Structure-Guided Identification of a Small Molecule That Inhibits Anaerobic Choline Metabolism by Human Gut Bacteria

Article abstract of DOI:10.1021/jacs.8b04883

The anaerobic gut microbial pathway that converts choline into trimethylamine (TMA) is broadly linked to human disease. Here, we describe the discovery that betaine aldehyde inhibits TMA production from choline by human gut bacterial isolates and a complex gut community. In vitro assays and a crystal structure suggest betaine aldehyde targets the gut microbial enzyme choline TMA-lyase (CutC). In our system, we do not observe activity for the previously reported CutC inhibitor 3,3-dimethylbutanol (DMB). The workflow we establish for identifying and characterizing betaine aldehyde provides a framework for developing additional inhibitors of gut microbial choline metabolism, including therapeutic candidates.

Full text of DOI:10.1021/jacs.8b04883

Communication
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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Structure-Guided Identification of a Small Molecule That Inhibits
Anaerobic Choline Metabolism by Human Gut Bacteria
Marina Orman,† Smaranda Bodea,† Michael A. Funk,‡ Ana Martínez-del Campo,† Maud Bollenbach,†
Catherine L. Drennan,‡,§,∥,⊥ and Emily P. Balskus*,†
†Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United
States
§
∥
⊥
‡Department of Chemistry, Department of Biology, Howard Hughes Medical Institute, and Center for Environmental Health
Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
which choline-degrading gut microbes affect host physiology
remain largely uncharacterized.
ABSTRACT: The anaerobic gut microbial pathway that
converts choline into trimethylamine (TMA) is broadly
linked to human disease. Here, we describe the discovery
that betaine aldehyde inhibits TMA production from
choline by human gut bacterial isolates and a complex gut
community. In vitro assays and a crystal structure suggest
betaine aldehyde targets the gut microbial enzyme choline
TMA-lyase (CutC). In our system, we do not observe
activity for the previously reported CutC inhibitor 3,3-
dimethylbutanol (DMB). The workflow we establish for
identifying and characterizing betaine aldehyde provides a
framework for developing additional inhibitors of gut
microbial choline metabolism, including therapeutic
candidates.
The choline utilization (cut) gene cluster encodes the
enzymes involved in anaerobic microbial choline metabolism,
including the glycyl radical enzyme choline TMA-lyase (CutC)
and its corresponding radical SAM activating protein
(CutD).18−20 CutD postranslationally modifies CutC, instal-
ling a radical on a conserved glycine residue and enabling
catalysis of the key C−N bond cleavage reaction that generates
TMA. We gained insights into the mechanism of this
transformation from structural studies and mutagenesis (Figure
1B,C).20 The glycyl radical of CutC is proposed to abstract a
hydrogen atom from an adjacent cysteine (Cys489),
generating a thiyl radical intermediate that initiates the
reaction with choline. Within the polar active site of CutC,
choline is oriented such that its C1 hydroxyl engages in
hydrogen bonding with Glu491. The primary substrate-specific
contacts are an array of CH−O hydrogen bonds between the
polarized methyl groups of the trimethylammonium moiety
and the side-chain oxygen atoms of Tyr208, Tyr506, Asp216,
and a bound water molecule. We propose the thiyl radical
abstracts the pro-S hydrogen atom from C1 of choline.
Subsequent deprotonation of the C1-OH by Glu491, followed
by C−N bond cleavage via a “spin-center shift,” would
eliminate trimethylamine. The resulting product-based radical
could reabstract the hydrogen atom from Cys489, reforming
the thiyl radical and generating acetaldehyde. Proton transfer
from Glu491 to Asp216 via Thr502 could reset the CutC
active site for the next round of catalysis.
These observations informed our search for small molecule
inhibitors of CutC. We identified a series of choline analogs
that would preserve many of the key interactions with CutC
active site residues, including the CH−O hydrogen bonds.
These molecules were tested for inhibition of anaerobic
choline metabolism by the cut gene cluster-containing human
gut isolate Escherichia coli MS 200-1 (Figure 2A). Cultures
were incubated anaerobically in a rich medium containing 1
mM of (trimethyl-d9)-choline (d9-choline) and three concen-
trations (1, 5, and 10 mM) of compound. Production of d9-
TMA was analyzed by LC-MS/MS. Thiocholine (2) and
icroorganisms living within the human gastrointestinal
M(GI) tract (the gut microbiota) produce a diverse
repertoire of small molecules, some of which are associated
with disease.1,2 Deciphering the roles of these metabolites in
disease development and progression has proven challenging.
Most methods for manipulating the gut microbiota (anti-
biotics, probiotics, and prebiotics) alter multiple microbial
activities. However, elucidating the mechanisms by which gut
microbial metabolites affect host biology requires modulating
specific transformations within this community. The develop-
ment of small molecules that selectively inhibit gut microbial
enzymes is a promising but underexplored approach for
addressing this challenge.
Anaerobic metabolism of the essential nutrient choline is a
prominent, disease-associated gut microbial activity.3 Gut
anaerobes use choline as a source of carbon and energy,
cleaving its C−N bond to generate trimethylamine (TMA) and
acetaldehyde (Figure 1A). TMA is metabolized in the liver to
trimethylamine-N-oxide (TMAO) by flavin monooxygenases
(FMOs) and excreted.4,5 Elevated levels of plasma TMAO in
humans are linked to nonalcoholic fatty liver disease,6,7
cardiovascular disease,8−11 chronic kidney disease,12,13 and
type 2 diabetes.14 Additionally, patients with the inherited
metabolic disorder trimethylaminuria exhibit decreased FMO3
activity and excrete TMA.15−17 Despite the many connections
between this activity and human health, the mechanisms by
Received: May 9, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b04883
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Journal of the American Chemical Society
Communication
Figure 2. Betaine aldehyde inhibits TMA formation in E. coli and in
vitro. (A) Choline analogs were tested for inhibition of anaerobic
choline metabolism by E. coli MS 200-1. Bacteria were incubated in
BHI medium with 1 mM d9-choline and 1, 5, or 10 mM of inhibitor at
37 °C. Production of d9-TMA was measured by LC-MS/MS after 3.5
h. Dose−response curves were fit using nonlinear regression to
determine EC50 values. (B) Betaine aldehyde inhibits anaerobic d9-
TMA formation in E. coli MS 200-1 in a dose-dependent manner.
Bacteria were incubated in BHI medium with 1 mM d9-choline and
inhibitor (1 μM to 30 mM) at 37 °C. Production of d9-TMA was
measured by LC-MS/MS after 3.5 h. The data were fit using
nonlinear regression. Error bars represent the mean SD of three
replicates. (C) Betaine aldehyde inhibits the activity of CutC in vitro.
Betaine aldehyde (0.30 μM to 1 mM) was added immediately before
choline. The activity of CutC was coupled to that of NADH-
dependent yeast alcohol dehydrogenase and the absorbance of
NADH at 340 nm was recorded over time. The data were fit using
nonlinear regression. Error bars represent the mean SD of three
replicates.
Figure 1. Anaerobic choline metabolism is a disease-linked gut
microbial activity. (A) Microbial generation of TMA from choline and
its subsequent processing in the human body. (B) Crystal structure of
CutC (PBD = 5FAU) and (C) proposed mechanism for choline
cleavage.
betaine aldehyde (9) emerged as promising inhibitors.
Notably, we observed no inhibition with DMB (12), a
previously reported CutC inhibitor, and only modest inhibition
with its oxidized analog 3,3-dimethylbutyric aldehyde
(DMBA) (13).21
We chose to further examine the activity and mechanism of
inhibition of betaine aldehyde. This compound exhibited an
EC50 value of 0.4 mM in the whole cell assay (Figure 2B). To
assess whether the reduction in TMA resulted from inhibition
of CutC, we performed in vitro assays. Recombinant wild-type
CutC and CutD from D. alaskensis G20 were expressed and
purified.19,20 Using a 7-point dilution series, the IC50 of betaine
aldehyde for CutC was determined to be 26 2 μM (Figure
2C). Thus, betaine aldehyde’s ability to inhibit TMA
production by E. coli may arise from direct targeting of
CutC. DMB did not inhibit CutC, even at the highest
concentrations tested (Figure S1).
(Tables S1−S2) was determined to 2.35-Å resolution and
showed a large continuous patch of density around Cys489.
We modeled a thiohemiacetal adduct of betaine aldehyde at
Cys489 (Figure 3A) and validated this assignment through
composite omit electron density maps (Figure 3B). This
betaine aldehyde adduct interacts with CutC active site
residues in a similar manner to choline, with the thiohemiacetal
alcohol hydrogen bonding with Glu491 (2.5 Å) and the
backbone amide of Cys489 (2.7 Å) (Figure 3C). The
trimethylammonium moiety is shifted slightly away from the
rear of the active site, where residues Asp216, Tyr208, Tyr506,
and a water molecule make CH−O hydrogen bonds to the
methyl groups, slightly altering the distances from those
observed in the choline-bound structure (Table S3). Also, the
α-carbon of betaine aldehyde is shifted closer to Phe395 (3.5 Å
vs 3.8 Å), a residue thought to make a cation−π-like
interaction with the substrate. Overall, betaine aldehyde
appears to be well accommodated in this active site. The
observed covalent interaction could explain betaine aldehyde’s
ability to inhibit CutC and the apparent decrease in IC50 upon
preincubation of inhibitor with the enzyme (Figure S2).
Alternatively, inhibition could result from a long dwell time of
To gain information about how betaine aldehyde inhibits
CutC, we performed X-ray crystallographic studies on the
unactivated enzyme−inhibitor complex. After preincubating
wild-type CutC with 10 mM of betaine aldehyde, we obtained
a homogeneous solution that was crystallized using conditions
similar to those reported previously.20 The resulting structure
B
DOI: 10.1021/jacs.8b04883
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Journal of the American Chemical Society
Communication
Figure 3. Betaine aldehyde binding to CutC appears to involve a
covalent, thiohemiacetal linkage. (A) Crystal structure of unactivated
CutC soaked with betaine aldehyde (10 mM) reveals interactions
between CutC active site residues and bound inhibitor (cyan). Shown
are traditional hydrogen bonds (2.5−3.2 Å, black dashes), CH−O
hydrogen bonds (3.2−3.7 Å, yellow dashes), a cation−π-like
interaction (gray dashes), and distance between Gly821 and catalytic
Cys (red dashed line). (B) Composite omit 2Fo − Fc density (light
blue) contoured at 1.8σ. Density surrounding betaine aldehyde-
modified Cys493 is highlighted (yellow). (C) Comparison of
inhibitor-bound (green/cyan) and choline-bound (gray) CutC
structures. Hydrogen bonding interactions with the Cys loop (black
dashes) are shown.
Figure 4. Betaine aldehyde inhibits anaerobic growth only on choline
and prevents choline metabolism in both gut bacterial cultures and a
human fecal suspension. (A) Growth of E. coli MS 69-1 on minimal
media containing 30 mM choline or 30 mM glycerol and on BHI, a
rich medium. Cultures were supplemented with either phosphate
buffer (no inhibitor) or betaine aldehyde and grown under N2. (B)
Betaine aldehyde inhibits d9-TMA formation from d9-choline in cut
gene cluster-harboring gut bacteria, including Proteus mirabilis
BB2000, Escherichia coli MS 69-1, Clostridium sporogenes ATCC
15579, and Klebsiella sp. MS 93-2. Production of d9-TMA was
this molecule in the active site due to these favorable
interactions.
measured by LC-MS/MS. Error bars represent the mean
SD of
CutC is essential for the anaerobic growth of cut gene
cluster-containing E. coli when choline is provided as the sole
carbon source and fumarate is a terminal electron acceptor.22
We thus explored whether inhibition of CutC by betaine
aldehyde would affect anaerobic growth of the choline-
metabolizing strain E. coli MS 69-1 in minimal medium
containing 30 mM choline as the sole carbon source. Under a
N2 atmosphere, 20 mM of betaine aldehyde effectively
abolished growth on choline but had no impact on growth
in minimal medium containing 30 mM glycerol as the sole
carbon source or growth in BHI, a complex medium
containing 2 g/L of glucose (Figure 4A), suggesting that
betaine aldehyde may specifically target anaerobic choline
metabolism. This result, along with previous work in
gnotobiotic animal models,22 indicates that anaerobic choline
metabolism is nonessential when alternative carbon sources are
present and suggests that inhibiting CutC activity may not
dramatically alter gut microbiota composition. Finally, as
betaine aldehyde is endogenously produced in bacteria as an
intermediate in the conversion of choline to glycine betaine,
we monitored its stability in cultures grown anaerobically on
choline (Figure S3) and BHI media (Figure S4). We did not
observe metabolism of d9-betaine aldehyde in either condition.
The cut genes are widely distributed among human gut
bacteria and likely encode a major pathway for choline
utilization in the GI tract.23 We therefore tested whether
betaine aldehyde could inhibit TMA production by additional
three replicates. (C) Betaine aldehyde inhibits d9-TMA formation
from d9-choline by a human fecal suspension. Production of d9-TMA
was measured by LC-MS/MS. Error bars represent the mean SD of
two replicates.
Gram-positive (Clostridium sporogenes ATCC 15579) and
Gram-negative (Proteus mirabilis BB2000, Escherichia coli MS
69-1, Klebsiella sp. MS 93-2) choline-metabolizing human gut
isolates. Betaine aldehyde was effective against all strains
assayed, with EC50 values ranging from 1 to 5 mM (Figure 4B).
Finally, betaine aldehyde inhibited the conversion of choline to
TMA by a human fecal suspension ex vivo (Figure 4C),
confirming its efficacy in a complex setting more reminiscent of
the gut microbiota. Overall, these findings demonstrate the
feasibility of inhibiting anaerobic choline metabolism across a
range of bacteria and in a complex mixture.
Previous work has explored inhibiting microbial choline
metabolism as a potential treatment for atherosclerosis.21 The
lead compound from this study, DMB (12), attenuated plasma
TMAO levels and reduced the size of aortic lesions in mice fed
a high-choline diet. Described as a specific inhibitor of TMA
production, DMB has been used in additional animal
studies.24,25 Unexpectedly, we found DMB did not reduce
TMA production from choline by CutC in vitro or in E. coli.
This result may be explained by the inability of DMB to engage
in critical hydrogen bonding interactions within CutC’s polar
C
DOI: 10.1021/jacs.8b04883
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Journal of the American Chemical Society
Communication
active site. As DMB is rapidly metabolized in vivo by liver
alcohol dehydrogenase,21 we wondered whether an oxidized
metabolite could be responsible for the phenotypes observed
in mice. However, the corresponding aldehyde, DMBA (13),
was a relatively poor inhibitor of TMA generation by E. coli.
Other aldehydes lacking a trimethylammonium moiety were
also poor inhibitors of CutC activity in vitro (Figure S5). Using
the same P. mirabilis strain employed by Wang et al.,21 we
observed minimal inhibition of TMA production by whole
cells with DMB and DMBA (5 mM), whereas betaine aldehyde
had an EC50 of 2.3−11.1 μM (Figure S6). DMB was also
ineffective at inhibiting TMA generation by P. mirabilis cell
lysates (Figure S7). We therefore conclude that DMB does not
directly target anaerobic choline metabolism under these
conditions and may not be an appropriate tool compound for
studying this microbial activity. These findings also suggest the
TMAO-lowering activity of DMB in vivo may not originate
from inhibiting CutC.
In summary, we have identified a small molecule that
inhibits the gut bacterial choline-metabolizing enzyme CutC.
This compound and the workflow used to characterize its
activity will inform future inhibitor development. Although
small molecules that specifically disrupt pathogen−host
interactions have been discovered,26 the use of such
compounds to modulate activities from the human gut
microbiota remains largely unexplored. Notable exceptions
are gut bacterial β-glucuronidase inhibitors that prevent
reactivation of glucuronide−drug conjugates.27 Small mole-
cules that selectively modulate additional gut microbial
activities would aid in deciphering microbe−microbe and
microbe−host interactions that influence host biology and may
provide a novel approach for treating disease.
ern Collaborative Access Team beamlines, which are
supported by Award GM103403 from the National Center
for Research Resources at the National Institute of Health. The
Pilatus 6M detector on 24-ID-C beamline is funded by the
Office of Research Infrastructure Programs High-End
Instrumentation Grant No. S10 RR029205. Use of the
Advanced Photon Source is supported by the U.S. Department
of Energy, Office of Basic Energy Sciences, under Contract No.
DE-AC02-06CH11357. We acknowledge Peter Turnbaugh
(University of California San Francisco) for supplying the fecal
sample.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b04883.
■
S
Experimental data and procedures, crystallographic data
(PDF)
AUTHOR INFORMATION
Corresponding Author
*balskus@chemistry.harvard.edu
■
ORCID
Michael A. Funk: 0000-0003-1898-9008
Catherine L. Drennan: 0000-0001-5486-2755
Emily P. Balskus: 0000-0001-5985-5714
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge support from the Packard Fellowship for
Science and Engineering (E.P.B.), the National Science
Foundation (EAGER MCB-1650086) (E.P.B.), and a
Blavatnik Biomedical Accelerator Award (E.P.B.). S.B. is
supported by an HHMI International Student Research
Fellowship. M.A.F. is supported in part by the National
Science Foundation Graduate Research Fellowship under
Grant No. 0645960. C.L.D. is a Howard Hughes Medical
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