In vitro studies of maleidride-forming enzymes

Article abstract of DOI:10.1039/d1ra02118d

In vitro assays of enzymes involved in the biosynthesis of maleidrides from polyketides in fungi were performed. The results show that the enzymes are closely related to primary metabolism enzymes of the citric acid cycle in terms of stereochemical preferences, but with an expanded substrate selectivity. A key citrate synthase can react both saturated and unsaturated acyl CoA substrates to give solely anti substituted citrates. This undergoes anti-dehydration to afford an unsaturated precursor which is cyclised in vitro by ketosteroid-isomerase-like enzymes to give byssochlamic acid. This journal is

Full text of DOI:10.1039/d1ra02118d

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In vitro studies of maleidride-forming enzymes†
*
Sen Yin, Steffen Friedrich, Vjaceslavs Hrupins and Russell J. Cox
Cite this: RSC Adv., 2021, 11, 14922
In vitro assays of enzymes involved in the biosynthesis of maleidrides from polyketides in fungi were
performed. The results show that the enzymes are closely related to primary metabolism enzymes of the
citric acid cycle in terms of stereochemical preferences, but with an expanded substrate selectivity. A key
citrate synthase can react both saturated and unsaturated acyl CoA substrates to give solely anti
substituted citrates. This undergoes anti-dehydration to afford an unsaturated precursor which is
cyclised in vitro by ketosteroid-isomerase-like enzymes to give byssochlamic acid.
Received 17th March 2021
Accepted 15th April 2021
DOI: 10.1039/d1ra02118d
rsc.li/rsc-advances
These experiments suggest a series of events in which a pol-
yketide synthase produces a linear acyl group attached to its acyl
Introduction
carrier protein (ACP) 14. This must be released (e.g. 15) and
reacted with oxaloacetate to form a vinyl citrate 16 by the citrate
synthase enzyme (Scheme 1). Dehydration by the 2MCDH
enzyme would then provide the observed substituted maleic
Alkyl citrates are a class of fungal secondary metabolites derived
from a polyketide or fatty acid component and oxaloacetic acid.
They include relatively simple monomeric compounds such as 1
and 2, piliformic acid 3,1 oryzines A and B 4–5,2 sporothriolide
6,3 CJ-13,981 7 (ref. 4 and 5) and hexylitaconic acids6 known
from Aspergillus niger (Fig. 1). More complex examples include
compounds such as viridiofungin A 8 (ref. 7) and squalestatin
9
S1 9 (ref. 8 and ) which are potent inhibitors of squalene syn-
thase. In many cases dehydration of the alkyl citrate affords
a maleic anhydride as observed in 1 and 2, and dimerisation of
these monomers leads to the formation of compounds with
large alicyclic rings known as heptadrides, octadrides and
nonadrides.10 Collectively such compounds are known as mal-
eidrides.11 These include agnestadride A 10,11 zopellein 11 (ref.
12 and 13) byssochlamic acid 12 (ref. 14) and the selective
herbicide cornexistin 13.15,16 These types of compounds are
wide-spread in fungi (Fig. 1).17
To-date the main evidence for the biochemical reactions
involved in the biosynthesis of alkyl citrates and maleidrides
has come indirectly from in vivo genetic knockout or heterolo-
gous expression experiments. Thus, in the case of byssochlamic
acid 12, for example, we showed that co-expression in the host
Aspergillus oryzae of genes encoding: a fungal highly-reducing
polyketide synthase (hr-PKS); an ab-hydrolase;
a citrate
synthase-like protein (CS); and a homolog of 2-methylcitrate
dehydratase (2MCDH) results in production of 1 and 2. Like-
wise, Oikawa and coworkers expressed homologous genes from
the phomoidride BGC from unidentied fungus ATCC 74256
and related genes from Talaromyces stipitatus and showed
production of anhydride monomers related to 1 and 2.18
OCI, BMWZ, Leibniz University of Hannover, Schneiderberg 38, 30167, Hannover,
Germany. E-mail: russell.cox@oci.uni-hannover.de
Fig. 1 Structures mentioned in the text. Bold bonds indicate atoms
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ra02118d
derived from oxaloacetate.
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Table 1 Summary of protein production. bf, genes from B. fulva; pv,
genes from P. variotii; I, insoluble protein; 3, soluble protein observed;
7, no protein observed; -, experiment not tried; † cell-free extract
used; green shading, functional protein
induction) with an N-terminal his6-tag were successful for
BfL1 (hydrolase), BfL2 (citrate synthase) and BfL9 (PEBP) only
(Table 1), giving soluble protein. In other cases either insol-
uble protein was detected, or no signicant expressed protein
was detected. In the cases of BfL5, BfL6 and BfL10 (PEBP and
KI) putative signal peptides sequences were detected at their
N-termini (SignalP 5.0 server).20 These were removed by
cloning N-truncated versions into pET28a and expression
trials were set up in E. coli BL21(DE3). This resulted observable
production of protein, but in the insoluble fraction in each
case. The truncated bfL5, bfL6 and BfL10 genes were then
individually co expressed in the presence of the pG-KJE8
chaperone system in E. coli, resulting in observation of
soluble protein in each case. The nal genes, bfL3 and pvL2,
encoding putative dehydratases could not be expressed in E.
coli under any of the previously tried conditions. These genes
were cloned into pESC-URA vectors and expressed in Saccha-
romyces cerevisiae strain W303-1b, induced by the addition of
galactose. Soluble protein was detected in each case by SDS-
PAGE analysis.
The ACP region of the B. fulva PKS was synthesised in E. coli
optimised form (S2511 – E2618) in pET28a by a commercial
supplier. The fragment was expressed in E. coli BL21 DE3 and
soluble apo-protein was puried by nickel affinity chromatog-
raphy and converted to holo-ACP using the Sfp phosphopante-
theine transferase in vitro.21 Both apo and holo-proteins were
conrmed by mass spectrometry (see ESI† for details).
Hydrolase
Previous in vivo results indicate that BfPKS builds the hex-2-
enoyl intermediate required for construction of monomeric
maleic anhydrides (Scheme 1). We assumed that release of the
hexenoyl unit from the PKS is achieved by the BfL1 hydrolase,
and observations from coexpression studies were in agreement
with this hypothesis.
Scheme 1 Proposed pathway to the maleidride byssochlamic acid 12.
In order to probe the selectivity of the BfL1 hydrolase we
synthesised SNAC, pantetheine, CoA and ACP-bound potential
substrates (Scheme 2).22,23 In our previous work we assumed
that the acyl group should be unsaturated (e.g. an E-hex-2-enoyl
substrate) but this was unproven,10 so we made both E-hex-2-
enoyl 14a–d and hexanoyl 15a–d substrates. Initial experi-
ments showed that direct formation of thiolesters of a,b-
unsaturated species (e.g. in carbodiimide type couplings) was
accompanied by unwanted Michael addition, the products of
which could not be easily removed. This was overcome by
formation of the ylides 19c and 20c from the corresponding
thiols 19a and 20a. These reacted smoothly with aldehydes to
give the desired 14b and 14c aer deprotection (Scheme 2). CoA
thiolesters were formed more easily by direct coupling of mixed
anyhydrides with CoASH itself, and in turn these were loaded
onto the isolated apo-ACP of the B. fulva PKS using the phospho-
pantetheinyl transferase (PPTase) enzyme Sfp.24,25 Finally,
saturated SNACs and panthetheins 15b and 15c were made by
standard EDCI coupling reactions.
acids such as 17 and its equilibraiting anhydride from 1.
Anhydride 1 decarboxylates spontaneously to give 2, which is
also in equilibrium with a diacid form 18.19 We also showed that
1 appears to be a substrate for a pair of enzymes with homol-
ogies to ketosteroid isomerase-like (KI) and phosphatidyl-
ethanolamine-binding protein (PEBP) which then produce the
alicylic rings of dimeric maleidrides 10, 12 and 13 by an as-yet
unknown mechanism. In order to probe these reactions and
enzymes in more detail we chose to study them in vitro with the
aim of determining their selectivities. Since the byssochlamic
acid 12 and cornexistin 13 pathways were previously described
in our hands, we focussed our efforts on the study of enzymes
from these systems.
Results
Gene cloning, expression and protein purication
Initial attempts to clone codon optimised fungal whole-length
In order to assess the substrate selectivity of the BfL1
ORFs in E. coli expression systems (pRSETA, Rosetta, IPTG hydrolase we incubated it with hex-2-enoyl and hexanoyl
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Scheme 3 In vitro assays of substrate analogs. A, results of incubation
with the BfL1 hydrolase; B, results of incubation with the BfL2 citrate
synthase and oxaloacetic acid.
Citrate synthase
Citrate synthase [EC 2.3.3.1] is a key enzyme of primary
metabolism and is known to react acetyl CoA with oxaloacetic
acid to produce citric acid. The enzyme performs both the
condensation reaction and the hydrolytic-release of CoA. The
gene bfL2 from the byssochlamic acid BGC in B. fulva encodes
a citrate synthase homolog which is 20% identical to the
mitochondrial (presumed primary metabolism) CS of B. fulva.10
In vitro assays were set up containing puried BfL2, oxaloacetic
Scheme
2 Synthesis of hex(en/an)oyl substrates. Reagents and
conditions: (a) BrCH2CO2H, EDCI, DMAP, CH2Cl2, RT; (b) PPh3,
toluene, 80 ꢀC, then aq. NaOH; (c) 19c, CH2Cl2, RT, 72 h; (d) 20c,
CH2Cl2, 40 ꢀC, 3 d then THF/CF3CO2H, RT, 60 min; (e) ClCO2Et,
CH2Cl2, Et3N, 0 ꢀC; (f) CoASH, aq. NaHCO3; (g) 19a, EDCI, DMAP,
CH2Cl2; (h) 20a, EDCI, DMAP, CH2Cl2, then THF/CF3CO2H, RT, 60 min.
thiolester species 14b–d and 15b–d and acyl-BfACP 14a and 15a.
Assays were set up in vitro and contained substrate anꢀd puried
BfL1 in buffer only. Reactions were incubated at 30 C in Tris
buffer containing MgCl2, NaCl and the non-nucleophilic
reducing agent tris(2-carboxyethyl)phosphine (TCEP, 1 mM),
then quenched by addition of 1 volume of CH3CN. Protein was
precipitated by centrifugation and the residue analysed directly
by LCMS.
In the case of the ACP species 14a and 15a, the acyl ACP was
directly monitored by ESIMS. In all cases 14a–d and 15a–d we
observed very rapid hydrolysis to the respective carboxylic acids,
and by direct observation of the formation of holo-BfACP
(Scheme 3A, see ESI† for bfACP mass data).
Fig. 2 LCMS results for incubation of saturated and unsaturated CoAs
with oxaloacetic acid and BfL2 (CS), ELSD traces. A, hex-2-enoyl CoA
14d; B, 14d + BfL2 + oxaloacetic acid; C, hexanoyl CoA 15d; D, 15d +
BfL2 + oxaloacetic acid.
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acid and substrates 14a–d and 15a–d. The reactions were fol- bromohexanoate 25 or its 3-unsaturated congener 27 (itself
lowed by LCMS. ACPs, SNACs and pantetheines were not derived from 26) in a Reformatsky reaction in DMF.26 This
substrates for BfL3, but both CoA thiolesters 14d and 15d were afforded a 2 : 1 mixture of diastereomers of the required tri-
converted to the corresponding citrates 16 and 30 (Scheme 3, methyl esters 28 (unsaturated) and 29 (saturated). These were
and Fig. 2).
hydrolysed by extended reux in aqueous HCl (3 days) to afford
A synthesis was developed for the production of the good yields of the required triacids 16 (unsaturated) and 30
proposed substituted-citrate products of the CS (Scheme 4). The (saturated) – again as 2 : 1 mixtures of diastereomers.
synthesis involves a Reformatsky reaction with an oxalate
Previous work by Barrett and coworkers showed that similar
diester. Diethy oxaloacetate is commercially available, and it reactions are anti-selective27,28 and this was conrmed by 1H
reacts satisfactorily to give citrate products, however the ethyl NMR analysis of the mixture of diastereomers of 16 which
citrates could not be hydrolysed in our hands. We therefore showed that the major compound is the 2,3-anti (3S*, 4R*)
required dimethyl oxalate 24. This innocuous compound is not diastereomer 16a. The 1H chemical shis of H-4 are particularly
commercially available and attempts to produce it by esteri- diagnostic, with H-4syn consistently resonating at lower eld
cation of oxaloacetic acid, or transesterication of diethylox- than H-4anti (see ESI† for details). HPLC analysis showed that
alate
were
unsuccessful.
However,
starting
from the major anti diastereomer 16a elutes slightly before the minor
dimethylacetylene dicarboxylate (DMAD) 21, selective hydration syn diastereomer 16b.
of the alkyne was achieved in two steps by the controlled
The individual diastereomers of synthetic 16 were separated
addition of morpholine 22 to give the intermediate 23, followed by HPLC to yield the pure racemic stereoisomers. NMR and
by hydrolysis in the presence of oxalic acid. This yielded 24 as LCMS analysis also revealed the presence of equilibrating
yellow crystals.
mixtures of the corresponding cyclic anhydrides 31, again with
Although this material did not give satisfactory spectro- a prevalence of the 3,4 anti diastereomer (3S*, 4R*, ESI Fig
scopic analysis, it did react smoothly and as expected in the S5.1†). Chromatographic comparison of the pure synthetic
following step in which it was reacted with either methyl-a- diastereomers with the products of the in vitro assay of BfL2
showed that the enzyme product is exclusively the anti diaste-
reomer 16a (ESI Fig S5.3†).
Structures of many primary metabolism citrate synthases
have been determined at high resolution. In particular the
structures of CS from Escherichia coli,29 Thermus thermophilus,30
Pyrococcus furiosus31 and Sus scrofa32 are understood in signi-
cant detail. Multiple sequence alignment between BfL2 and CS
from these organisms showed that the known active site resi-
dues D377, H284 and R332 are fully conserved (BfL2
numbering, see ESI†).
Other residues known to be involved in binding oxaloacetate
(e.g. G322, H323, R402 and R422*) and acyl CoA (e.g. K/R317, L/
I318, G320, R324, R/K370 and N375) are also conserved. N249 is
replaced by histidine in the other organisms, but modelling
(vide infra) shows that this residue forms an auxiliary hydrogen
bond to the C-4 carboxylate of oxaloacetate which can be ach-
ieved by either histidine or asparagine.
We built a 3D model of BfL2 using the SwissModel server33
and CS from Acetobacter aceti34 as a template (pdb 2H12,
Scheme 5B, see ESI† for details). The A. aceti structure was ob-
tained in complex with oxaloacetate and an acetyl CoA mimic
which allows the determination of residues which bind these
substrates.33 Overlay of the BfL2 model and the A. aceti structure
shows that all of the residues involved in catalysis and binding
oxaloacetate and acyl CoA are structurally highly conserved
(Scheme 5B). The model shows that the expected Si face of the
˚
oxaloacetate ketone is within 3.7 A of the nucleophilic carbon,
consistent with the formation of an S-congured tertiary
alcohol upon carbon–carbon bond formation.
Since the oxaloacetate-binding and catalytic residues of the
BfL2 model and the structure of 2H12 are identical, and overlay
˚
to within 1.15 A RMSD over 13 residues, it is highly likely that
the BfL2 alkyl citrate synthase also creates a 2S-stereocentre.
Since we already know that BfL2 synthesises exclusively the anti
Scheme 4 Synthesis of citrates. Abbreviations: DBPO, dibenzoylper-
oxide; NBS, N-bromosuccinimide; DMSO, dimethylsulfoxide.
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We next tested the anti 16a and syn 16b diastereomers of the
citrate substrate separately. The anti diastereomer 16a was
converted to the new peak, while the syn diastereomer 16b was
unreactive. The new compound 17 elutes with the same reten-
tion time as the syn diastereomer of the citrate 16b. Extended
reaction never reduced the amount of (ꢁ) starting material
below 50% suggesting that the enzyme is selective for a single
enantiomer, presumably the 2S,3R stereoisomer. Although the
UV and MS analysis of the product of the reaction were
consistent with formation of the expected 2,3-unsaturated
system, the in vitro assays did not yield enough material for full
NMR characterisation (Fig. 4).
In order to fully characterise 1 it was re-isolated from
a fermentation of A. oryzae expressing bfPKS, bfL1 (hydrolase),
bfL2 (CS) and b3 (dehydratase). A time course experiment
Scheme 5 Conserved residues in citrate synthase: A, mechanism of
citrate synthase; B, model of BfL2 (cyan) overlayed with crystal
structure data from A. aceti citrate synthase 2H12 (green). Residue
numbering from BfL2 throughout. Oxaloacetate and an acetyl CoA
mimic bound to 2H12 are shown in grey. * ¼ residue from other dimer
subunit.
diastereomer, then we conclude that the product of BfL2
possesses 2S,3R conguration.
Dehydratase
Dehydratase assays were also set up in vitro and monitored by
analytical LCMS. In initial reactions, the synthetic mixture of E-
1-butenyl-citrate diastereomers 16ab was incubated with the
BfL3 and PvL2 dehydratase enzymes in separate reactions.
When observed by diode array detector (200–300 nm), an
increase in the area of the later-running peak corresponding to
the syn citrate was noted. The citrates themselves have no
signicant UV absorbtion above 220 nm, but the new peak has
a maximum UV absorbtion at 270 nm consistent with the
formation of the expected unsaturated product 17 which co-
elutes with 16b (Fig. 3).
Observation of the reaction at 270 nm more clearly showed
the increase in this species. Mass analysis also indicated a loss
of 18 mass units, consistent with the expected dehydration.
PvL2 was more active that BfL3 and was therefore used for all
further assays.
Fig. 3 LCMS analysis of PvL2 and BfL3 reactions in vitro. A, 16 alone; B,
16 + BfL3; C, 16 + PvL2; D, 16 alone; E, 16 + BfL3; F, 16 + PvL2.
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Scheme 6 Reactions catalysed by BfL3 and Pvl2 dehydratases, and
facile decarboxylation.
anhydride 1. Attempts to purify either compound always led to
recovery of mixtures.
KI and PEBP
Our previous in vitro work showed that expression of KI genes
with the earlier pathway genes led to dimerisation of 1 and
formation of byssochlamic acid, while addition of the PEBP
genes increased the titre. However, we were unable to conclude
whether the PEBP proteins played a role in the catalysis or,
alternatively, provided an uncharacterised resistance mecha-
nism to byssochlamic acid 12 which is known to be toxic to
fungi.35 Here we tested the effects of the isolated proteins with
the proposed substrate 1 in vitro, once again following the
reactions by analytical LCMS.
Initial work focussed on attempts to detect conversion of
maleic anhydride monomer 1 to bysochlamic acid 12 using
BfL5, BfL6, BfL9 and BfL10 obtained by E. coli expression of the
genes (lacking signal peptides and containing N-terminal his6
tags) either singly or in combination. However despite
numerous attempts and varied conditions we could never
observe any activity. We then attempted to express the full-
length genes in yeast using the pESC system already
described. In these cases we were not able to observe signicant
amounts of new soluble proteins by SDS-PAGE, or purify the
proteins because they were cloned without his6 tags. However,
we tested cell-free extracts of the induced yeast strains and
compared the effects vs. control strains lacking the expression
plasmids. In these cases we could clearly observe the conversion
of 1 to byssochlamic acid 12 by a combination of all 4 protein
extracts. The same results were observed for the combination of
the two KI components, and for the KI components tested
singly. The PEBP proteins appeared to be inactive in all condi-
tions tested. They did not form 12 either alone or in combina-
tion, and their addition to the KI proteins did not seem to
signicantly increase the amount of 12 formed (Scheme 7).
Finally, decarboxylated maleic anhydride 2 was also tested as
a substrate in the dimerisation assay. However no conversion to
byssochlamic acid 12 was observed, and 2 and its hydrolysed
Fig. 4 LCMS analysis of the indicated DH reactions. Top (2R,3S/2S,3R)
anti substrate 16a reacted with enzyme: A, BfL3; B, PvL2; and C, empty
vector control respectively. Bottom (2R,3R/2S,3S) syn substrate 16b
reacted with enzyme: D, BfL3; E, PvL2; and F, empty vector control.
determined that 1 reaches maximum concentration aer 4 days
of fermentation. Aer this time cells were collected, and
extracted with EtOAc. Extended contact with organic solvents
such as CHCl3 and DMSO causes 1 to decarboxylate to give 2.
Similarly, freeze-drying procedures also decarboxylated 1.
However, rapid purication by preparative HPLC in CH3CN/
H2O mixtures, followed by removal of CH3CN under mild
vacuum led to stable aqueous solutions of 1 which could be
analysed by NMR and used for further in vitro assays (vide infra).
The product of the in vitro reaction of PvL2 and BfL3 was thus
proven to be identical to 1 isolated from fermentation (Scheme
6).
LCMS analysis also showed that the maleic acid 17 (UV max
268 nm) and maleic anhydride 1 (UV max 313 nm) forms are in
equilibrium. The diacid 17b elutes signicantly earlier than the
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congener 18 were the only compounds observed by LCMS mechanisms are known in other fungal PKS systems such as
analysis (see ESI† for details).
phaeospelide A.37 However, BfL1 also appears to rapidly
hydrolyse other thiolesters, so how its selectivity is controlled in
vivo is not yet known. This release mechanism differs from that
used by fungal FAS (e.g. SpoFas)3 where the malonyl-palmitoyl
transferase (MPT) domain is known to both load malonyl CoA
extenders, and release products directly as CoA thiolesters.38
The next step of the pathway is also closely related to primary
metabolism. We showed that the citrate synthase BfL2 selec-
tively reacts acyl CoAs 14d and 15d with oxaloacetate. This
differs from the suggestion by Tang and coworkers that similar
CS enzymes react directly with ACP-bound acyl groups, for
example during the biosynthesis of squalestatins.39 This reac-
tion does not occur in the case of the byssochlamic acid
pathway where we showed that acyl-ACPs (i.e. 14a and 15a) are
not substrates for BfL2.
The requirement for CoA substrates by the CS raises an
interesting question. In the case of FAS-based pathways acyl
CoAs are released directly by the FAS, but for the PKS + hydro-
lase pathways the released carboxylic acids must be activated to
CoA thiolesters, however the required CoA synthetase does not
appear to be encoded within the Bf BGC. Presumably such
short-chain thiolesters could be synthesised by endogenous
primary metabolism enzymes.
Discussion
Alkyl citrates are a class of fungal secondary metabolites which
are closely related to primary metabolites. In the case of the
maleidrides byssochlamic acid 12 and cornexistin 13 the
pathway starts with a highly-reducing polyketide synthase (hr-
PKS),36 but in many other cases such as the sporothriolides3 the
pathway begins with a typical fungal fatty acid synthase (FAS).
The function of these synthases at the start of the pathways is
subtly different.
hr-PKS such as BfPKS1 can control chain-length and func-
tionalisation of the acyl chain, but these hr-PKS usually lack an
integrated release mechanism. Thus BfPKS1 appears to syn-
thesise a hex-2-enoyl product which is released by a dedicated
trans-acting hydrolase BfL1 as a carboxylic acid. This is
demonstrated in vitro here where we showed that BfL1 can
hydrolyse acyl-BfPKS-ACP species. Similar hydrolytic release
The BfL2 CS can react both saturated and E-unsaturated
substrates. In the case of E-unsaturated substrates the alkene
migrates during the reaction. Since it is known that the active
site base of CS is a highly conserved aspartate (D377, Scheme 8),
Scheme 7 LCMS analysis of reactions catalysed by KI enzymes BfL6
and BfL10, and PEBP proteins BfL5 and BfL9: A, two KSI and two PEBP;
B, two KI; C, BfL6; D, BfL10; E, empty vector control; F, only substrate; Scheme 8 A, Likely mechanism of BfL2 during the synthesis of alkyl
G, producing A. orzyae transformant. * mixture of diacid forms of 12. citrates; B, adapted mechanism for the synthesis of vinyl citrates via
Note that data is qualitative rather than quantitative.
a dienol intermediate.
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we speculate that this residue could be involved in both the heptadride 10 was not observed in vitro, raising the question
processes, forming a dienol intermediate during the synthesis of how this is formed in vivo.
of 16a (Scheme 8B). Thus-far we have not experimented with Z-
alkenes as substrates, but this would be an interesting future tive. The B. fulva 12 BGC encodes two pairs of KI and PEBP
goal. enzymes, but most other maleidride clusters encode only
The PEBP proteins studied here appeared catalytically inac-
We showed that the BfL2 CS produces exclusively 3,4-anti a single pair, so it seems likely that the B. fulva system has
products, and structural considerations strongly suggest this is undergone gene duplication. Lack of catalytic activity of PEBP
the 3S,4R stereoisomer. In the cases of other known citrate- proteins supports previous suggestions that these may be part
derived metabolites (and in primary metabolism) the 3S of resistance systems as they are encoded by most maleidride
conguration is also observed, but in the known cases of BGC, however another function cannot be ruled out.
squalestatin S1 9, viridiofungin 8 and CJ-13,981 7, for example,
Thus the in vitro studies of the function and stereoselectivity
the CS products are 3S,4S.7,40,41 Differences in conguration at of enzymes involved in fungal alkyl citrate biosynthesis show
the 4-position must be controlled by the geometry of the enoyl that the pathway is very closely related to primary metabolism.
CoA intermediate, and in-turn this may be related to the ability Indeed simple compounds like CJ-13981 7 should require only
of the BfL2 CS to react 2,3-unsaturated substrates. Detailed a fatty acid synthase and an alkylcitrate synthase, both
structural work will be required to probe this question further. presumably duplicated from primary metabolism, to produce
The family of secondary metabolite alkyl citrate synthases a compound with useful activity as an inhibitor of squalene
resemble methylcitrate synthases involved in propionate synthase.4,5 Exchange of the FAS for an hr-PKS (and hydrolase)
metabolism. Recent work by Reddick and coworkers has reas- allows more complex chains to be built – for example squales-
signed the product of these enzymes as 3S*,4R* in agreement tatin precursors which are also squalene synthase inhibi-
with our stereochemical assignment.42
tors.46–48 Finally, gain of a dehydratase (again almost directly
In the next step the dehydratase BfL3 removes a water from primary metabolism) and a KI-like protein then allows the
molecule. This step is closely related to the primary metabolism pathway to make maleidrides. Other pathways like those
enzyme 2-methylcitrate dehydratase (2MCDH). Again the ster- involved in squalestatin or sporothriolide diversify by gain of
eoselectivity of this step by the primary metabolism 2-methyl- more obviously secondary metabolism steps involving C–H
citrate dehydratase has been debated, but Reddick's results41 activation by oxidation. The close relationship of such
conclusively show that the enzyme takes 3S*,4R* isomer to give secondary metabolism pathways to key primary metabolic
Z-methyl aconitate – i.e. anti dehydration.43 Again, the BfL3 and enzymes shows how complex secondary metabolic pathways
PvL2 dehydratases follow exactly the same stereochemical have likely evolved. However, for the same reasons, this makes it
course, emphasising the similarity of the primary and difficult for bioinformatic systems trained to nd secondary
secondary metabolism pathways. The dehydrated products metabolism genes and pathways to recognise these pathways.
spontaneously equilibrate between diacid 17 and anhydride 1
forms. Reddick showed that the primary metabolism 2MCDH
enzymes can dehydrate both the anti and syn diastereomers of
2-methylcitrate, but the BfL3 enzyme cannot dehydrate the syn
substrate diastereomer 16b.
Experimental details
All experimental details and characterisations of individual
compounds and proteins is contained in the ESI.†
Finally, we showed that ketosteroid-isomerase-like (KI-like)
enzymes BfL6 and BfL10 catalyse the dimerisation of 1 to the
nonadride byssochlamic acid 12. They appear to act alone and
VH cloned the BfPKS ACP, prepared acylated ACPs and con-
both are individually active. Interestingly, decarboxylated
congener 2 is not a substrate for the enzyme catalysed reaction.
In previous in vitro work Baldwin44 and others45 have shown that
related decarboxylated compounds can give low yields of
dimeric products in organic solvents in the presence of medium
Author contributions
ducted assays with these species, and worked with Sfp. SY
cloned all other genes, expressed all other proteins and per-
formed all other in vitro assays. SF synthesised all small mole-
cules and performed hydrolase assays. RJC devised the study,
gained funding and wrote the paper.
to strong bases which are likely to be able to form low
concentrations of anionic intermediates. However our results
suggest that while such intermediates could be involved in
dimerisation, they are likely to be generated by enzyme-
Conflicts of interest
There are no conicts to declare.
catalysed decarboxylation of 1.
We could not obtain high yields of the KI-like enzymes in
soluble form, and only cell-free extracts produced in yeast
showed any tangible activity. We were therefore unable to
Acknowledgements
perform more detailed studies of the mechanisms of these We thank Ben Schleibaum for assistance with the synthetic
intriguing enzymes, but never-the-less this is the rst report of chemistry. The China Scholarship Council is thanked for
their activity in vitro and sets the scene for future more detailed funding (SY, CSC201606210136). DFG is thanked for analytical
studies. Also unexpected is the observation that the KI proteins instrumentation (INST 187/686-1) and funding for VH (CO 1328/
appear to make a single nonadride product in vitro: formation of 3-1). Prof. Jianqiang Kong is thanked for sending us yeast
© 2021 The Author(s). Published by the Royal Society of Chemistry
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plasmids and strains. The publication of this article was funded
C. P. Butts, C. L. Willis, T. J. Simpson and R. J. Cox, Chem.
Commun., 2017, 53, 7965–7968.
¨
by the Open Access Fund of the Leibniz Universitat Hannover.
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K. Gomi and H. Oikawa, Org. Lett., 2015, 17, 5658–5661.
19 A. J. Szwalbe, PhD thesis, University of Bristol, 2016.
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RSC Adv., 2021, 11, 14922–14931 | 14931