Insights into β-ketoacyl-chain recognition for β-ketoacyl-ACP utilizing AHL synthases

Article abstract of DOI:10.1039/c8cc04532a

Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and acyl-homoserine lactone biosynthetic pathways are important targets for developing antimicrobial, anticancer and antiparasitic compounds. Published reports on successful isolation of beta-ketoacyl-ACPs in a laboratory remain scarce to date and thus most beta-ketoacyl-ACP utilizing enzymes are routinely characterized using small molecule substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the systematic investigation into the electronic, geometric and spatial aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl-ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum signal synthases.

Full text of DOI:10.1039/c8cc04532a

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Insights into b-ketoacyl-chain recognition
for b-ketoacyl-ACP utilizing AHL synthases†
Cite this:DOI: 10.1039/c8cc04532a
a
a
a
ab
Mila Nhu Lam, Dastagiri Dudekula,‡ Bri Durham, Noah Collingwood,§
a
a
Received 6th June 2018,
Accepted 11th July 2018
Eric C. Brown and Rajesh Nagarajan
*
DOI: 10.1039/c8cc04532a
rsc.li/chemcomm
Beta-ketoacyl-ACP utilizing enzymes in fatty acid, polyketide and laboratory, despite multiple attempts, we were unable to synthe-
acyl-homoserine lactone biosynthetic pathways are important targets size b-ketoacyl-ACPs in the amounts required for routine enzyme
for developing antimicrobial, anticancer and antiparasitic compounds. assays. Although we do not claim that beta-ketoacyl-ACPs are
Published reports on successful isolation of beta-ketoacyl-ACPs in a impossible to make, based on the observations discussed above,
laboratory remain scarce to date and thus most beta-ketoacyl-ACP we surmise that the direct functionalization of coenzyme A
utilizing enzymes are routinely characterized using small molecule derivatives with an enolizable b-keto moiety will be a synthetically
substrates in lieu of the bonafide 3-oxoacyl-ACPs. We report the challenging task to accomplish. We therefore decided to circumvent
systematic investigation into the electronic, geometric and spatial this issue by (a) not installing the ‘‘b-keto’’ moiety but instead mimic
aspects of beta-ketoacyl-chain recognition to develop 3-oxoacyl- this functionality in the acyl-chain of b-ketoacyl-ACPs or (b) suppress
ACP substrate mimics for two beta-ketoacyl-ACP utilizing quorum enolization by masking the a-hydrogens. To develop stable and
signal synthases.
catalytically active b-ketoacyl-ACP mimics, we describe the design,
synthesis and characterization of a library of molecular probes to
Beta-ketoacyl-acyl carrier proteins are important cellular systematically investigate the electronic, geometric and spatial
metabolites synthesized by a ketosynthase catalyzed Claisen characteristics of the ‘‘b-keto’’ functional group recognition for
condensation of malonyl-acyl carrier protein and an unsubstituted two b-ketoacyl-ACP utilizing enzymes.
acyl-acyl carrier protein during fatty acid and polyketide
The following guiding principles were considered while
1
biosynthesis. b-Ketoacyl-ACP utilizing enzymes in these pathways designing the b-ketoacyl-ACP mimics: (i) presence of a hetero-
are prime targets for developing medicines to treat bacterial atom at or around the b-carbon in the acyl-chain, (ii) reduction
2
infections, parasitic infections, cancer, obesity etc. Most b-ketoacyl- or elimination of b-ketoenolate tautomer formation, and
ACP utilizing enzymes are often characterized using N-acetylcyste- (iii) commercial availability or ease of synthesis of the carboxylic
amine or CoA-based small molecule substrates in lieu of ACP-based acid starting materials. Based on the limited structural information
substrates, perhaps due to the relatively easier access (cost, time and on b-ketoacyl-substrate utilizing enzymes available to date, it
yield) to these compounds compared to protein-based substrates. appears that the active site usually has a complementary hydrogen
Unfortunately, biochemical investigations on this class of enzymes bond donor residue such as threonine to specifically aid in b-keto
4
with small molecule substrates miss out valuable information on moiety recognition. We therefore placed high emphasis to include
the role of protein–protein recognition and dynamics in enzyme a heteroatom at or around the b-carbon in the acyl-chain of
catalysis. Despite more than three decades of literature on the the substrate (Fig. 1). For instance, 2-furanacetyl-ACP (10-ACP)
2
synthesis of unsubstituted acyl-ACPs, peer-reviewed publications includes an oxygen atom attached to C3 that is also sp
on the successful isolation of 3-oxoacyl-ACPs, especially the long- hybridized. So, we hypothesized this substrate would most
3
chain b-ketoacyl-ACPs, are surprisingly hard to find. In our own likely be a close mimic of a b-ketoacyl-ACP. Nevertheless, we
were concerned about the effect of having one of the lone pairs
a
of electrons on oxygen becoming involved in the (4n + 2) aromatic
system and whether its participation in p-electron delocalization
would potentially disrupt the ability of the remaining ring oxygen
atom lone pair to hydrogen bond with a complementary active site
residue. Furthermore, this substrate should reveal the importance
of enolizability of the b-keto oxygen in enzyme-substrate recognition.
In the fully reduced ring of 2-tetrahydrofuranacetyl-ACP (11-ACP),
Department of Chemistry and Biochemistry, Boise State University,
910 University Dr, Boise, ID 83725, USA. E-mail: rajnagarajan@boisestate.edu
1
b
Renaissance High School, 1307 E Central Drive, Meridian, ID 83642, USA
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc04532a
Current address: CanAm Bioresearch Inc., 6-1200 Waverly Street, Winnipeg,
†
‡
Manitoba, R3T 0P4, Canada.
Current address: USC Viterbi School of Engineering, 3650 McClintock Ave, Los
§
Angeles, CA 90089, USA.
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Fig. 2 Catalytic efficiencies of b-ketoacyl-ACP substrate analogs in 3-oxo-
AHL synthesis. (A) The native substrate for P. stewartii EsaI and Y. pestis YspI,
respectively, are 3-oxohexanoyl-ACP and 3-oxooctanoyl-ACP. (B) Catalytic
efficiencies of b-ketoacyl-ACP substrate analogs with EsaI and YspI enzymes.
Fig. 1 Chemical structures of b-ketoacyl-ACP substrate analogs used in this
study. The structural attributes of each substrate is described in Fig. S84 (ESI†).
the oxygen lone pair is not delocalized; however, the b-carbon
3
is sp hybridized. The sulfur atom in 2-thiopheneacetyl-ACP acyl-substrate and S-adenosyl-L-methionine as the acyl-acceptor
7
(
12-ACP) has a larger van der Waals radius (longer C–S bond), to synthesize AHL quorum sensing signals (Fig. 2). Since signal
increased electron pair delocalization and decreased hydrogen synthesis play a vital role in microbial communication, AHL
5
bonding potential compared to oxygen in 2-furanacetyl-ACP.
Unlike thiophene, the nitrogen atom in 2-pyridylacetyl-ACP sensing in several pathogenic bacteria that use a b-ketoacyl-ACP
13-ACP) is part of a larger 6-membered ring and since the utilizing 3-oxo-AHL synthase. Some of these enzymes include
synthase enzymes are bonafide targets for inhibiting quorum
(
nitrogen lone pair is not delocalized, it can participate in hydrogen 3-oxododecanoyl-ACP utilizing Pseudomonas aeruginosa LasI
bonding interactions with a suitable hydrogen bond donor in the (causative agent for pneumonia, cystic fibrosis, bacterial meningitis
active site. 2-Benzofuranacetyl-ACP (15-ACP) includes a second ring etc.), 3-oxooctanoyl-ACP utilizing Yersinia pestis YspI (bubonic plague
intended to gain additional binding energy with enzymes that have pathogen) and 3-oxohexanoyl-ACP utilizing Pantoea stewartii EsaI
4
a deeper acyl-chain pocket. In order to assess the importance of the (plant pathogen that causes Stewart’s wilt and leaf blight disease).
position of oxygen atom in the acyl-chain, we synthesized the With the exception of a nice paper from the Greenberg laboratory
4-oxoacyl-ACP (g-position; substrates 6-ACP and 7-ACP), 5-oxoacyl- on a few YspI inhibitors discovered through a high throughput
ACP (d-position; substrates 8-ACP and 9-ACP) and 2-furoyl-ACP screen, there is little progress in AHL synthase inhibitor discovery
8
(
a
a-position; 14-ACP) substrates. The pK of a-hydrogens in for this class of enzymes. This deficiency in the literature could
b-ketoacyl(thio)esters are reportedly lower compared to simple be attributed to most 3-oxo-AHL synthases still remaining
6
oxo or thioesters. Under soft enolization conditions, we wondered uncharacterized, perhaps due to challenges in the successful
if the enolate ion could promote intermolecular displacement of isolation of b-ketoacyl-ACPs and its analogs, discussed above.
the thiol (over a period of time) to decrease substrate stability? To test the feasibility of our approach towards developing stable
We therefore replaced the two a-hydrogen atoms with two and active b-ketoacyl-ACP mimics that can be readily synthesized
methyl-groups in substrates 4-ACP and 5-ACP. Unsubstituted in sufficient yields for routine enzymological investigations, we
acyl-ACPs (1-ACP, 2-ACP and 3-ACP) were included as a control synthesized and tested the activity of analogs 1-ACP–15-ACP on
in this study to determine the effect of removing the b-keto EsaI and YspI AHL synthases.
oxygen on substrate activity.
Tables S1 and S2 (ESI†) summarizes the kinetic data for EsaI
The N-acyl-L-homoserine lactone synthases aka AHL synthases and YspI determined using lactonization (MTA) and acylation
in Gram-negative bacteria use acyl-ACP or acyl-CoA as the (holo-ACP) HPLC assays (Fig. 2). The catalytic efficiencies of
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these substrates with EsaI and YspI were measured indepen- a gatekeeper residue in ensuring the selective recognition of
1
0
dently using both HPLC assays as described in the ESI† section 3-oxoacyl-ACP over unsubstituted acyl-ACP (Fig. S79, ESI†).
of this paper (Fig. S75 and S76). The kinetic constants
9
Substrate activity trends in YspI resemble the trends in
obtained from both acylation and lactonization assays were substrate catalytic efficiencies with EsaI. Since YspI synthesizes
comparable, which further validates the robustness of the HPLC 3-oxooctanoyl-homoserine lactone autoinducers for Y. pestis, its
assay used in this study. The kinetic constants for b-ketohexanoyl- preference for longer acyl-chains is clearly seen in substrate
4
c
ACP mimics with EsaI are represented in Table S1 (see also activity trends shown in Table S2 (Fig. S80 and S81, ESI†). For
Fig. S77 and S78, ESI†). The acyl-chain binding pocket in 3-oxo- instance, while EsaI’s most active substrate is 2-furanacetyl-ACP,
AHL synthases typically have a complementary hydrogen bond the two best substrates for YspI are 2-benzofuranacetyl-ACP and
donor residue (such as Thr140 in EsaI) to recognize the b-keto 2,2-dimethyl-3-oxooctanoyl-ACP (Tables S1, S2 (ESI†) and Fig. 3).
oxygen. As discussed earlier, 2-furanacetyl-ACP (10-ACP) has an The 2-pyridylacetyl-ACP substrate is more active with YspI
2
sp hybridized oxygen atom at the b-position and therefore is suggesting the larger pyridine ring faced less steric clashes with
the likely candidate to mimic the native substrate. Indeed, this a deeper acyl-chain pocket of YspI. Substrates with heteroatoms
m
substrate has the highest kcat/K with EsaI among all substrates at a, g and d positions are far worse active in YspI compared to
À1
À1
in our focused library (0.37 Æ 0.05 mM min ; Table S1, ESI†). EsaI revealing a more stringent requirement for the heteroatom
This result also seem to suggest that enolization of the ‘‘b-keto’’ position in this enzyme. Irrespective of acyl-chain lengths,
moiety is not essential for substrate recognition in EsaI. unsubstituted acyl-ACPs fare worse again reiterating the impor-
Although the lone pair of electrons on the heteroatom could tance of a hydrogen bond acceptor at the b-position. Overall, it
donate two electrons towards the 6p electrons in the ring, it appears YspI is more selective in substrate preference compared
seems the reduced aromatic character of furan compared to to EsaI.
other 5-membered heterocycle such as pyrrole or thiophene
Finally, 2-thiopheneacetyl-ACP is the only substrate in our
could have contributed towards enhancing the activity of this library to display substrate inhibition with both EsaI and
substrate. 2-Tetrahydrofuranacetyl-ACP is almost as active as YspI (Fig. S77, S78, S80 and S81 (ESI†); see Fig. S77 (ESI†)
3
2
-furanacetyl-ACP despite sp hybridization at the b-position legend for details on substrate inhibition mechanism). The K
m
À1
À1
(k
cat/K
m
= 0.27 Æ 0.04 mM min ). Although the above result
seems to hint that EsaI doesn’t appear to discriminate between
2
3
sp and sp hybridized centers at the b-carbon, this outcome
must be interpreted with caution because the substrate selectivity
differences between furan and tetrahydrofuran side-chains with
EsaI may not truly resemble the reactivity differences between
3-oxoacyl-ACP and 3-hydroxyacyl-ACPs with the enzyme. While
the K is unaltered between 2-pyridylacetyl-ACP and 2-furanacetyl-
m
ACP, the loss of substrate activity is due to a lower kcat value, which
indicate that [EÁ2-pyridylacetyl-ACPÁSAM] ternary complex is
in a non-optimal, less-productive conformation compared to
[
EÁ2-furanacetyl-ACPÁSAM] for the chemistry and/or product
release steps in AHL synthesis. The catalytic efficiencies of
-furoylacetyl-ACP, 4-oxohexanoyl-ACP and 5-oxohexanoyl-ACP
2
are about 4–20 fold lower than 2-furanacetyl-ACP with EsaI high-
lighting the importance of the heteroatom position in the acyl-chain.
Displacement of the heteroatom from b to a, g, and d positions in
m
the acyl-chain increased the K and decreased kcat relative to the
most active substrate, 2-furanacetyl-ACP. The a-dimethylated sub-
strate was the second most active substrate in the series suggesting
that the methyl substitution doesn’t seem to affect the substrate’s
À1
À1
catalytic efficiency (kcat/K
m
= 0.30 Æ 0.05 mM min ).
EsaI has a narrow acyl-chain pocket that restricts acyl-chain
4
a
lengths to 6 carbons or less. The low catalytic efficiencies for
larger/longer acyl-chain substrates (2-benzofuranacetyl-ACP,
octanoyl-ACP, 4-oxooctanoyl-ACP and 5-oxooctanoyl-ACP) indicate
that larger substrates are probably occluded from the narrow acyl- Fig. 3 The b-ketoacyl-ACP mimic for EsaI and YspI. (A) Bar diagram
comparing the catalytic efficiencies of 2-furanacetyl-ACP substrate for
EsaI and 2-benzofuranacetyl-ACP for YspI (both shown in red bars) with
chain cavity in EsaI. Hexanoyl-ACP, a 6-carbon substrate was one of
the worst substrates for EsaI wildtype enzyme clearly underscoring
the importance of a heteroatom at the b-position. Furthermore,
native substrates of a few well-characterized AHL synthases. (B) Structural
similarities between 2-furanacetyl-ACP (EsaI)/2-benzofuranacetyl-ACP
the 50–60 fold increase in the activity of hexanoyl-ACP with EsaI
(
YspI) and b-ketoacyl-ACPs. The part of the substrate that closely mimics
T140A mutant over wild-type enzyme reiterates the role of T140 as the b-ketoacyl-ACPs is drawn in red.
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of 2-thiopheneacetyl-ACP with both enzymes is also the lowest to inhibit both ketosynthase and ketoreductase is particularly
among all substrates in our library suggesting a tightly bound appealing to develop potent fatty acid synthase inhibitors, develop
[enzyme–substrate] complex for this substrate (Tables S1 and mechanistic probes that could arrest polyketide synthesis at specific
S2, ESI†). Although commonly found in proteins, hydrogen steps and discover novel antivirulent compounds that inactivate
bonding interactions with sulfur are proposed to be weaker 3-oxoacyl-homoserine lactone synthases from pathogenic bacteria.
compared to oxygen and the increased aromatic character of
Financial support for this project came from Boise State
thiophene should further diminish the potential of sulfur atom University start-up funds (RN), NIH 1R15GM117323-01 (RN),
to accept a hydrogen bond from a donor residue in the active NIH INBRE grants P20 RR016454 and P20 GM103408. Noah
1
1
site. Nevertheless, sulfur also participates in ‘‘sigma hole’’ Collingwood was supported by the American Chemical Society
1
2
interactions with electronegative atoms such as N, O etc.
Project SEED fellowship.
Quantum mechanical calculations reveal that the magnitude
of interaction energies at the sigma hole could match typical
hydrogen bond energies (22 to 30 kJ mol ). In addition, the
À1 13
Conflicts of interest
size of two sigma holes on the divalent sulfur are larger on
aromatic rings such as thiophene. It appears these additional
sigma hole interactions for sulfur could have likely contributed
There are no conflicts of interests to declare.
1
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Notes and references
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of 2-thiopheneacetyl-ACP with EsaI and YspI. In
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is about 3-fold
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2
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(
Table S3, ESI†). Interestingly, the kcat is also 4–5 fold lower
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-furanacetyl-ACP for EsaI or 2-benzofuranacetyl-ACP for YspI
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2
(
[
EÁ2-thiopheneacetyl-ACPÁSAM] ternary complexes in both enzymes
are perhaps in a non-optimal mode for chemistry and product
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class of enzymes.
3
4
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for substrate activity with b-ketoacyl-ACP utilizing AHL synthase
enzymes such as EsaI and YspI. In this study, we have uncovered
5
6
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(
(
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8
9
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ACP utilizing enzymes in therapeutically important biosynthetic
1
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ketosynthase product inhibitors, the inert, ring substrate analogs
such as 2-furanacetyl-ACP could be used to develop competitive
inhibitors for ketoreductase. The potential of b-ketoacyl-ACP mimics
1
Chem. Commun.
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