Pac13 is a Small, Monomeric Dehydratase that Mediates the Formation of the 3′-Deoxy Nucleoside of Pacidamycins

Article abstract of DOI:10.1002/anie.201705639

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3′-deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5′-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3′-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.

Full text of DOI:10.1002/anie.201705639

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Short and Sweet: Pac13 is a Small, Monomeric Dehydratase
that Mediates the Formation of the 3′- Deoxy Nucleoside of
Pacidamycins.
Authors: Freideriki Michailidou, Chun-wa Chung, Murray J. B. Brown,
Andrew F. Bent, James H. Naismith, William J. Leavens,
Sean M. Lynn, Sunil V. Sharma, and Rebecca Jane Miriam
Goss
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201705639
Angew. Chem. 10.1002/ange.201705639
Link to VoR: http://dx.doi.org/10.1002/anie.201705639
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COMMUNICATION
Short and Sweet: Pac13 is a Small, Monomeric Dehydratase that
Mediates the Formation of the 3′- Deoxy Nucleoside of
Pacidamycins.
[
a,b]
[b]
[b]
[a]
Freideriki Michailidou,
Chun-wa Chung, Murray J. B. Brown, Andrew F. Bent, James H.
[
a]
[b]
[b]
[a]
[a]
Naismith, William J. Leavens, Sean M. Lynn, Sunil V. Sharma, and Rebecca J. M. Goss*
Abstract: The uridyl peptide antibiotics (UPAs), of which
pacidamycin is a member, have a clinically unexploited mode of
action and an unusual assembly. Perhaps the most striking feature
of these molecules is the biosynthetically unique 3′-deoxyuridine that
they share. This moiety is generated by an unusual, small and
monomeric dehydratase, Pac13, which catalyses the dehydration of
uridine-5’-aldehyde. Here we report the structural characterisation of
Pac13 with a series of ligands, and gain insight into the enzyme’s
mechanism demonstrating that H42 is critical to the enzyme’s
activity and that the reaction is likely to proceed via an E1cB
mechanism. The resemblance of the 3′-deoxy pacidamycin moiety
with the synthetic anti-retrovirals, presents a potential opportunity for
the utilisation of Pac13 in the biocatalytic generation of antiviral
compounds.
most characterised dehydratases, Pac13 is small (121 aa),
monomeric, co-factor independent and utilises a non-activated
[
6]
nucleoside, rather than a free monosaccharide, as a substrate .
The biosynthetic uniqueness coupled to the potential synthetic
utility inspired us to investigate Pac13’s structure and
mechanism. This is the first mechanistic study of the formation
of the 3′- deoxynucleosides in natural product biosynthesis. We
demonstrate that not only is Pac13 unusually small, monomeric
and cofactor independent, but that it is also mechanistically
distinctive.
Nucleic acids play
a central role in nature and modified
nucleosides are present in a wide range of anti-viral, anti-cancer
[
1]
drugs and antibiotics. Though a variety of naturally occurring
nucleic acid analogues exist, few include modifications to the
ribose or deoxyribose ring. The uridyl peptide antibiotics (UPAs)
pacidamycin, naspamycin, mureidomycin and sansanmycin,
[
2]
attract much attention with a clinically unexploited mode of
[
1]
[3]
action and an unusual biosynthetic assembly. Intriguingly, the
UPAs) contain a biosynthetically distinct 3′-deoxyuridine that
resembles the synthetic anti-retrovirals such as stavudine 4,
abacavir (Figure 1) and the cytotoxic natural product
cordycepin 6, the biosynthesis of which has not yet been
(
5
[
4]
determined.
A detailed mechanistic understanding of the
individual enzymes employed in the generation of the 3′-
deoxyuridine core is required in order to facilitate their future
biotransformative potential.
Figure 1. Medicinally relevant compounds containing 3′-deoxy-nucleoside
moieties: A biogenesis of the pacidamycin nucleoside motif; Pac13 catalyses
dehydration of uridine-5′-aldehyde 1 to form 3′-deoxy-3′,4′-didehydrouridine-5′-
aldehyde 2,
B pacidamycin, n uridyl peptide antibiotic: (highlighted: the
In pacidamycin 3, biosynthesis the 3′-deoxy moiety is mediated
by Pac13, an enzyme found to catalyze the key dehydration of
uridine-5′-aldehyde 1 to form 3′-deoxy-3′,4′-didehydrouridine-5′-
pacidamycin nucleoside core, the characteristic 3′-deoxy uridine), C stavudine
4 and abacavir 5, widely used anti-virals and cordycepin 6, cytotoxic natural
product.
[
5]
aldehyde 2. Previously proposed as
a
dehydratase
Dehydratases are important enzymes in primary and secondary
metabolism and have been shown to mediate catalysis via a
investigations into the structure, kinetics and mechanism of this
unusual enzyme remained to be performed. In stark contrast to
[7]
variety of mechanisms including metal-dependent, acid-base,
[
8]
[9a]
radical and covalent
mechanisms which we summarise in
the SI (Figure S15). All dehydratases studied so far that are
involved in carbohydrate processing, to the best of our
knowledge, have been shown to be large, multimeric and in
general to require co-factors (commonly metal ions or
nucleotide)or needing covalent interactions for catalysis. A
handful of dehydratases studied so far utilise a more unusual
E1cb mechanism, these include the well-studied dTDP-D-
[
a]
b]
Freideriki Michailidou, Andrew F. Bent,, James. H. Naismith, Sunil
V. Sharma, Rebecca J. M. Goss, School of Chemistry, University of
St Andrews
North Haugh, St Andrews, Fife, KY16 9ST (UK)
E-mail: rjmg@st-andrews.ac.uk
[
Chun-wa. Chung, Murray J. B. Brown, William J. Leavens, Sean M.
Lynn, GSK, Stevenage, SG1 2NY UK
[
6a]
Supporting information for this article is given via a link at the end of
the document
glucose 4,6-dehydratase RmlB
and the UDP-GlcNAc 5,6-
[
6b]
dehydratase TunA, both nicotinamide-dependent
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COMMUNICATION
(
Figures S15 and S16, SI p.21). RmlB and TunB mediate an
+
NAD assisted oxidation of
a hydroxyl group within their
A
nucleotide substrates, followed by the enzymes’ mediation of an
E1cb dehydration to generate the respective enones, and a
subsequent reduction of the resultant conjugated C=C double
[
6a]
bond in the case of RmlB (Figure S16).
3-dehydroquinate
dehydratase I (DHQ I) is a dimeric enzyme which catalyses the
third step of the shikimate pathway in a variety of organisms. It
relies upon an essential Schiff base formation (Figure S16)
between the substrate and a conserved lysine for catalysis that
[
9b]
leads to an E1cb elmination.
[
10]
[11]
Bioinformatic analysis of Pac13 using BLAST and HHpred,
reinforced Pac13’s similarity to both metal dependent and metal
[
12]
independent cupins, including lyases and isomerases (Tables
S5-S7). Heterologous expression, purification and crystallisation
resulted in Pac13 wt crystals that diffracted to 1.55 Å. Solution of
the structure required the preparation of the seleno-methionine
derivative (SeMet-Pac13) and demonstrated that the enzyme
was indeed a cupin (Figure 2). Pac13 is the only monomeric,
metal-free cupin dehydratase characterised to date (Table S8).
Cupins generally occur as components of multimeric complexes
B
[
12b]
and are frequently accompanied by metal ions,
in contrast
Pac13 is small, discrete and metal independent. Comparison of
the Pac13 coordinates with the PDB archive using the Dali
[
13]
server
are cupins of non-assigned function, while the third is the lyase
(Table S6) revealed that the two closest homologues
[
14]
KdgF
dimeric, cupin (Figure S18) requiring a M for the conversion of
,5-unsaturated galacturonate (ΔGalUA) to 5-keto-4-
deoxyuronate (DKI), a reaction distinct to Pac13’s chemistry.
(PDB: 5fpx, Z-score 13.4, RMSD 2.1). KdgF is a
2
+
4
C
[
12]
Whilst the cupin fold
is common for isomerases and
as shown in the CATH database
[
15])
epimerases (eg RmlC
(
(
(
Figure S18) it is not common for lyases and ectoine synthase
EtcC) is the only other cupin shown function as a dehydratase,
Table S8, Figure S21). Perhaps such dehydratase activity will
be seen to be very rare for cupins as the open β-barrel does not
assist in the protection of the product from water. EtcC is dimeric
2
+
and Fe dependent, catalysing the formation of ectoine by ring
closure of the substrate N-γ-acetyl-L-2,4-diaminobutyric acid.
The open active site of wt Pac13 did not allow us to locate the
substrate binding site with confidence. Initial soaks with uridine-
5
′-aldehyde 1, the natural substrate, were problematic due to
both turnover and the inherent instability of this molecule. In
order to overcome this problem we used substrate analogues,
uridine 7 and uridine 5′-uronic acid 8, which enabled us to obtain
liganded complexes of the enzyme, providing the first clues of
the location and structural determinants of the active site (Figure
D
1
). Both ligands occupied the same binding pocket in their
respective co-crystal structures (Figure S20). Comparison of the
apo- and ligand-bound structures revealed that small
movements in residues such as K16, F103 and L21 create a
pocket that encloses the ribose ring of the substrate analogues
but leave one side of the uracil group solvent exposed (Figure 1).
Within the ribose pocket, the 5′ oxygen lies almost equidistant
(2.5-2.7 Å) between Y55 and Y89, with a slightly shorter
distance to Y89, whereas the 4′ proton and 3′ hydroxyl are 2.7-3
Å from H42.
Figure 2. A Crystal structure of Pac13 (PDB: 5OO5), refined as the monomer
to 1.55 Å, MolProbity score of 96%. Cartoon representation of Pac13 with
uridine 5′-uronic acid 8. B Active site ‘pocket’ of Pac13 with uridine 5′-
uronic acid 8. Residues K16, L21, F103 and the ligand are shown as stick
representation. Pac13 is shown as cartoon and surface. C Active site of Pac13
with uridine 5′-uronic acid 8. The active site residues Y89, Y55, K16, E108
and F103 and their distances from the ligand functional groups are shown.
V36 and L96 are also shown. D Compounds that were successfully used for
crystal soaks: uridine 7, uridine 5′-uronic acid 8.
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COMMUNICATION
3
′ hydroxyl group of the substrate and thus generating a better
leaving group. Alternatively, as informed by the crystal structures
of the Pac13 complex, E108 could play a dual role: substrate
binding and acting as the active-site acid, through a coordinated
water molecule (Figure S19). Electrons moving from the enolate
could lead to water elimination at 3′ position and generation of
the desired product. Residues F103 and K16 could simply be
involved in the orientation and binding of the substrate, rather
than in catalysis. This proposed mechanism is perhaps closest
to the E1cb mechanism postulated to be utilised by DHQ II, a
[
9b]
dodecameric enzyme with a flavodoxin-like fold
(Scheme 2),
but distinctive with Y89 and Y55 potentially acting to stabilise the
enolate anion and H42 implicated as possibly playing the role
the role of both acid and base.
Tyr 28
Tyr 28
O
HO
HO
H
HO
-O2C
-
O2
C
OH
O
OH
O
Ala 82
O
H
H
H
Ala 82
Asn 16
N
O
H
H
N
H
H
N
O
H
N
O
H
N
O
H
O
H2N
NH
Asn 16
N
H
O
O
H2N
N
O
O
His 106
79Asn
N
H
His 106
79Asn
N
N
H
N
Pro15
Pro15
Scheme 1. Proposed mechanism for Pac13-mediated dehydration of uridine-
5
′-aldehyde 1 to to form 3′-deoxy-3′,4′-didehydrouridine-5′-aldehyde 2. Initially
Scheme 2. Proposed E1cb mechanism for DHQ II, depicting Tyr28 acting as
base to catalyse enolate formation, with the enolate being stabilised by
hydrogen bonding to regions oft he peptide backbone, via a bridging water
speculated Pac13-mediated dehydratase mechanisms; A E1cB mechanism; B
carbocation formation; C radical formation.
[
9b
molecule. Figure adapted from
This leaves the 2′ hydroxyl of the ribose ring coordinated by the
side chains of K16 and E108. In contrast to this extensive
network of direct polar interactions the uracil ring makes a single
H-bond to K16 at the lip of the active site, with more Van der
Waals (L21, V34, V99) and π-stacking interactions (F103)
characterising recognition of this part of the substrate. Notably,
lipophilic residues L97 and V36 border the active site, creating a
hydrophobic environment and perhaps sheltering the substrate
or dehydrated product from attack from water. Pac13’s
substrate-binding site is distinct to the active site environment of
To probe Pac13 function, the reaction of Pac13 with uridine-5′-
aldehyde 1 was monitored by LC-MS. A species with m/z 225,
corresponding
to
the
3´-deoxy-3´,4´-didehydrouridine-5´-
aldehyde 2 was detected. No conversion could be seen in
enzyme free controls. In aqueous conditions, 1 exists completely
[
3c]
1
as its hydrate form . Next, we followed the reaction by H NMR
and observed the disappearance of the 4′ proton multiplet at 4.0
ppm. The characteristic 3′ proton could be seen to shift
downfield, appearing as a doublet of doublets at 5.4 ppm,
consistent with generation of an olefinic bond, and enabling
reaction monitoring. Unlike other dehydratase-mediated
[
15]
the most well characterised cupin isomerases, such as RmlC
Scheme S1), suggesting a unique mechanism.
(
[
7]
reactions that exist in equilibrium, we demonstrate here that
the Pac13-catalysed dehydration of uridine-5′-aldehyde reaches
completion. However, rapid abstraction of the 4′ proton in
comparison to the depletion of other substrate protons such as
H5 and H6, indicative of an E1Cb mechanism, was not observed
The observed electronic environment in the active site caused
us to disfavour series of mechanisms. An E2 mechanism is
unlikely; the substrate’s stereochemistry prevents the required
antiperiplanar relationship between the proton being abstracted
and the exiting hydroxyl. It is unlikely that an E1 mechanism
(
Figure S24-5, SI p.33). COSY and HSQC NMR of the
enzymatic preparation allowed us to confirm the structure of the
expected product. The optimal conditions for Pac13 activity were
(
Scheme 1B) operates, as there is no highly acidic residue
proximal to the 3′ hydroxyl group that could assist the
stabilisation of carbocation. dehydroquinase type I
2
then examined. NADH, NADPH, MgCl were found not to be
a
A
required. Based on extensive crystallographic and biochemical
analysis, including enzymatic assay in presence of 100 mM
EDTA, requirement of a metal for either catalysis or structural
integrity of the protein could be confidently excluded. Kinetic
constants were determined by following the course of the
reaction by HPLC; these studies revealed a relatively low
mechanism (Figure S16) can be eliminated as the possibility of a
covalent lysine linkage does not exist. Furthermore a radical
mechanism (Scheme 1C) could be excluded due to the absence
of a metal ion or prosthetic group. An E1cB mechanism can be
postulated from the structure to be likely (Scheme 1A): in the
first step, H42 could act as base extracting the activated 4′
proton, creating an enolate 10. The oxyanion could then be
stabilised by hydrogen bonding by Y55 and Y89, and the
protonated H42 could then act as the acid, donating a proton to
-
1
reaction rate with a high K
.9 mM). Whilst this slow rate is consistent with those reported
for other
M M
(kcat = 3.63 ± 0.3 min , K = 5.86 ±
0
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COMMUNICATION
)
A
B
C
D
Figure 3. A and C Complex of H42Q with uridine (colored in purple, PDB: 5OO8) superimposed with Pac13 wt complex with uridine (colored in yellow and gree,
PDB: 5OO4) B and D Complex of Y89F with uridine (colored in purple, PDB:5OOA) superimposed with Pac13 wt complex with uridine (colored in yellow and
green).
[
16]
enzymes involved in secondary metabolism,
it may also
effects seen are specific to interactions with each mutated
residue rather than global effects on protein folding and structure.
reflect the low concentration of aldehyde present as the
substrate uridine-5′-aldehyde 1 exists in equilibrium with its
hydrate form in the aqueous condition of the enzymatic assay. In
Table 1. Kinetic Constants for Pac13 WT and mutants
M
more native conditions, the K may be significantly lower as it is
postulated that the aldehyde substrate of Pac13 could be
channelled from the previous biosynthetic enzyme and protected
from bulk solvent.
A detailed pH profile analysis demonstrated that the enzyme
was completely inactive below pH 5.4 and above pH 10. The
profile followed a bell-shaped curve (Figure S13B), with pKa
1
and pKa values of 6.2 and 8.6 respectively. Since the substrate
2
does not have any ionisable groups in this pH range, this result
supports our proposal of a histidine acting as a general base to
[
17]
abstract the 4′ proton . There is no obvious residue positioned
to protonate the leaving hydroxide ion and this role may be
fulfilled by H42 or by a solvent molecule mediated through E108
to eventual His deprotonation. Though unusual, a histidine
residue mediating as a general base and acid in two sequential
[
18]
steps, has been previously reported (Figure S16 and SI p.33)
.
To gain further confidence with regards to our proposed
mechanism, we carried out a series of site-directed mutagenesis
(
SDM) studies. Mutants E108Q, K16R, H42Q, Y55F and Y89F
were produced and purified, affording diffracting crystals of the
same morphology and space group as wt Pac13, which raised
our confidence that the mutants folded correctly and that the
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COMMUNICATION
functionality, these changes are small and consistent with our
hypothesis that decreased catalytic efficiency of these mutants
is largely due to inability of the phenyl mutants to stabilize the
transient 5′ oxo-anion that forms during catalysis, rather than
having a major effect on binding competency.
-
1
Enzyme
K
M
(mM)
k
cat (min )
k
cat/K
M
%
-
1
-1
(
min mM )
conversion
at 1 mM [S]
WT
5.86 ±
.9
3.63 ± 0.3
0.79 ± 0.1
0.71 ± 0.1
0.62
43
24
19
0
This study constitutes the first structural and mechanistic
investigation into the biosynthesis of the intriguing 3′-deoxy
K16R
Y55F
6.40 ±
.3
0.12
nucleoside moiety of the UPAs. Through
a
series of
1
crystallographic and biochemical experiments, supported by
SDM and kinetics, we provide insight into the mechanism of the
unique C=C bond formation orchestrated by Pac13. We
demonstrate here that the reaction most likely proceeds via an
E1Cb mechanism, with H42 acting as an active site base,
however, unlike many other dehydratases, no metal or cofactor
is required for activity. Our deepened understanding of this
biosynthesis paves the way toward a biosynthetic alternative to
highly valuable yet, synthetically challenging 3′-modified
nucleosides. Our next goal is to further explore and challenge
Pac13 so as to develop a promising biocatalyst.
8.13 ±
.2
0.09
1
1
Y89F
E108Q
H42Q
n.d.
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
10
4
0
The mutation of H42 to a Q completely abolished activity, further
supporting our hypothesis that H42 acts as a base for the
abstraction of the 4′ proton and possibly for the protonation of
the 3′ hydroxyl group. The kinetic measurements for the activity
of Y55F and K16R demonstrated lower affinity and catalytic
efficiency than wt Pac13 (Table 1). Mutation of K16 to an R did
not abolish activity, further demonstrating that K16 is not an
essential lysine involved in a Schiff-base formation; hence a
DHQ I – type mechanism could be further excluded with
confidence. Y89F demonstrated a significant loss in activity
compared to the wt (Table 1). Mutation of the E108 resulted in
almost no activity; a result that could perhaps be attributed to the
importance of E108 both in stabilizing substrate binding and
having a role in catalysis. Indeed, given the open active site of
Pac13, as observed in the crystal structure, the existence of a
residue that would maintain the substrate in a productive
orientation appears to be essential. Finally, the significant loss of
activity with mutants Y89F and Y55F is consistent with their
proposed involvement in stabilizing the oxyanion and underlines
their important role for catalysis.
Acknowledgements
This work was supported by the EPSRC council (Grant number
1
398501), Wellcome Trust (Investigator Award) and
GlaxoSmithKline. We thank Diamond Light Source for
synchrotron time. We thank the the EPSRC UK National Mass
Spectrometry Facility at Swansea University for mass
spectrometric analyses. We are extremely grateful to Dr Pam
Thomas for discussions and assistance with the bioinformatic
analysis.
Keywords: nucleoside • dehydratase • UPA • enzymology •
structural biology
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and Y55 and Y89 remained the same as in the WT-uridine
structure, additionally the hydrogen bond between K16 and
E108 with 2′- OH, as well as the π-stacking of the uracil ring with
F103 are all preserved. Q42 appears to have the same distance
from the 4′ proton and the overall fold of the protein does not
seem to be impacted, all of which suggests that the H42Q
mutation affects the key catalytic step and not substrate binding.
Similarly, on examining the Y89F-uridine complex (Figure 3B,
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and uracil rings, of the substrate, and in the orientation of the 5′
[
7]
1
5
26-546.
n.d. = not determined
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COMMUNICATION
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