dideoxy-D-glucose
deaminase, DesII),29−31 AprD4 is the only radical SAM
hydroxy-lyase that has been characterized to date. Recent
studies have also demonstrated that AprD4 along with AprD3
can catalyze C3′ deoxygenation of kanamycin C, kanamycin B,
and neamine (see Figure S1) via related pathways.28 The
resulting absence of the C3′-hydroxyl group in each of these
antibiotics leaves them inert to modification by 3′-phospho-
transferases, which is a common mechanism of aminoglyco-
side-resistance in some bacterial strains.32,33 Moreover, AprD4
may also be involved in the biosynthesis of tobramycin,34
nebramycin 5′,35 and lividomycin B36 given the structural
resemblance of their pseudodisaccharide cores to that of
paromamine. Therefore, a more complete understanding of the
mechanism of AprD4 and its substrate specificity is anticipated
to serve as a useful foundation for the development of more
effective aminoglycoside antibiotic regimens.
RESULTS AND DISCUSSION
■
Like other members of the radical SAM enzyme superfamily,
AprD4 harbors a characteristic [4Fe-4S] cluster in the active
site. However, unlike most members of this superfamily,
AprD4 is characterized by a unique CX3CX3C [4Fe-4S]
binding motif rather than the canonical CX3CX2C motif.37,38
The reaction of a typical radical SAM enzyme is initiated by
electron transfer from the reduced [4Fe-4S]1+ cluster to SAM
leading to reductive homolysis of the latter (5) concomitant
with the generation of methionine (6) and a 5′-deoxyadenosyl
radical (5′-dAdoCH2•) equivalent (7). The latter serves to
abstract a hydrogen atom from the substrate thereby activating
the substrate for its subsequent radical-mediated trans-
formation to 4′-oxo-lividamine (3) (Scheme 2).39−42 The
well-characterized DesII is a closely related enzyme, which
catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-
glucose (49) to the corresponding 4,6-dideoxy-3-ketoglucose
(53) (see Scheme 4).29−31
Two mechanisms have been proposed to account for the
AprD4-catalyzed dehydration of 1 to 4′-oxo-lividamine (3) as
shown in Scheme 2.26 Both mechanisms involve initial
hydrogen-atom abstraction from the C4′ position of parom-
amine (1) by the 5′-deoxyadenosyl radical equivalent 7.26−28
The resulting substrate radical (8) may be deprotonated to
form a ketyl radical (10/10′) that subsequently undergoes β-
elimination of the 3′−OH group to yield the keto/enol radical
(11/11′) (Scheme 2, pathway A). One-electron reduction of
11/11′ would then complete the dehydration. This mechanism
is similar to those of (R)-2-hydroxyacyl-CoA dehydratase24,25
as well as glycyl radical-dependent diol-dehydratase19,22,23 and
has been argued for on the basis of the AprD4 crystal structure
with bound substrate.43 Alternatively, the initially formed
substrate radical (8) may undergo a radical-induced 1,2-
hydroxyl migration to form the gem-diol radical 12 (Scheme 2,
pathway B), a process analogous to that of the B12-dependent
a
For the sake of brevity, the aminocyclitol 2-deoxystreptamine (2-
DOS) (2) unit is not shown in some compounds in Scheme 2.
diol-dehydratases and ethanolamine ammonia lyases.15−19
After reduction, the gem-diol intermediate 13 could then
eliminate water to form 4′-oxo-lividamine (3). Although initial
hydrogen-atom abstraction from the C4′ position has been
established based on deuterium transfer from [4′-2H]-parom-
amine to 7 to yield [5′-2H]-5′-dAdo (9),26 a complete picture
of the AprD4 catalytic cycle remains to be described. A third
mechanism suggested by a reviewer can also be envisioned that
involves ring flipping of 8 followed by abstraction of a
hydrogen atom from 5′-dAdo (9) by a 3′-hydroxy radical
leaving group from 8′ to yield an enol product (3-enol) and
regenerate 7 (Scheme 2, pathway C). However, because
multiple incorporation of deuterium into 5′-dAdo is not
observed with [4′-2H]-paromamine in vitro,26 SAM is not
regenerated from the 5′-dAdo radical (7), which would need
to be reduced to 5′-dAdo.
To investigate the mechanism of AprD4, several substrate
analogues (14−21, Figure 1A) were synthesized. Compound
14 was designed to distinguish pathways A and B, because the
C4′-methoxy group will prevent ionization of the initial
substrate radical thereby impeding formation of a ketyl radical
intermediate (vis-a-vis 8 → 10/10′). In contrast, the C4′-
methoxy group should have much less of an impact on radical-
induced 1,2-migration of the C3′-hydroxyl group to give 12. As
shown in Figure 1C (trace b), when 1 mM 14 was incubated
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Table 1. Deuterium Incorporation into 5′-dAdo (9) during
the Incubation of Substrate Analogues with AprD4/AprD3
a
b
Entry
Substrate
obs. A253/A252
% Fraction of labeled 5′-dAdo
1
2
3
4
5
6
1
14
15
0.105 0.001
0.101 0.001
0.113 0.000
0.179 0.001
0.276 0.003
0.474 0.005
NA
NA
0.8 0.1
6.9 0.1
14.6 0.3
27.0 0.4
15 (5 mM)
16
16 (5 mM)
a
The MS signal for protonated 5′-dAdo at 253.1 m/z (m + 1) was
integrated and divided by that at 252.1 m/z (m) to obtain the
observed signal intensity ratio A253/A252. This ratio largely reflects
natural abundance 13C in the absence of labeled substrate (entry 1).
Therefore, the ratio R of C5′-deuterated vs unlabeled 5′-dAdo
observed in the presence of labeled substrate can be obtained by
b
subtracting from the observed A253/A252 ratio that of entry 1.45 The
fraction of labeled 5′-dAdo is then given by R/(R+1) and is listed as a
percentage (NA indicates no significant labeling). All ratios were
measured from three separate assays each.
(Table 1, entry 4; and Figure S2E), and only the low-level
formation of unlabeled 14 was observed instead of lividamine
(see Figure S3A,B). These results imply that the majority of 5′-
dAdo produced in the presence of 14 and 15 is due to
uncoupled reduction of SAM, which is commonly observed
among the radical SAM enzymes.39−42,44 According to the
AprD4 structure recently reported by Zhang and Nicolet,43 the
main chain amide N-atom of the active site residue Ala27 is
positioned near the 4′−OH group of paromamine (1). This
residue may therefore engage in a steric clash with the bulky
4′-OMe group of 14/15 thereby hampering hydrogen atom
abstraction from C4′.
In response to these complications, the analogue 16 carrying
both a deuterium and a less sterically hindered fluoro
substituent at C4′ was also prepared (Scheme 3A). The key
steps included fluorination with diethylaminosulfur trifluoride
(DAST) and coupling with 26 to form the pseudo-disaccharide
27, which after alkaline hydrolysis and hydrogenation gave
16.46 It was expected that redistribution of electron density on
the initially formed substrate radical (i.e., 8 → 10/10′),
necessary to promote elimination of the C3′-hydroxyl group,
would be prohibited by the 4′-fluoro group, whereas 1,2-
migration (i.e., 8 → 12) leading to 4′-oxo-lividamine via the
4′-fluoro-4′-hydroxyl intermediate should not be markedly
affected.47 Once again, no discernible formation of lividamine
was observed (Figure 1E, trace b); however, in contrast to the
experiments with 14 and 15, enrichment of 5′-dAdo (9) with
deuterium was much more robust (Table 1, entries 5 and 6;
and Figure S2F,G), though not as significant as in the case of
[4′-2H]-paromamine, where in a previous work nearly all the
observed 5′-dAdo had been found to become labeled.26
Furthermore, formation of unlabeled 16 was detected by LC-
MS (see Figure S3D,E). These findings imply that a substrate
radical is indeed generated during the reaction of AprD4 with
16 but does not undergo conversion to 4′-oxo-lividamine.
To further evaluate the mechanistic alternatives, (3′S)-3′-
deoxy-3′-fluoro-paromamine (17) and (3′R)-3′-deoxy-3′-fluo-
ro-paromamine (18) were prepared according to Scheme
3B,C. The fluoro substituent at C3′ in both cases was
introduced in a nucleophilic substitution reaction (29 → 30 or
35 → 36) using tetra-n-butylammonium fluoride (TBAF) as
the fluorine donor.48 Due to the increased electronegativity of
the fluoro substituent, the C3′-F moiety of 17 and 18 was
Figure 1. Mechanistic study of 1,2-diol dehydration catalyzed by
AprD4. (A) Structures and exact masses of paromamine (1), substrate
analogues 14−21 and lividamine (4). (B−G) LC-MS analysis of
AprD4 and AprD3 assay shown with (a) extracted ion chromatogram
(EIC) of [M + Na]+ corresponding to the respective substrate in each
assay; (b) EIC of [M + Na]+ = 330.16 (corresponding to 4) in the
assay; (c) EIC of [M + Na]+ corresponding to substrate standard; (d)
EIC of [M + Na]+ = 330.16 for lividamine (4) standard. AprD4 and
AprD3 were incubated with (B) paromamine (1), (C) 14, (D) 15,
(E) 16, (F) 17, or (G) 18.
with 0.05 mM AprD4, 0.05 mM AprD3, 2 mM SAM, 1 mM
NADPH, 10 mM DTT, and 2 mM sodium dithionite in 50
mM NH4HCO3 (pH 7.8) for 16 h, neither the 4′-hydroxy-4′-
methoxy hemiacetal nor lividamine product was observed.
Both enzymes were active, as formation of 4 was observed
when incubated with paromamine (1) (Figure 1B, traces a and
b).
However, this result could also be a consequence of
interference of the C4′ methoxy group with the H atom
abstraction step. Indeed, little deuterium was transferred to 5′-
dAdo (9) when the C4′ deuterated isotopolog 15 was used in
place of 14 (Figure 1D, trace b; Table 1, entry 3; and Figure
S2D). In this experiment, any deuterium transfer is assumed to
be from the C4′ of 15 to the 5′-methyl group of 5′-dAdo (9).
Moreover, increasing the concentration of 15 to 5 mM had
only a small effect on the deuterium enrichment of 5′-dAdo
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Scheme 3. Synthetic schemes for the preparation of [4′-2H]-
4′-deoxy-4′-fluoro-paromamine (16), (3′S)-3′-deoxy-3′-
fluoro-paromamine (17), and (3′R)-3′-deoxy-3′-fluoro-
paromamine (18)
Scheme 4. Comparison of Proposed Mechanisms for AprD4
and DesII
anticipated using 19 as the substrate. However, if the reaction
proceeds via a 1,2-migration (pathway B), then partial
retention of the 18O label would be expected due to the
16O,18O-gem-diol intermediate. Excess AprD3 was used in the
assays to minimize the extent of solvent exchange into the
ketone moiety of 3. Incubation of 19 with various ratios of
AprD3/AprD4 in H2O resulted in no retention of the 18O-
label in lividamine (Table S1, entries 1−4). Furthermore,
incubation of unlabeled paromamine (1) in buffered H218O
(>90% enriched) showed minimal (<2%) wash-in of H218O
(Table S1, entries 5−8). In contrast, incubation of 20 with
AprD4 and AprD3 led to significant (>97%) retention of 18O
under the same assay conditions (Table S1, entries 9−12)
indicating that the C4′−O bond of paromamine remains intact
during the catalytic cycle. While 1,2-migration and stereo-
selective elimination of the C3′ hydroxyl group from C4′ of
the putative gem-diol 12 in the enzyme active site cannot be
definitively ruled out, such a mechanism would not explain the
lack of AprD4 activity with the C4′-fluoro analog 16.
Furthermore, if the reaction were to proceed via a gem-diol
intermediate, then at least one of the two diastereomers 17 or
18 would likely have failed to produce 4′-oxo-lividamine.
The final step of the AprD4 catalyzed reaction involves one-
electron reduction of the product radical (e.g., 11/11′) to
afford 4′-oxo-lividamine (3) and may involve hydrogen atom
transfer to C3′ from an active site residue such as tyrosine or
cysteine.50 When [3′-2H]-paromamine (21, Figure 1A) was
prepared and reacted with AprD4/AprD3, the C3′ of the
product (22) was found to have the S-configuration such that
the radical substitution proceeds with the hydrogen atom
adding to the same face from which the C3′−OH leaves
(Figure S5). This result is in agreement with a recent report
that a deuterium is incorporated equatorially at C3′ of
lividamine when the reaction is run in buffered D2O.28 This
is also consistent with the proposal that Tyr216 of AprD4 is
responsible for the transfer of the solvent-exchangeable
hydrogen atom to C3′ of the radical intermediate based on
the crystal structure of AprD4 and experiments demonstrating
the reduced activity of the Y216F and Y216H mutants.43
anticipated to be less prone to 1,2-migration compared to the
C3′−OH group of 1 (see Scheme 4, 46 → 48 → 12).49
However, the fluoro substituent was expected to be a good
leaving group thereby facilitating the conversion of 17 and/or
18 to 4′-oxo-lividamine (3) via an E1cB-type elimination
analogous to pathway A. Indeed, when either 17 or 18 was
incubated with AprD4 and AprD3, lividamine (4) production
was observed (Figure 1F,G, trace b) based on LC-MS analysis
and derivatization with benzyl chloroformate (see Figure S4).
More lividamine appeared to be produced in the assay of 18 vs
17, consistent with 18 more closely matching the stereo-
chemistry of paromamine. These observations collectively
appear to be more suggestive of a mechanism involving a C4′
ketyl radical intermediate as opposed to a 1,2-migration.
Furthermore, the conversion of 17 to 4′-oxo-lividamine is also
inconsistent with the mechanism in Pathway C of Scheme 2,
because this analog lacks an oxygen or a fluorine as the H atom
acceptor with the requisite stereochemistry at C3′.
Finally, compounds 19 and 20 with 18O-labeling at C3′ and
C4′, respectively, were also prepared. If the reaction proceeds
via a ketyl radical intermediate (Scheme 2, pathway A), then
no retention of the 18O labeling in lividamine would be
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Notes
CONCLUSION
■
The authors declare no competing financial interest.
In summary, AprD4 catalyzed dehydration of paramomine is
more consistent with a ketyl radical intermediate (pathway A)
rather than a radical-mediated 1,2-migration (pathway B).
Elimination of 18O from C3′-18O paromamine and retention of
18O during dehydration of C4′-18O paromamine demonstrate
that the C3′ hydroxyl group is selectively eliminated during
this enzymatic reaction. Incubation of AprD4 with the
substrate analogues 14−16 further show that the hydroxyl
group at the C4′ position of paromamine (1) is necessary for
elimination of the C3′ hydroxyl group despite not being
necessary for substrate radical generation. These observations
favor direct elimination the C3′ hydroxyl group from a ketyl
radical intermediate, which is also consistent with the
observation that AprD4 can function as an effective
dehydrofluorinase when presented with the 3′-deoxy-3′-fluoro
analogs of paromamine (17 and 18). This mechanistic model
involving a ketyl radical intermediate is likewise consistent with
the conclusions drawn from the AprD4 structural analysis43 as
well as the mechanisms proposed for several other radical-
mediated dehydratases including ribonucleotide reductase,20,21
1,2-propanediol dehydratases,19 and hydroxyacyl-CoA dehy-
dratases.24,25
ACKNOWLEDGMENTS
■
We thank an anonymous reviewer for suggesting an alternative
mechanism (pathway C in Scheme 2). We also thank Dr. Mark
Ruszczycky for his critical review and help in preparing the
manuscript. This work was supported by grants from the
National Institutes of Health (GM035906) and the Welch
Foundation (F-1511).
ABBREVIATIONS
■
DesII, TDP-4-amino-4,6-dideoxy-D-glucose deaminase or
TDP-4-amino-4,6-dideoxy-D-glucose 4-amino-lyase; AprD4,
paromamine 3′-dehydratase or paromamine 3′-hydroxy-lyase;
AprD3, 4′-oxo-lividamine 4′-reductase
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ASSOCIATED CONTENT
* Supporting Information
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https://pubs.acs.org/doi/10.1021/jacs.1c00076.
Experimental details, including synthesis of compounds,
activity assays, analytic methods, and NMR spectra
(PDF)
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AUTHOR INFORMATION
Corresponding Author
■
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; orcid.org/0000-0001-8953-4794;
Email: h.w.liu@mail.utexas.edu
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biosynthesis and the acyl carrier protein. Biochem. J. 2010, 430, 1−19.
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in carbohydrate metabolism. Chem. Rev. 2000, 100, 4615−4661.
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Authors
Yu-Cheng Yeh − Department of Chemistry, University of
Texas at Austin, Austin, Texas 78712, United States
Hak Joong Kim − Department of Chemistry, University of
Texas at Austin, Austin, Texas 78712, United States;
orcid.org/0000-0002-9265-9181
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c00076
Author Contributions
$Y.-C.Y. and H.J.K. contributed equally to this work.
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