Research Article
MedChemComm
Adenylating enzymes use ATP as a mechanism to activate
the fatty acid in two independent half reactions (Fig. 2). The
first half reaction is attack of the deprotonated carboxylic acid
on the α-phosphate of ATP creating an acyl-adenylate intermedi-
amino acids to generate the corresponding N-acyl amides.
We then applied this to the production of the recently discov-
ered class of N-acyl amide NPs from Legionella pneumophila.
The versatility of the TamA ANL domain suggests it is a use-
ful addition to the biocatalytic toolbox.
ate which is driven by liberation of the PP product. The incom-
i
ing nucleophile can subsequently attack the tightly bound acyl-
adenylate intermediate, releasing adenosine monophosphate
Results and discussion
(AMP), which is the driving force for the second half reaction.
This approach is often used by nature to combine carboxylic
acids with a range of nucleophiles to produce esters, amides,
Since adenylating enzymes activate carboxylic acids for nucle-
ophilic attack, once the TamA ANL domain generated the key
acyl-adenylate intermediate from the fatty acid substrate and
ATP, we began by exploring whether this could be captured
with a range of nucleophilic amines. We wished to use only
the catalytic TamA ANL domain so this was prepared by de-
leting the C-terminal ACP domain and a short segment
thought to be the inter-domain linker sequence (Fig. S1†). In
the absence of an X-ray crystal structure of TamA, construc-
tion of an active TamA ANL domain was guided by sequence
alignment with the fatty acid AMP ligase (FAAL) from
Legionella pneumophila and a homology model which predicts
that the domain spans residues (1-571) in the N-terminus
16,17
and thioesters.
The adenylating superfamily contains a se-
ries of amide bond synthetases (ABS) which catalyse the for-
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mation of various amide-containing NPs. Included in this
family are the firefly luciferases involved in biolumines-
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20
cence and the acyl-CoA synthetases which channel carbox-
ylic acids into their respective metabolic pathway. Also,
the recently discovered McbA, from Marinactinospora
thermotolerans, has been shown to catalyse amide bond for-
mation on a broad range of β-carboline substrates which can
be exploited for the sustainable synthesis of amides by the
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1,22
pharmaceutical industry.
These enzymes illustrate the
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vast potential of the adenylate-forming enzymes for biocataly-
sis. Recently we showed that TamA plays a key role in
tambjamine YP1 biosynthesis; the ANL domain selects and ac-
tivates the fatty acid substrate and delivers it to its ACP do-
main as a thioester. This ACP-bound product is the substrate
for downstream processing to the corresponding amines
(Fig. S2 and S3†). This also suggests the residues that are
most likely involved in the acyl chain length specificity (I195,
F199 and F266). We prepared four constructs of different
lengths with N-terminal His-tags and based on solubility, the
shortest version (TamA ANL576) was isolated in good yield
and used in all subsequent biocatalysis experiments (Fig.
S4†). Gel filtration chromatography (GFC) and LC ESI-MS
analysis of the TamA ANL domain confirmed the protein was
monomeric with a deconvoluted mass of 64 653 Da ± 1 Da
consistent with the predicted mass of 64 651 Da (Fig. S5†).
We also isolated full length TamA according to Marchetti
(Fig. 1). We also discovered that as well as activating the C12
lauric acid found in YP1, TamA could also use C6–C14 sub-
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strates suggesting that it displays some substrate flexibility.
An interesting family of recently discovered biocatalysts
are the carboxylic acid reductases (CARs) which catalyse the
conversion of carboxylic acids to aldehydes. A number of dif-
ferent CAR isoforms have been characterised but they are all
composed of three domains; ANL, carrier protein (CP) and
NADPH-dependent reductase (R), which select, activate and
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3
et al. for comparative analysis (Fig. S6†).
With the TamA ANL domain in hand we began by exploring
primary amide formation with a range of long chain fatty acids
(C2–C16) and ammonia, following established reaction condi-
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4–27
23,28
reduce a broad range of different acids.
Four members
tions.
The amide products were detected by LC ESI-MS
of the CAR family have recently been investigated by Flitsch
and coworkers to produce a number of primary, secondary
with an increase in retention time corresponding to an in-
creasing chain length (Fig. 3 and S7†). Under these conditions
TamA ANL generates the amide products from the correspond-
ing C9–C14 acids which is similar to the chain length specific-
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and tertiary amides.
Since ANL domain enzymes are
gaining attention we sought to explore the synthetic capabil-
ity of TamA. We found that the TamA ANL domain alone
could be used to couple various fatty acids with several
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ity that we observed for the full length TamA didomain.
We then selected the C12 acid as a substrate to investigate
the amine scope of the TamA ANL-catalysed reaction. We
picked enthanolamine and benzylamine, as well as amino
acids to test whether the enzyme could be used to generate
the corresponding N-acyl amino acid secondary amides. We
focussed on L- and D-histidine for three reasons; firstly, to
prepare the N-acyl histidine targets which have recently been
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discovered in L. pneumophila, a human pathogen. These
metabolites are upregulated during the L. pneumophila infec-
tion cycle and could regulate human signalling pathways,
analogously to other identified acyl amides. Secondly, the
C12 N-acyl histidine has also been investigated for its surfac-
Fig. 2 The TamA ANL domain catalyses the ATP-dependent formation
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1
tant properties. Therefore these molecules are important
for studying human-pathogen interactions as well as having
2
of the C12-adenylate intermediate. This reacts with an amine (H N-R)
to generate the N-acyl amide.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2019
Marchetti, Piera M.
