Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer

Article abstract of DOI:10.1074/jbc.RA120.016019

Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease, and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail α3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located >40 A apart in the same monomer but adjacent in two neighboring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in intersubunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π- cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

Full text of DOI:10.1074/jbc.RA120.016019

JBC Papers in Press. Published on November 6, 2020 as Manuscript RA120.016019
The latest version is at https://www.jbc.org/cgi/doi/10.1074/jbc.RA120.016019
Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition and
inter-subunit electron transfer
1
2
1
1
Mussa Quareshy , Muralidharan Shanmugam , Eleanor Townsend , Eleanor Jameson ,
3
1
1
Timothy D.H. Bugg , Alexander D. Cameron , Yin Chen
1
School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, U.K.
Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street,
2
Manchester, M1 7DN, U.K.
3
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, U.K.
Correspondence to Dr. M Quareshy (mussaquareshy@gmail.com) or Prof. Y Chen
(
y.chen.25@warwick.ac.uk, 00442476528976)
Running title: structure and inhibition of CntA
Keywords: gut microbiota, carnitine oxygenase, CntA, EPR, inhibitor
1

Abstract
wellbeing and disease states. There is a
considerable interest in the past decade to
investigate the formation of methylated
amines in human health, particularly with
regard to cardiovascular diseases and non-
alcoholic fatty liver diseases (1-5). Dietary
intake of quaternary amines such as
choline and carnitine, both of which are
prevalent in the human diet, can be
processed by gut microbiota to produce
Microbial metabolism of carnitine to
trimethylamine (TMA) in the gut can
accelerate atherosclerosis and heart
disease and these TMA-producing
enzymes are therefore important drug
targets. Here, we report the first structures
of the carnitine oxygenase CntA, an
enzyme of the Rieske oxygenase family.
CntA exists in a head-to-tail  trimeric
3
structure. The two functional domains (the
Rieske and the catalytic mononuclear iron
domains) are located > 40 Å apart in the
same monomer but adjacent in two
small
methylated
amines
(e.g.
trimethylamine, TMA), which enter
vascular circulation leading to subsequent
hepatic oxidation to trimethylamine oxide
(TMAO). TMAO is linked to cardiovascular
disease, kidney disease, diabetes and
various forms of cancers (6-8). These TMA-
producing and metabolising enzymes from
gut microbiota therefore represent
promising new drug targets (9-11).
neighbouring
determination of CntA and subsequent
electron paramagnetic resonance
monomers.
Structural
measurements uncover the molecular
basis of the so-called bridging glutamate
(
E205) residue in inter-subunit electron
transfer. The structures of the substrate-
bound CntA help to define the substrate
pocket. Importantly, a tyrosine residue
The key microbial enzymes responsible for
TMA formation from choline and carnitine
have only been identified relatively
recently (12-14). Choline-TMA lyases
belong to a large family of proteins,
attacking the carbon-nitrogen (C-N) bond
in choline using a radical species generated
from a conserved glycine residue. The
structure of choline-TMA lyase CutC has
been solved recently (16-17), aiding the
development of inhibitors (substrate
analogues) for attenuating TMA formation
from choline (18-21). The carnitine
monooxygenase (CntA) and associated
reductase (CntB) responsible for carnitine
oxidation to TMA (Figure 1A) was originally
identified in Acinetobacter spp. but was
subsequently found to be present in a
range of gut microbiota species (13-15).
CntA belongs to a large group of non-heme
iron-containing Rieske oxygenase family
(
Y203) is essential for ligand recognition
through a π-cation interaction with the
quaternary ammonium group. This
interaction between an aromatic residue
and quaternary amine substrates allows us
to delineate a subgroup of Rieske
oxygenases (group V) from the prototype
ring-hydroxylating Rieske oxygenases
involved in bioremediation of aromatic
pollutants
in
the
environment.
Furthermore, we report the discovery of
the first known CntA inhibitors and solve
the structure of CntA in complex with the
inhibitor, demonstrating the pivotal role of
Y203 through a π-π stacking interaction
with the inhibitor. Our study provides the
structural and molecular basis for future
discovery of drugs targeting this TMA-
producing enzyme in human gut.
(
13). Rieske oxygenases are typically
multicomponent enzyme systems
Introduction
involving an oxygenase, a reductase and
sometimes a separate flavin cofactor (22).
The
vast
array
of
co-habiting
microorganisms in the human gut impose
discernible influence on human
The oxygenase component has
a
a
2

conserved
coordinated by two cysteine and two
histidine residues and catalytic
[2Fe-2S]
Rieske
centre
provides novel insights into the mode of
inhibition and the inter-/intra- subunit
electron transfer in carnitine oxidation.
Our work therefore provides the structural
and molecular basis for future discovery of
drugs targeting this TMA-producing
enzyme in human gut.
a
mononuclear iron (Fe) centre (23-24). A
reductase component reduces pyridine
nucleotides, generating electrons which
are ultimately transferred to the Rieske
oxygenase for substrate oxidation. The
archetypal Rieske oxygenases, also known
as ring-hydroxylating Rieske oxygenases,
catalyse the oxidation of a range of
aromatic and polyaromatic substrates; as
such, they are important in bioremediation
of environmental pollutants (24-25).
Structural determination of several ring-
hydroxylating Rieske oxygenases (e.g.
naphthalene dioxygenase and biphenyl
dioxygenase) reveals that the Rieske
centre and the catalytic mononuclear Fe
centre are usually far apart in the same
subunit (>40 Å) and effective electron
transfer can only occur across the interface
of two neighbouring subunits (23, 26, 27).
Results
Structure determination of CntA
We set out to solve the structure of CntA
from A. baumannii and we successfully
obtained structures of CntA in the apo
protein form (2.1 Å, PDB 6Y8J) and ligand-
bound forms with two substrates, carnitine
(2.0 Å, PDB 6Y9D) and -butyrobetaine
((gBB), 1.6 Å, PDB 6Y8S) (Table 1). We
observed a head-to-tail 3 trimeric
structure for CntA (Figure 1B), with the
Rieske [2Fe-2S] cluster and mononuclear
Fe centre at opposing regions of the
monomer 44 Å apart (Figure 1C). The
observed assembly of  homotrimer is in
3
agreement with the analyses by native
protein gel (13) and analytic gel filtration
It has becoming increasingly clear that
quaternary
amines-oxidising
Rieske
(
Figure S1). The two CntA substrate bound
oxygenases represent a new emerging
clade of the Rieske oxygenases family that
is distinct from the archetypal ring-
hydroxylating aromatic Rieske oxygenases
(
13, 27-29). The carnitine-degrading CntA
enzyme is a promising new drug target,
given that gut microbial metabolism of
carnitine is known to increase plasma
TMAO, leading to the subsequent
development of atherosclerosis and
cardiovascular diseases (9). However, no
structure of CntA or description of possible
inhibitors have been published. In this
study, we report high-resolution crystal
structures of CntA with and without
substrates-bound and report the first
known inhibitors for this enzyme and the
crystal structure of an inhibitor-bound
CntA. Structure, biochemical and electron
paramagnetic
resonance
(EPR)
characterisation CntA and mutants
3

histidine ligands (His 213, His 208) and a
bidentate aspartate residue (Asp 323) in a
well-documented His-His-Asp catalytic
triad (31), co-ordinating one face of the Fe
centre (Figure 1E). A pair of reduced Cys
apart in the same subunit. As such,
electron transfer to the catalytic
mononuclear Fe centre is likely to occur
only at the interface of two adjacent CntA
subunits (12.2 Å apart) and facilitated by
the so-called bridging glutamate (E205,
Figure 2A) (13). Electron transfer from
NADH to the carnitine oxygenase CntA is
mediate through an flavin mononucleotide
(FMN)-containing reductase CntB, which
was essential for the enzyme activity (13).
To monitor electron transfer pathway
(Figure 2B) from the reductant NADH via
the flavin-containing reductase CntB to the
catalytic domain in the oxygenase CntA,
continuous wave (cw)-EPR was used.
(
Cys206, Cys209) is found near the
mononuclear Fe in CntA whereas in
stachydrine demethylase Stc2 the
corresponding Cys formed a disulphide
bridge (28). A water molecule and a
-
thiocyanate [SCN] anion occupy the two
co-ordination sites, which are 2.2 Å and 2.3
Å away from the mononuclear Fe centre in
CntA, respectively (Figure 1E, Figure S2C).
-
Both the water molecule and [SCN] are
positioned 3.7 Å below the C adjacent to
the carnitine ammonium group, which is
expected to be the site of hydroxylation
catalysed by this enzyme (Figure 1E). SCN-
Purified CntB is EPR-silent (Figure
2C, black trace); however, the reduced
ferredoxin-centre in CntB is readily
detectable once NADH is added (red trace).
The EPR spectrum of reduced CntB has a
characteristic reduced ferredoxin [2Fe-
bound at the O site likely resulted in the
2
stabilisation of CntA co-crystalized with the
substrates. The catalytic mononuclear Fe
2+
+1
in purified CntA is in a ferrous (Fe ) state,
showing no EPR signals associated with
either high-spin (S = 5/2) or low-spin (S = ½)
2S] signal [g tensor 2.031, 1.937, 1.899]
and a radical signal resulted from flavin
semiquinone gave=2.0015. Similar flavin
semiquinone radicals have been observed
previously in other Rieske oxygenase
reductase as well as monoamine oxidase
(35, 36). Reduced CntB spectrum was
simulated by combination of two different
S = ½ spin states (97 % for the reduced
ferredoxin and 3 % for a flavin
semiquinone radical) (Table 2), confirming
electron transfer from the reductant NADH
to the ferredoxin domain in CntB (steps 1-
3 in Figure 2B). The purified CntA (blue
trace) showed a very weak anisotropic EPR
signal with gave < 2.0 [g tensor 2.011, 1.916,
1.757], indicating that the Rieske centre in
CntA is predominantly in the oxidized,
3+
ferric centre (Fe ) despite purifying the
protein under aerobic conditions. This is
confirmed by the addition of nitric oxide
(
NO) to CntA, showing an EPR signal from a
2+
S = ½ species of the Fe -NO adduct (Figure
F). This is contrast to the other non-heme
1
oxygenase enzymes, where NO binding to
the mononuclear iron centre often led to
the formation of high-spin, S = 3/2 signal
(
32). This plausibly suggests that the
mononuclear iron ion exists as an S = 0 in
its ferrous state. The observed EPR signal
2+
for the Fe -NO adduct is similar to the
previously reported heme/non-heme-NO
adducts (33-34).
2+
diferric [2Fe-2S] state (Table 2) (37-38).
Addition of an oxidizing agent (H ) to
Structural basis of long-distance electron
transfer and EPR spectroscopy
Structural determination of CntA revealed
that the two functional domains in CntA
O
2 2
CntA has minimal effect on the overall
spectrum (Figure S3A), however, the
Rieske centre in CntA can be readily
reduced by dithionite (Figure S3B). CntA
also appears capable of oxidising the
(
the Rieske domain and the catalytic
mononuclear Fe domain) are located 44 Å
4

substrate (carnitine) using H
2
O
2
in the
and the isotropic signal at 3350 G is likely
originated from a new organic radical
species, which is formed during CntA
catalysis in the presence of the substrate.
The EPR simulation from these two S = ½
species (reduced Rieske cluster and a new
organic radical species) reproduced the
observed spectra (Table 2, Figure S4). The
complete loss of reduced ferredoxin EPR
signal from CntB, together with the
formation of a novel organic radical species
(isotropic signal seen at 3350 G) in
CntA+CntB+NADH+carnitine suggests an
efficient electron transfer to the catalytic
mononuclear Fe in the wild-type CntA
(step 5 in Figure 2B).
absence of CntB/NADH (Figure S3A),
consistent with the peroxide shunt-
mechanism known in other Rieske
oxygenases (39-40). In order to monitor
electron flow from CntB to CntA, the
spectrum of CntA +NADH+CntB was
recorded (magenta trace). This complex
spectrum can be simulated by the
combination of three different S = ½ spin
states (Table 2, Figures S4). Thus, the
addition of CntB+NADH to CntA allowed
reduction of the Rieske centre in CntA,
confirming cross-subunit electron transfer
from CntB to CntA (step 4 in Figure 2B).
We next sought to confirm the
electron flow from the reduced Rieske
cluster to the catalytic mononuclear Fe
centre in CntA and investigate the critical
role of the bridging E205 in electron
transfer as suggested by the CntA structure
To further support this proposed
electron transfer pathway, we compared
EPR spectra of the E205A mutant with the
wild-type CntA. Indeed, the EPR spectra
are significantly different between E205A
(Figure 2C, purple trace) and the wild-type
CntA (cyan trace). The spectrum of the
E205A mutant shows complex EPR signals
with 4 different S = ½, EPR active species
(Table 2). In the spectrum with the E205A
mutant, a significant amount (60 %) of the
(
step 5 in Figure 2B). The E205 residue
bridges the Rieske cluster on one subunit
and the mononuclear Fe centre in the
adjacent subunit (Figure 2A) (13, 41). None
of the E205 mutants that we generated in
this study were active (Figure 2D), with no
major perturbations to the overall
secondary structure as observed by
circular dichroism (Figure 2E). As it is
known that substrate-binding can be
required for the activation of the catalytic
mononuclear Fe centre to initiate catalysis
1+
EPR signal of the reduced [2Fe-2S]
ferredoxin in CntB was observed while this
was not found in the spectrum of the wild-
type CntA, suggesting that electron flow is
significantly impacted in this mutant.
Together, the EPR observations provide
strong evidence of each step involved in
the electron flow as proposed in Figure 2B
and confirm the role of the bridging
glutamate (E205) in cross-subunit electron
transfer as suggested by the CntA
structure.
(
42-43), the substrate (carnitine) was
added to the reaction and the EPR
spectrum was monitored (Figure 2C, cyan
trace). The spectrum shows no EPR signals
at low magnetic fields, ruling out the
presence of high-spin (S = 5/2 or 3/2) ferric
species in the sample. However, strong
isotropic (3350 G) and highly anisotropic
EPR signals are observed between the
magnetic fields of 3200-3900 G (Figure 2C).
The observed gave < 2.0 (Table 2) strongly
indicates that these EPR transitions likely
arise predominantly from one-electron
Structural basis of substrate binding and
inhibition
CntA can catalyse the oxidation of carnitine
and several substrate analogues (Figure
3A). The high resolution CntA structures
co-crystalized with the ligands (carnitine,
2.0 Å; gBB, 1.6 Å) allowed us to define the
+
1
reduced [2Fe-2S] Rieske cluster in CntA
5

substrate binding pockets. The ligands
were built into the clear density which was
much better defined for the carnitine
structure (Figure 3B). The substrate-
binding site is formed by a series of -
sheets and two -helices coordinating the
substrate through hydrophobic/steric
interactions involving residues Phe 258,
Tyr 315, and Phe 319 and polar
interactions with residues Tyr 225, Asn
on the presence or absence of the
signature aromatic residue at position 203
(Y203 or F203), group V Rieske oxygenases
can be further separated into two clades
Va and Vb where in clade Vb the Tyr (or Phe
in OxyBAC) is replaced by an Asn (Figure
4C). Substrates for the former clade
include carnitine (CntA, 13), choline
(CmoA, CmoS, CmoB, 45-47) and
benzalkonium (OxyBAC, 29) whereas the
latter clade Vb enzymes undertake
oxidative demethylation from the
ammonium group for glycine betaine
2
70, and Tyr 295. (Figure 3C, Figure S5).
The Phe 319 residue is positioned at the
kink of an observed -helix feature for
residues 306 to 337 (44). Importantly, we
also observe that Tyr 203 forms a -cation
interaction with the positively charged
ammonium group in the substrates (Figure
(
(
GbcA, BmoA, 27) and proline betaine
stachydrine, Stc2, 28).
Comparing
structures of CntA (group Va) and Stc2
group Vb) uncovers striking differences in
the
ligand-bound
(
3C). The importance of the binding site
the way quaternary amine substrates are
oriented (28). In Stc2 the substrate
analogue proline is ligated with the
catalytic Fe through the carboxylate group
residues was then tested by mutating them
to alanine. Indeed, all mutants decreased
or abolished activity (Figure 3D).
Interestingly, the Y203F mutant was active
(
Figure 4D), whereas in CntA, although a
(
Figure 3D), demonstrating a recovery of
carboxylate group is present, the
substrates were oriented via a -cation
interaction with the trimethylammonium
moiety and the carboxylate group is not
ligated with the mononuclear Fe (Figure
function and the pivotal role of the -
cation interaction with the ammonium
group.
The presence of an aromatic
residue in this position (Y203 or F203)
appears to be restricted to CntA and
several closely related enzymes. Indeed, a
comprehensive phylogenetic analysis of
Rieske oxygenases, including several other
recently defined Rieske enzymes involved
in the oxidation of quaternary ammonium
substrates clearly defined a group which
delineates quaternary ammonium Rieske
oxygenases (group V) from the well-
3C).
In order to understand the
substrate specificity for the enzyme, we
investigated the structure activity
relationship (SAR) of 22 substrate
analogues with differences on the aliphatic
chain, the carboxyl group or the amine
terminal groups (Figure 5A, Figure S6). In
addition to carnitine/-butyrobetaine
(
compound 1 and 2), CntA can also oxidise
studied
ring-hydroxylating
Rieske
meldonium (compound 3) and 1-butyl-
trimethylammonium (compound 4) albeit
with a lower affinity (Figure 3A, Figure 5A).
The lack of a carboxylic acid in compound
oxygenases (Figure 4A) (27, 29). While
group I-IV Rieske oxygenases primarily
catalyse the dihydroxylation of aromatic
and polyaromatic compounds that are
4
reduces the affinity for the substrate and
important
environmental pollutants, group V Rieske
oxygenases include enzymes that
in
bioremediation
of
substrates with longer alkyl chains or other
functional groups showed no activity
(
compounds 5-9). A further series of
principally oxidise quaternary ammonium
substrates (Figure 4B). Interestingly, based
compounds related to -butyrobetaine but
6

not
quaternary
ammonium
salts
of carnitine in CntA (Figure 5B), also play a
key role in binding to the inhibitor (Figure
S8). Tyr 203 form a π-π stacking interaction
with the 1,6‐naphthyridine core ring and
Tyr 295 forms polar interaction with the
oxygen on the methoxy group in the
inhibitor. Interestingly, the inhibitor is also
coordinated to the mononuclear Fe via a
nitrogen on the 1,6‐naphthyridine ring at
distance of 2.65Å and to an oxygen of the
carboxylic acid moiety of Asp 323 at 2.7Å
(
compounds 10- 12) did not serve as
substrates for CntA. Other quaternary
amine compounds tested were not
accepted as substrates (Figure S6).
Together, structural, mutagenesis and SAR
data therefore suggest that the substrates
for CntA are primarily co-ordinated
through a -cation interaction between
Y203 and the trimethylammonium group,
which positions the substrate in place
above the mononuclear Fe site for
oxidative cleavage to occur (Figure 5B).
Although there is some tolerance for the
lack of a carboxylic acid, increases in the
length of the molecule and increased steric
bulk are not favoured for enzyme activity
as they likely disturb the polar interaction
with Tyr 295.
(
Figure S8). Other π-π interactions are also
observed between Phe 319 and the phenyl
methoxy group and between Phe 258/ Phe
2
47 and the imidazole group. When the
binding pockets of carnitine and MMV12
are compared (Figure 6E & F), Phe 258
moves to accommodate the imidazole
group of the inhibitor whilst the loop
containing Phe 216 is drawn in closer to the
active site to form the π-π interaction with
the inhibitor.
A competitive inhibitor binds to the same
substrate recognition pocket through π-π
stacking interactions
The two other inhibitors MMV1
Encouraged by previous success in the
identification of substrate mimics as
potent inhibitors for the choline-TMA lyase
CutC (18-21), we also screened these
carnitine analogues (Figure 5A, Figure S6)
as potential inhibitors of CntA in
competition assays. However, none of
these substrate analogues inhibited CntA
activity. We thus used a random approach
by screening drug libraries and successfully
obtained 3 inhibitor compounds MMV1,
MMV3 and MMV12 (Figure 6A). Their
inhibitory IC50 values are in the range of
and MMV3 share similar structural
features to MMV12. MMV1 has a core
bicyclic fused aromatic scaffold with two
branching cyclic groups. MMV3 has an
imidazole group with an aliphatic amine
linker group. The remaining pair of
inhibitors show more complex patterns of
inhibition for CntA. Using the structure of
CntA co-crystalized with MMV12, we
applied in silico docking of MMV1 and
MMV3 to observe if these molecules could
adopt comparable poses (Figure S9). The
results showed that both compounds show
logical poses in the binding site with
docking scores of -10.576 (kcal/mol) and -
1
.1-5.8 μM (Figure 6B). From competition
assay data (Figure S7) we were able to
derive a K (1.09 μM) for MMV12 (ID,
i
11.169 (kcal/mol), respectively. These are
MMV020670), indicating that it is likely a
competitive inhibitor.
comparable to a score of -11.7 (kcal/mol)
when MMV12 is docked into the empty
pocket in CntA. Both docked compounds
demonstrate likely coordination to the Fe
centre via a nitrogen atom and similar π-π
stacking interactions.
We successfully solved the
structure of MMV12 with CntA (PDB, 6ZGP)
and indeed, the inhibitor occupied the
substrate binding pocket (Figure 6D).
Interestingly, Tyr 203 and Tyr 295, both of
which are important for the coordination
In order to assess the activity of
these inhibitors, we monitored the impact
7

of these inhibitors on the production of
TMA in A. baumannii cells pre-grown on
carnitine. Indeed, we observed a decrease
in TMA production in the presence of
MMV3 and MMV12 (Figure 6C), whilst
MMV1 did not inhibit TMA formation in
the bacterial culture. MMV3 has almost
completely inhibited TMA formation while
MMV12 caused ~20% reduction in TMA
production. It is worth mentioning that
MMV3 was originally developed for
treating tuberculosis and the differences in
inhibitor activity towards A. baumannii
cells may reflect their ability to penetrate
bacterial cell membranes.
newly discovered inhibitors. The structure
and EPR analysis also allow us to better
understand the structural basis of long-
range electron transfer across the
interface of CntA subunits.
Rieske oxygenases are challenging
proteins to study and this was no different
in the case of CntA. Different expression
strategies or the choice of buffers and
additives may have contributed to the
apparent differences in enzyme activities
among studies of CntA protein (13, 50).
Due to sensitivity to oxygen, considerable
effort was made in our work in order to
obtain CntA crystals. It is worth noting that
the presence of SCN in the crystallisation
media was essential to obtain CntA
crystals. Given that we observe the SCN
coordinated to the Fe centre in CntA-
carnitine and CntA-gBB complex, we
investigated the effect of additional SCN on
the activity of CntA. Indeed, SCN inhibited
CntA activity with the half maximal
inhibitory concentration (IC50) of 35 mM
(Figure S10A) and EPR analysis showed that
the addition of SCN may have affected
inter-/intra subunit electron transfer
(Figure S10B). It is therefore likely that SCN
affects CntA functionality by coordinating
to the Fe centre and preventing oxygen
binding, which in turn helps to stabilise the
protein for crystallization.
Discussion
A major breakthrough in gut microbiome
research over the past decade is the
realization that gut microbiota play an
integral role in human health and diseases
(
48). This is exemplified in the study of
TMA-formation from gut microbiota and
its implication in the development of
atherosclerosis
and
cardiovascular
diseases (9-11). Recent progress using
experimental animals and human clinical
studies have shown that TMA precursors
such as choline, carnitine and -
butyrobetaine promote atherosclerosis via
the TMAO pathway through the interplay
between gut microbiota and host
metabolism (49). The identification of the
key microbial enzymes, including the
choline-TMA lyase (12) and CntA (13)
provides promising new drug targets for
attenuating TMA release from gut
microbial metabolism of TMA-precursors.
Although much has been learnt from the
structures of choline-TMA lyase and
subsequent development of selective
Our study shows that CntA forms a
head-to-tail
homotrimer
and
the
substrates appear to be poised in place
next to the mononuclear Fe centre ready
for cleavage. Despite both substrates (i.e.
carnitine and -butyrobetaine) possessing
carboxylate moieties, these are not
coordinated to the mononuclear Fe centre
as might be expected and that has been
observed for group Vb Rieske oxygenase
such as proline-bound stachydrine
demethylase Stc2 (28). The Y203 residue is
strictly conserved in all CntA sequences
which is key for a -cation interaction to
coordinate the ammonium moiety of the
inhibitors
for
choline-to-TMA
transformation (18-21), little progress has
been made on CntA due to the lack of CntA
structures. Here in this study, we provide
the structural basis of substrate co-
ordination and mode of inhibition by the
8

substrate and for a - stacking interaction
to the inhibitor (Figure 6). This
corresponding aromatic residue is also
present in other group Va Rieske
methyl‐3,6‐dihydro‐2H‐pyridin‐3‐ol (18-
21). FMC is reported as the most potent
inhibitor with an EC of 56 nM by forming
an irreversible covalent bond in the CutC
catalytic site (20). Encouragingly and
particularly relevant to CntA is the
development of inhibitors based on the
structure of -butyrobetaine against the
50
oxygenases,
monooxygenase (tyrosine) and OxyBAC
phenylalanine) (Figure 4), suggesting that
such
as
choline
(
such a -cation interaction is commonly
used for orientating choline and
benzalkonium during catalysis. Conversely,
when compared to the stachydrine and
glycine betaine degrading enzymes, Stc2
and BmoA/GbcA respectively, there is not
an aromatic residue present in the same
position. The structure for Stc2 showed
that stachydrine coordinates to the
mononuclear Fe through the carboxylate
group and the suggested mechanism
requires this binding mode in order to
cleave the methyl substituent (28). This
insight suggests a differentiation amongst
the Group V members and thus a
distinctive way to classify quaternary
ammonium catalysis by such Rieske
oxygenases.
carnitine-producing
-butyrobetaine
hydroxylase from human (52). The
inhibitors were derivatives of the
trimethylammonium, aliphatic chain and
carboxylate
region
with
some
demonstrating a similar binding mode to
the native substrate but with higher
affinities. The structure of CntA, together
with the information inferred from the
substrate mimics and the competitive
inhibitors that we reported here in this
study, could be investigated in a rational
drug design approach to formulate more
potent CntA inhibitors for future
exploitation. The data presented here
suggest CntA substrate analogues are less
likely to inhibit its activity and rational drug
design should perhaps take advantage of
the presence of several aromatic residues
in the binding pocket to utilize a -
stacking interaction as we have
demonstrated in the inhibitor compound
Among the substrate analogues
tested for CntA, activity was only seen in
compounds containing a quaternary
ammonium group, In agreement with the
pivotal role of the π-cation interaction for
substrate coordination. Given the link
(
Figure 6).
Taking advantage of the CntA
between
TMA
production
and
cardiovascular diseases, targeting CntA
with small molecule inhibitors could have a
therapeutic potential. Inhibitors for CntA
may also be useful to treat individuals with
structure we solved in this study, we
employed EPR analysis to track the
activation and progression of the electron
transfer pathway involving the carnitine
oxygenase complex. We propose that
during catalysis, CntA and its associated
reductase, CntB approach and electron
transfer from the one-electron reduced
a
socially
challenging
condition
trimethylaminuria (TMAU) or commonly
referred to as the “fish odour syndrome”
(
51). An elegant example of such rational
+
1
designed inhibitors starting from the
endogenous substrate has been reported
for the choline-TMA lyase, CutC with DMB
ferredoxin [2Fe-2S]
oxidized Rieske [2Fe-2S] effectively takes
place. Efficient electron transfer from the
in CntB to the
2+
1+
(
3,3-dimethyl-1-butanol), DMBA (3,3-
dimethylbutyric aldehyde), FMC (N-
fluoromethyl)-2-hydroxy-N,N-
dimethylethan-1-aminium) and (3S) ‐ 1 ‐
reduced Rieske [2Fe-2S] centre to the
catalytic mononuclear Fe centre in CntA
requires both substrate, carnitine and the
crucial bridging glutamate reside E205,
(
9

mutation of which significantly alters
electron flow (Figure 2C), resulting in the
loss of enzyme activity (Figure 2D). This
was also independently validated by a
recent study showing that both E205D and
E205Q mutants lost enzyme activity and
the ability for electron transfer to the
active centre (13, 50). Binding of the
substrate to the catalytic mononuclear Fe
basis to facilitate rational design and
further optimization of specific inhibitors
targeting carnitine-to-TMA transformation
by gut microbiota.
Methods
Cloning of CntAB and site-directed
mutagenesis of CntA. The plasmids for
expressing CntA and CntB of A. baumannii
were constructed previously (13). All A.
baumannii CntA mutants were chemically
synthesized by GenScript and cloned into
the pET28a(+) expression vector using the
NdeI and HindIII sites. All plasmids were
transformed into BL21(DE3)pLysS cells for
protein expression as described previously
(13).
centre caused a shift in g
3
tensor from
3
810 G to 3750 G (Figure 2C). This suggests
that substrate entrance might influence
the redox-potential of the mononuclear Fe
centre in CntA, possibly influencing the
electron transfer from the reduced Rieske
centre to the mononuclear Fe centre in
CntA although its impact on the one
electron reduced Rieske centre cannot be
completely ruled out. The terminal step of
the electron transfer pathway is the
formation of an organic radical species
Protein expression and purification. For
each respective protein, recombinant E.
coli strains were streaked from glycerol
stocks onto LB agar plates with kanamycin
(50 µg/mL) and incubated overnight at
37°C. Recombinant protein expression was
induced with 0.2 mM IPTG into liquid
culture in LB medium. After 18 hours
incubation the cells were collected by
centrifugation at 6,000 rpm at 4°C for 10
minutes and the pellet was resuspended in
20mL PBS + 0.3M NaCl on ice, aliquoted
and frozen at -80°C. For purification the
standard buffer conditions were 20 mM
Tris-HCl pH 7.6, 250 mM NaCl and 0.5 mM
tris(2-carboxyethyl)-phosphine (TCEP). To
purify CntA and CntB proteins, cells were
broken by sonication in the standard buffer
and the cell-free extract was loaded on a
Roche Complete resin for affinity
purification. The resin was then washed
with standard buffer + 5 mM imidazole and
the target protein was eluted with the
standard buffer + 250 mM imidazole + 10%
(
Table 2) which is likely to be crucial for
CntA catalysis. Its identity, however, has
yet to be elucidated. Potential active
intermediates in other well studied Rieske
oxygenases include Fe(III)-(hydro)peroxy
followed by a rearrangement leading to a
high valent hydroxo-Fe(V)=O or Fe(IV)=O
species (53-55). The active high valent
catalytic Fe in CntA catalysis certainly
warrants further investigations. However,
the methodology established here may
also facilitate the discovery of novel CntA
inhibitors targeting the electron transfer
pathway.
To sum up, this work provides the
structural basis of carnitine degradation by
CntA. Structural determination of CntA and
spectroscopic characterisation using EPR
provides structural and mechanistic insight
into the role of bridging glutamate (E205)
in inter-subunit electron transfer. Co-
crystallisation of the substrates and the
competitive inhibitor with CntA unveils the
role of the π-π stacking interaction as the
key mode of inhibition. The work
presented here provides the structural
(v/v)
glycerol. The
elution was
concentrated in a Vivaspin 20 10,000 kDa
cut-off to 2.2 mL before loading onto a
Superdex 200 16/600 column using an
ÄKTA purifier and buffer exchanging into
10

1
0 mM HEPES, 250 mM NaCl, 0.5 mM TCEP
and 10% glycerol (v/v) buffer for storage or
0 mM HEPES pH 7.6, 10 mM NaCl and 0.5
Crystals were cryoprotected in an
equivalent solution to the mother liquor
supplemented with 5% glycerol and flash
1
mM TCEP for crystal growth. For storage,
the protein was concentrated to 2mg/mL
in a Vivaspin 6 10,000 kDa and aliquots
were kept frozen at -20°C, for
cooled
in
liquid
nitrogen.
For
CntA+MMV020670, MMV020670 was
added to a final concentration of 250 µM
in the crystallisation condition buffer drops
that were streaked and yielded the same
red hexagonal crystals 36-48 hours after
setting up. The crystals were collected and
stored as mentioned above.
crystallography
concentrated to the desired amount
between 5 to 50 mg/mL) and used
the
protein
was
(
immediately in crystallisation setups.
Purified CntB was estimated to have ~ 0.85
FMN per CntB using the method of Batie et
al. (1987) by measuring absorbance at 462
nm against FMN standards ranging from
The crystals were mounted robotically on
the i24 beamline (apo, carnitine and -
butyrobetaine)
and
i04
beamline
0
.3 to 321.5 µM (35).
(CntA+MMV020670) at the Diamond Light
Source (Harwell Science and Innovation
Campus, Didcot, UK). In the apo and -
butyrobetaine collections, we observed
split spots and streaking of spots on some
images. The apo, carnitine and
MMV020670 data were auto-processed
with Dials (56) using the Xia2 pipeline at
Diamond Light Source followed by scaling
with Aimless (57). For the -butyrobetaine
dataset we processed the data using the
Dials User Interface (DUI) followed by
scaling with Aimless in the CCP4i2 suite of
tools (58). An initial model was derived
from PDB 3VCP. Molecular replacement
was performed in PHASER. Autobuilding in
phenix.autobuild was followed by iterative
rounds of manual building in COOT (59)
interspersed with refinement in PHENIX
Protein crystallography. CntA crystals
were seeded and obtained at 22°C using
the hanging drop method. All drops
contained 1µL of protein material and 1µL
crystallisation
crystallisation conditions were obtained
condition.
Initial
from sparse matrix screening using JCSG-
plus
TM
(Molecular Dimensions) and
optimised using the Additive Screen
Hampton Research). Seeding material was
(
generated with protein at 10-20 mg/mL in
drops containing 100 mM HEPES, 0.2 M
NaSCN, 20% (w/v) PEG 3350, 10 mM NaCl
and 0.5 mM TCEP where large irregular
crystals formed over 48 hours at 22°C.
These crystals were scratched with
acupuncture needles and then streaked
through drops containing protein at 7.5
mg/mL 100 mM HEPES, 0.2 M NaSCN, 18%
(
60). Within Coot, monomers for L-
carnitine (PDB code 152), -butyrobetaine
PDB code NM2), glycerol (GOL),
(
w/v) PEG 3350, 10 mM NaCl and 0.5 mM
(
TCEP with substrates (carnitine and -
butyrobetaine) also present at a final
concentration of 1 mM, the needle was run
through 2 consecutive drops with a third
drop on the cover slip as a control. Single
red hexagonal crystals developed between
thiocyanate (SCN) and HEPES (EPE) were
added into clear density that was evident
in both 2fo-fc and fo-fc maps consistent
with the respective ligands. In order to
assign the coordinates for MMV020670,
Polder OMIT maps (61) were generated in
PHENIX. Geometry restraint information as
2
4 and 36 hours at 22°C and were collected
immediately thereafter (prolonged
.cif file for MMV020670 was generated in
incubation of the crystals was found to be
detrimental to their crystal quality).
eLBOW (62) within the PHENIX suite of
programmes and the MMV020670
11

inhibitor was added into the model guided
by Polder OMIT maps. The structure model
coordinates can be accessed from the PDB
repository with the following accession
codes: CntA Apo (6Y8J), CntA + carnitine
Super High-Q resonator as reported
previously (63-64). All EPR samples were
prepared in the quartz capillary tubes
(outer diameter; 4.0 mm, inner diameter
3.0 mm) and flash-frozen immediately and
(
6Y9D), CntA
+
gBB (6Y8S) and
stored in liquid N until analysis. The X-
2
CntA+MMV020670 (6ZGP).
band EPR tubes were then transferred into
the EPR probehead, which was pre-cooled
to 20 K. The low-temperature EPR spectra
were measured at 20 K as a frozen
solution. A microwave power of 30 dB (0.2
mW) and modulation of 5 G appear to be
optimal for recording the EPR spectrum of
the CntA/CntB (oxygenase/reductase)
UV/VIS Enzyme assays. All activity assays
were performed on a BMG FLOUstar
Omega 96 well plate reader, set at
wavelength 340 nm with a sample volume
of 200 µL. The assay comprised of
combining mixture A 60 µg CntA and 60 µg
CntB with mixture B 0.25 mM L-carnitine
and 0.25 mM NADH in a 10 mM HEPES
buffer, pH 7.6 with 250 mM NaCl and 0.5
mM TCEP. The enzyme does not accept
NADPH as the electron donor and the
additional flavin (FAD or FMN) to the
enzyme assay mixture had no major impact
on enzyme activities (Figure S1). The
reactions were measured for 5 minutes
recording the linear decrease of signal at
domains
experimental
prepared
under
various
The
conditions.
concentration of the CntA/CntB domains
was 200 μM in all the samples. All
experiments were carried out in 10 mM
HEPES, pH 7.6, 250 mM NaCl, 10 % Glycerol
and 0.5 mM TCEP, with additional NADH
and or L-carnitine at a final concentration
of 75 mM added accordingly. The analysis
of the cw-EPR spectra and simulations
were performed using EasySpin toolbox
(5.2.18) for the Matlab program package
(65).
3
40 nm for NADH oxidation. The slope of
the line was measured between 60 and
20 seconds to yield the rate.
1
Michaelis–Menten kinetics. Michaelis–
Menten kinetic parameters were derived
using the UV/VIS Enzyme assays
Circular Dichroism. The proteins were
buffer exchanged on a PD-10 column into a
0.2M sodium phosphate buffer with 0.01M
NaCl at pH 7.0 and prepared at 0.1mg/mL
final concentration. The samples were
analysed on a JASCO J-1500 at 20°C using a
0.1mm path length quartz cuvette. Data
was collected between 260 to 180 nm with
8 scans per sample collected.
methodology
above.
Substrate
concentrations were varied from a top
concentration of 2 mM and decreasing in a
2
-fold manner for 10 successive dilutions.
Observed rates were obtained from 4
independent measurements and fitted to a
nonlinear regression Michaelis-Menten
model in Prism 8 for macOS v8.2.0.
Inhibitor screening assay. Compounds in
the
Pathogen
Box
Electron Paramagnetic Resonance (EPR)
analysis. EPR measurements were carried
out using a Bruker ELEXSYS-500/580 X-
band EPR spectrometer operating in both
continuous-wave (cw) and pulsed modes,
equipped with an Oxford variable-
temperature unit and ESR900 cryostat with
(https://www.mmv.org/mmv-
open/pathogen-box/about-pathogen-box)
were received as 10 mM stocks dissolved
in dimethylsulfoxide (DMSO). The
compounds were added such that the final
concentration was 50 µM in the 200 µL
reaction. As per the “UV/VIS Enzyme
12

assays” methodology above, the protein in
mixture A was incubated with inhibitor
compounds for 20 minutes before
commencing the assay. Compounds that
demonstrated 25% or better inhibition
relative to an untreated and equivalent
DMSO control were further validated in
two successive independent assays to
confirm the inhibition activity to CntA.
aliquoted into 5mL per replicate and
carnitine was added to final
a
concentration of 1 mM with/without the
addition of the inhibitors (i.e. MMV1,
MMV3 and MMV12) at a concentration of
200 µM. A corresponding DMSO only
control was also included. These cells with
appropriate treatments were incubated at
37°C for 1 hour. The supernatant was then
obtained by filtering through a 0.2 µm filter
and analysed by ion chromatography by
quantifying TMA production and carnitine
degradation as described previously (13).
Inhibitory IC50 determination. From a 25
mM stock solution of compound material
dissolved in DMSO, a 3-fold, 10 step serial
dilution was prepared and the final
concentration of each compound ranges
from 300 µM to 0.015 µM. The assay was
carried out as per the “UV/VIS Enzyme
Chemical structures and protein structure
depictions. MarvinSketch was used for
drawing, displaying and characterizing
chemical structures (Marvin v19.10.0 for
assays”
and
“Inhibitor
screening”
methodology above and each compound
was incubated with the protein material
for 20 minutes prior to assaying. For each
concentration, 4 replicates were carried
out. IC50 values were determined using a
built-in “Dose Response Inhibition” –
Mac,
2019,
ChemAxon
Reaction
http://www.chemaxon.com).
summaries were drawn in ChemDraw
V17.0 for Mac. Molecular graphics and
analyses were performed with UCSF
Chimera (66), Chimera X (67) and Coot
(59). Protein-ligand interaction maps were
generated in Maestro (Academic)
v11.9.010 Release 2019-1 – Schrodinger.
[
Inhibitor] vs. response model in Prism 8
for macOS v8.2.0.
Inhibitor Competition assays. As per the
Michaelis Menton kinetics methodology
above, we carried a series of 4 runs in the
presence of (i) 0x (control), (ii)0.25x, (iii)1x
and (iv)4x of the IC50 value for each
inhibitor compound with 4 independent
replicates for each run and fitted to a
nonlinear regression Michaelis-Menten
model in Prism 8 for macOS v8.2.0.
in silico docking. Structures were prepared
in MarvinSketch, saved as .sdf files an
imported in to Flare (Cresset) (v3.0). The
CntA+MMV020670 pdb file was imported
into Flare and minimised using the
standard built-in protocols. The ligands
were docked using the built-in docking
engine using the MMV020670 ligand pose
as a template.
Evaluation of inhibitors in A. baumannii
cell culture. A. baumannii cells were
cultured overnight in the M9 minimal
media at 37°C with 10 mM L-carnitine as
the sole carbon source. The cells were then
collected by centrifugation and washed
twice in a buffer containing 10 mM HEPES,
Acknowledgements This work was
supported by a Leverhulme trust research
grant (RPG-2016-307). MS acknowledges
The University of Manchester and MIB for
financial supports. We acknowledge the
generous support from the Malaria
Medicines Venture for providing the
Pathogen Box and follow up material for
this work and Cerith Harries and Caroline
1
mM NaCl (pH 7.6). After wash, the cells
were resuspended in the same buffer to a
density of OD600 of 0.4. The cells were
13

Stewart from the Media Preparation
Service at the University of Warwick for
help making various culture media and
reagents.
Author contributions Y.C, A.C and T.D.H.B
conceived the study. M.Q. performed
protein expression, purification, X-ray
crystallization, inhibitor design and testing.
M.S. performed EPR measurements. E.D.
and E. J. helped with inhibitor testing. M.Q.
wrote the manuscript with significant input
from all co-authors.
Data availability: The structures of CntA
and its ligand bound forms have been
submitted to the PDB database (accession
numbers 6Y8J, 6Y8S, 6Y9D and 6ZGP). All
other data have been included in the
manuscript.
Conflict of interest: The authors declare no
conflict of interest.
14

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19

Table 1 X-ray data collection and refinement statistics.
Name
CntA Apo
6Y8J
CntA + Carnitine
CntA + gBB
CntA+MMV12
PDB code
Wavelength (Å)
6Y8S
6Y9D
6ZGP
1
4
.7
1.3
1.3
0.9
0.1 - 2.05
81.42 - 1.97
(2.04 - 1.97)
39.78 - 1.63
(1.69 - 1.63)
78.64 - 2.01
(2.08 - 2.01)
Resolution range (Å)
(2.12 - 2.05)
P 63
P 1 21 1
P 63
P 1 21 1
Space group
9
1.6 91.6 82.96
91.59 177.77 158.8
90 90.17 90
91.16 91.16 81.47
90 90 120
91.07 173.77 157.29 90
90.15 90
Unit cell
90 90 120
2
2
9
9
7
24999 (13603)
4714 (2443)
.1 (5.6)
1180725 (105398)
353116 (35196)
3.3 (3.0)
203443 (9804)
47465 (4434)
4.3 (2.2)
2229436 (218489)
322879 (32095)
6.9 (6.8)
Total reflections
Unique reflections
Multiplicity
9.3 (99.2)
98.8 (98.4)
98.8 (93.4)
6.8 (1.13)
99.4 (99.1)
Completeness (%)
Mean I/sigma(I)
.41 (1.14)
5.2 (1.21)
6.9 (1.13)
2
38
31
24
33
Wilson B-factor (Å )
0
0
0
0
0
.189 (1.429)
0.149 (0.958)
0.178 (1.168)
0.096 (0.660)
0.984 (0.638)
0.996 (0.883)
0.134 (0.755)
0.152 (0.967)
0.070 (0.595)
0.982 (0.517)
0.996 (0.825)
0.165 (1.783)
0.179 (1.929)
0.068 (0.733)
0.996 (0.698)
0.999 (0.907)
R-merge
R-meas
R-pim
CC1/2
.200 (1.575)
.064 (0.651)
.994 (0.438)
.999 (0.781)
CC*
Reflections used in
refinement
24704 (2441)
1223 (101)
352719 (35160)
17582 (1828)
47464 (4434)
2374 (221)
321994 (31973)
16243 (1504)
Reflections used for R-
free
0
0
0
0
.201 (0.429)
.248 (0.530)
.958 (0.737)
.942 (0.821)
0.207 (0.338)
0.243 (0.371)
0.961 (0.805)
0.953 (0.760)
0.1704 (0.323)
0.193 (0.339)
0.963 (0.726)
0.911 (0.768)
0.216 (0.386)
0.254 (0.421)
0.961 (0.799)
0.948 (0.736)
R-work
R-free
CC(work)
CC(free)
Number of non-
hydrogen atoms
2874
35752
34344
3117
2832
36701
34901
2
803
macromolecules
20

4
6
3
0
1
408
31
588
ligands
7
1000
4224
0.009
1.10
254
350
0.007
1.05
1212
4328
0.009
1.32
solvent
45
Protein residues
RMS(bonds) (Å)
RMS(angles) (°)
Ramachandran favored
.008
.18
94.67
5.33
0.00
95.35
4.50
0.14
95.91
3.80
0.29
94.76
4.98
0.26
(
%)
Ramachandran allowed
%)
(
Ramachandran outliers
%)
(
0
5
.00
.86
0.87
0.00
0.85
Rotamer outliers (%)
Clashscore
9.36
3.41
13.20
51.59
51.60
2
55.95
56.24
45.44
45.40
35.71
34.94
Average B-factor (Å )
2
Macromolecules (Å )
[
2Fe-2S] Rieske
44.74
41.35
25.56
48.08
Centre
N/A
N/A
N/A
44.69
9
45.15
47.02
N/A
176.41
57.75
N/A
56.77
N/A
Fe
Substrate
Inhibitor
69.75
44.22
44
2
39.21
48
42.61
10
Solvent (Å )
Number of TLS groups
21

Table 2 Spin-Hamiltonian parameters used to model the EPR spectra.
Samples
EPR active signal
species
%
g
1
g
2
g
3
g
ave
As-isolated CntA
2.0105
1.9157
1.7574
1.8945
As-isolated CntB
CntB+NADH
No EPR active signal detected
[2Fe-2S]+1
Flavin radical
specA (reduced ferredoxin domain)
specB (Flavin radical)
93
7
2.0314
2.0015
1.9372
2.0015
1.8988
2.0015
1.9558
2.0015
+
1
CntA+CntB+NADH
[2Fe-2S] in CntB
Flavin radical
[
specA (reduced ferredoxin domain)
specB
specC (reduced Rieske centre)
61
2
37
2.0313
2.0010
2.0079
1.9376
2.0034
1.9153
1.8979
2.0039
1.7542
1.9556
2.0028
1.8924
+1
2Fe-2S] in CntA
CntA+CntB+NADH+Carnitine
an organic radical
specB
specC
34
66
2.0000
2.0071
2.0035
1.9077
2.0037
1.7986
2.0024
1.9045
+1
A
A
[2Fe-2S] in CntA
+1
E205A+CntB+NADH+Carnitine [2Fe-2S] in CntB
specA1
specA2
specB
40
20
8
2.0338
2.0449
2.0012
2.0070
1.9397
1.9493
2.0040
1.9077
1.8868
1.9037
2.0040
1.7896
1.9534
1.9660
2.0031
1.9014
+
1
[
2Fe-2S] in CntB
B
Flavin radical
+1
[
2Fe-2S] in CntA
specC
32
A
This EPR active signal may also contain an overlapping EPR-signals derived from the catalytic mononuclear iron centre; plausible EPR active
intermediates are ferric-(hydro)peroxy or a high-valent iron(V)-oxo species.
B
This may represent an overlay of both a flavin and an unidentified organic radical.
22

Figure 1 The overall structure of CntA and its catalytic centre.
23

A, The reaction of carnitine oxidation by CntAB to trimethylamine (TMA) and malic
semialdehyde.
B, CntA depicted as a homotrimer with the secondary structure represented in a ribbon
depiction in a translucent overall surface representation with each monomer in blue, red
and green, respectively.
C, An overlap of three monomer structure units (apo, carnitine-bound, gBB-bound), showing
no major changes to the tertiary structure of CntA with and without ligands bound. The C
and N labels refer to the sequence termini. The Rieske centre and the mononuclear Fe
centre are 44 Å apart in the same subunit.
D, The Rieske centre in CntA is coordinated by Cys86, Cys106, His88 and His109.
E, The catalytic mononuclear Fe centre in CntA is coordinated by a His-His-Asp catalytic triad
(
His208, His213, Asp323), a water and thiocyanate ion. Carnitine is shown above the
mononuclear Fe centre with a distance from the water molecule to the site of substrate
cleavage.
F, Electron paramagnetic resonance (EPR) spectra of as isolated CntA in the presence of nitric
2+
oxide (NO), showing the EPR-active, S = ½ species due to the formation of Fe -NO adduct.;
EPR conditions; microwave power 30 dB, modulation amplitude 5 G, time constant 81 ms,
conversion time 41 ms, sweep time 84 s, receiver gain 60 dB, average microwave frequency
9.384 GHz, temperature 20 K.
24

Figure 2 Structural basis of inter-subunit electron transfer and investigations by EPR.
A, Electron transfer in CntA between the Rieske centre and the mononuclear Fe centre across
the interface of two neighbouring CntA subunits (coloured in cyan and brown,
respectively) involved a key bridging glutamate reside (E205). Distances shown in Å.
B, Proposed electron transfer pathways from the reductant NADH to the catalytic
mononuclear Fe centre in CntA via the reductase CntB.
C, EPR spectra of a series of combinations of WT CntAB proteins, E205A mutant, NADH and L-
carnitine to track the propagation of EPR signal and thus the inter/intra subunit electron
transfer. NADH reduction in CntB (black trace to red trace; steps 2 and 3 in Fig. 2B); the
+
1
EPR active [2Fe-2S] species in the CntA Rieske centre with and without carnitine present
25

(
red trace to magenta trace and cyan trace; steps 4-6 in Fig. 2B). The E205A mutant (wine
red trace) demonstrates an EPR signal different to that of the wild-type CntA (cyan trace),
indicating a disruption to the electron transfer pathway; EPR conditions; as in Fig 1E.
D, Activity assays of CntA E205 mutants.
E, Circular dichroism measurement comparisons between CntA wild type and E205 mutant
enzymes showing no difference in secondary structure.
26

Figure 3 CntA substrate binding pocket.
A, Michaelis-Menten kinetics of CntA (n=4).
B, 2mF
o
ꢀ−ꢀDF map at 1.5 maps (Blue) of carnitine displayed at 3.0 and -butyrobetaine
c
(
gBB) displayed at 2.5.
C, Carnitine and -butyrobetaine interacting residues in the active site amongst which Y203
interacts with the substrate of CntA through the π-cation interaction.
D, Enzyme activity of site-directed mutants of key residues involved in substrate coordination
in CntA from N=3 independent replicates. The Y203F mutant in CntA regains activity
alluding to the crucial role of the aromatic system for a π-cation interaction.
27

Figure 4 CntA represents a novel group of Rieske oxygenases using quaternary amine
substrates (group V).
A, Phylogeny of Rieske oxygenases showing the five distinct groups. The evolutionary history
of Rieske oxygenases was inferred using the Neighbour-Joining method in MEGA7 (68).
The dashed circle grouping shows the clustering of CntA including Acinetobacter
baumannii (Ab), Escherichia coli SE11 (Ec), Klebsiella pneumonia (Kb) and Citrobacter
freundii (Cf) (See supplementary information, Appendix 1 for a list of gene accession ID’s).
Group V Rieske oxygenases oxidise quaternary amine substrates, including carnitine
(
CntA), choline (CmoA/B/C), benzalkonium (OxyBAC), glycine betaine (BmoA, GbcA) and
stachydrine (Stc2).
B, Chemical reactions catalysed by Rieske enzymes from the Group V Rieske oxygenases
involved in quaternary amine oxidations. Group Va includes carnitine oxygenase (CntA)
choline monooxygenase (CmoA, CmoS and CmoB) and benzalkonium oxygenase (OxyBAC).
Group Vb carries out oxidative demethylation reactions.
28

C, Sequence alignment of the substrate binding pocket residues of group V Rieske oxygenase
involved in quaternary amine oxidation. The aromatic residues for substrate coordination
through a π-cation in group Va and the corresponding position in group Vb are
highlighted by a red arrow. The Rieske centre is coded in blue, the catalytic triad of the
mononuclear Fe centre is shown in purple and the bridging carboxylate is highlighted in
pink.
D, The substrate binding site of the group Vb quaternary amine degrading enzyme Stc2. The
substrate mimic proline in Stc2 coordinates with the catalytic mononuclear Fe centre via
a carboxylic acid group.
29

Figure 5 Structure activity relationship of CntA substrate profiling and coordination during
catalysis.
A: A structure activity relationship scaffold hopping map of substrate analogues tested with
a single change between structures and the progression shown with arrows. Structures
in blue represent active substrates, whilst those in red are inactive. (1) Carnitine, (2) g-
BB, (3) Meldonium, (4) (1-Butyl)Trimethylammonium, (5) (1-Hexyl)Trimethylammonium,
(
6) (1-Octyl)Trimethylammonium, (7) (Cyclopropylmethyl)-Trimethylammonium, (8) (3-
Hydroxypropyl)Trimethylammonium, (9) (3-Oxobutyl)Trimethylammonium, (10) 4-
Dimethylamino)butyric acid, (11) 4-(Methylamino)butyric acid and (12) gamma-
(
Aminobutyric acid.
B, The coordination of quaternary amine substrates in the CntA binding site during catalysis
showing the role played by Tyr 203 and Tyr 295 in coordinating the substrates.
30