Enzyme-Catalyzed Intramolecular Enantioselective Hydroalkoxylation

Article abstract of DOI:10.1021/jacs.7b01089

Hydroalkoxylation is a powerful and efficient method of forming C-O bonds and cyclic ethers in synthetic chemistry. In studying the biosynthesis of the fungal natural product herqueinone, we identified an enzyme that can perform an intramolecular enantios

Full text of DOI:10.1021/jacs.7b01089

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Communication
Enzyme-Catalyzed Intramolecular Enantioselective Hydroalkoxylation
Shu-Shan Gao, Marc Garcia-Borràs, Joyann S. Barber, Yang
Hai, Abing Duan, Neil K. Garg, Kendall N. Houk, and Yi Tang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b01089 • Publication Date (Web): 27 Feb 2017
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Enzyme-Catalyzed Intramolecular Enantioselective Hydroalkoxylation
Shu-Shan Gao,§ Marc Garcia-Borràs, Joyann S. Barber, Yang Hai,§ Abing Duan, Neil K. Garg,,* K. N. Houk,,*
Yi Tang§,,*
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§Department of Chemical and Biomolecular Engineering and Department of Chemistry and Biochemistry, University of California,
Los Angeles, California 90095, United States
Supporting Information Placeholder
Of the cyclic ethers encountered in natural product structures,
the dihydrobenzofuran moiety attracted our attention. It is found in
the bacterial meroterpenoid neomarinone 1,9 the fungal polyketide
herqueinone 210 and the plant xanthone pruniflorone M 311 (Figure
1B), as well as numerous other natural products (see Figure S2).
The common structural component present in compounds 1–3 is
the 2,3,3-trisubstituted-dihydrobenzofuran. The presence of the
3,3-gem--dialkyl substitution suggests the dihydrofuran (DHF) ring
is formed from the 5-exo-trig addition of the phenolic oxygen to the
terminal olefin of a reverse prenyl unit as shown in Figure 1C. Inter-
estingly, all of these natural products are isolated as a single stereoi-
somer in the DHF rings, suggesting the cyclization step is most likely
enzyme-catalyzed. Motivated by the goal of identifying an enzyme
that can catalyze hydroalkoxylation, we performed biochemical
characterization of the enzymes involved in biosynthesis of 2. Here
we show that an enzyme with a conserved domain of unknown func-
tion (DUF) belonging to DUF3237 superfamily is indeed dedicated
to catalyze enantioselective cyclization to form the DHF ring of 2.
ABSTRACT: Hydroalkoxylation is a powerful and efficient method of
forming C–O bonds and cyclic ethers in synthetic chemistry. In study-
ing the biosynthesis of the fungal natural product herqueinone, we iden-
tified an enzyme that can perform an intramolecular enantioselective hy-
droalkoxylation reaction. PhnH catalyzes the addition of a phenol to the
terminal olefin of a reverse prenyl group to give a dihydrobenzofuran
product. The enzyme accelerates the reaction by 3 × 105-fold compared
to the uncatalyzed reaction. PhnH belongs to a superfamily of proteins
with a domain of unknown function (DUF3237), of which no member
has a previously verified function. The discovery of PhnH demonstrates
that enzymes can be used to promote the enantioselective hydroalkoxy-
lation reaction and form cyclic ethers.
Five and six-membered cyclic ethers are ubiquitous structural
fragments in natural products, and are important for their diverse bi-
ological activities.1 Notable examples include the topical antibiotic
mupirocin, which contains a tetrahydropyran pharmacophore that
binds to the target isoleucyl-tRNA synthetase,2a the anticancer drug
eribulin,2b and the antifungal drug griseofulvin.2c Biosynthetically,
formation of hydrofurans and hydropyrans can proceed via general
acid- and general base-catalyzed addition of alcohols to electrophiles
such as epoxides, carbonyls and Michael acceptors (Figure 1A).1a
Such reactions are also prevalent in synthetic chemistry.3
An alternative and perhaps more versatile method of forging a
new C–O bond involves the hydroalkoxylation of olefins to produce
Markovnikov addition products.4 The transformation utilizes simple
starting materials (i.e., an alcohol and an alkene), is atom economi-
cal, and can be used to form two bonds in one step without a change
in oxidation state.5 Brønsted acid catalysts, such as triflic acid6a and
imidazolium or triazolium ionic liquids,6b have been shown to cata-
lyze the nonstereoselective formation of cyclic ethers with very high
yields. Regarding enantioselective variants of hydroalkoxylation,
several transition metal-catalyzed reactions have been reported.7
However, a dedicated enzyme that can catalyze enantioselective hy-
droalkoxylation reaction has not been reported, which is surprising
given the prevalence of cyclic ether structures found in Nature. One
example of enzymatic hydroalkoxylation occurs in the last step of a
three-step transformation catalyzed by the monoterpene cyclase 1,8-
cineole synthase, which is proposed to convert the α-terpineol inter-
mediate to 1,8-cineole.8 Interestingly, 1,8-cineole synthase cannot
convert exogenously added α-terpineol to 1,8-cineole,8b,8c which sug-
gests formation of a tertiary carbocation during the overall terpene
cyclization cascade is required for the intramolecular alkylation of
the hydroxyl group.8c
Figure 1. Natural products containing cyclic ethers. (A) Routes to generate
cyclic ethers; (B) Natural products containing 2,3,3-trisubstituted-DHF
moiety from bacteria (1), fungi (2) and plant (3). See Figure S2 for more
examples. (C) Proposed cyclization steps required for the formation of
2,3,3-trisubstituted-DHF moiety. Reverse prenylation is followed by intra-
molecular hydroalkoxylation under general acid and general base catalysis.
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Figure 2. Characterization of the herqueinone pathway. (A) Key enzymes encoded in the phn cluster; (B) The complete biosynthetic pathway of herqueinone 2; (C)
Product profiles of wild type and phnF knockout mutant of P. herquei and of S. cerevisiae transformed with combinations of phn genes; (D) Product profiles of in vitro
assays using purified enzymes. *The spontaneous degradative product of 5.12 See SI for reaction conditions.
We recently identified the herqueinone (phn) biosynthetic
gene cluster from Penicillium herquei NRRL 1040 (Figure S3) and
showed the actions of a nonreducing polyketide synthase (NR-PKS
PhnA) and a flavin-dependent monooxygenase (FMO, PhnB) are
responsible for formation of phenalenone 5 (Figure 2B).12 In the
process of identifying the unknown steps that lead to formation of
the additional DHF ring in 2, we generated individual knockout mu-
tants of the pathway and characterized the products. Inactivation of
the predicted aromatic prenyltransferase PhnF led to abolishment of
2 and accumulation of 5 (Figure 2C ii and S5), indicating the role of
PhnF in catalyzing regioselective C6 alkylation of 5.
To verify the function of PhnF, the recombinant enzyme (48
kDa) was expressed and purified from Escherichia coli BL21(DE3)
(Figure S6). Due to instability of 5, we assayed using the immediate
precursor 4 in the presence of PhnB (with cofactors NADPH and
FAD, Figure 2D, i) together with PhnF (with dimethylallyl pyro-
phosphate (DMAPP) and MgCl2, Figure 2D, ii). Two new products
6a and 6b with the same molecular weights (MW = 342) were ob-
served. To scale up the reaction and determine the structures of 6a
and 6b, we coexpressed PhnA, PhnB and PhnF in Saccharomyces
cerevisiae BJ5464-NpgA, which led to the production of 6a and 6b
(Figure 2C, iv), whereas coexpression of PhnA and PhnB produced
5 only (Figure 2C, iii). During purification, it was observed that 6a
and 6b are interconverting tautomers; a final ratio of 1:2 of 6a to 6b
was found in the purified compound (Figure S13). NMR structural
analysis confirmed that 6a is the C6-reverse prenylated version of 5,
while 6b is the corresponding diketo tautomer (Figure 2B, Table S1
and Figures S17−S21). When the mixture of 6a and 6b was supple-
mented to the ΔphnA blocked mutant of P. herquei,12 the biosynthe-
sis of 2 was restored (Figure S4, iii), confirming the prenylated
phenalenone is an authentic intermediate of the pathway, which rep-
resents the immediate precursor to the proposed hydroalkoxylation
reaction.
The reverse C-prenylation of 5 by PhnF can occur by direct
electrophilic substitution at C6, or possibly via first a forward O-
prenylation of a neighboring phenol in 5, followed by a Claisen rear-
rangement to yield 6.13 Schmidt et al showed the forward C-prenyl-
ated tyrosine residue in cyanobactins is the product of a non-enzy-
matic Claisen rearrangement following a reverse O-prenylation cat-
alyzed by the prenyltransferase LynF.14 However, such rearrange-
ment has not been observed for the prenyltransferases catalyzing for-
ward O-prenylations, such as PagF15 and SirD.16 Indeed, density
functional theory (DFT) calculations show the Claisen rearrange-
ment after the forward O-prenylation at C7-OH of 5 would be very
slow at room temperature (ΔG‡ = 30.2 kcal/mol with predicted
298
t1/2 (25 °C) = 4.2 × 105 h, Figure S35). Thus, we conclude PhnF
must be a reverse C-prenyltransferase that directly alkylates 5.
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Figure 3. Proposed mechanism for PhnH-catalyzed hydroalkoxylation. (A) PhnH catalyzes 5-exo-trig cyclization via acid catalysis after the spontaneous deprotonation of
7-OH; (B) Transition state structure of the hydroalkoxylation reaction using acetic acid as a general acid; (C) Homology models of PhnH; The putative catalytic residue
E104 is highlighted in red.
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We next examined the stability of 6a and 6b, and whether a
spontaneous cyclization of the DHF ring can take place. When
placed in phosphate buffer at pH 7.4, rapid degradation of the com-
pounds was observed (Figure S9). In DMSO, ~ 40% of purified 6
(50 mM starting concentration) was converted to a new compound
8* in 23 days (Figure S10). The structure of the compounds was
determined to contain the racemic DHF ring as shown in Figure 3A.
We propose compound 8* should be derived from the autoxidation
of the cyclized product 7*, first as the1,2,3-tri-keto intermediate, fol-
lowed by water addition at C2.13 Similarly, 9* (Table S7 and Figures
S30−S33), the acetone adduct of oxidized 7* was observed when 6
was placed in a 1:1 mixture of acetone and water for 5 days (30%
conversion) (Figures 3A and S11). Both 8* and 9* displayed optical
rotation of [α]D = 0 (c 0.02, MeOH), thereby confirming the uncat-
alyzed hydroalkoxylation reaction is nonstereospecific. This obser-
vation, together with the slow rate of uncatalyzed reaction, indicate
an additional enzyme in the phn pathway is required for catalyzing
formation of the 2’-R-DHF in 2.
To search for the enzyme responsible, we performed combina-
torial expression of the remaining phn enzymes in S. cerevisiae (Ta-
ble S2). None of remaining enzymes in the cluster produced new
compounds (Figure S7). RT-PCR of genes immediately outside of
the initial cluster boundary revealed that only one gene is cotran-
scribed with the phn cluster (Figure S8). This gene, named phnH,
encodes a small protein (149 aa) that contains a conserved, but un-
characterized domain (DUF3237, Figures 2A and S3). Crystal
structures of several members of the DUF3237 family have been elu-
cidated to show a -barrel fold (PDB entries 4PUX, 3G7G, and
3C5O), although no function is known (Figures S38 and S39). To
determine the role of PhnH, we coexpressed PhnA, PhnB, PhnF and
PhnH in yeast, which led to the accumulation of a predominant new
product 7 (Figure 2C, v). The structure of 7 was determined to be
the known natural product atrovenetin17-19 (Figure S14) through ex-
tensive NMR analyses (Table S5 and Figures S22−S25). The abso-
lute stereochemistry at C2’ was assigned to be R by comparison of
the optical rotation measurement ([α]26D = +90.5 (c 0.02, MeOH))
to that reported for atrovenetin.20 When purified 7 was added to the
ΔphnA blocked mutant, production of 2 was restored, thereby veri-
fying it is indeed an on-pathway product (Figure S4, iv). Further-
more, the 2’R-isomer of the autoxidized product 8 ([α]30D = +81.3 (c
0.01, MeOH) (Table S6 and Figures S26−S29) was also purified
during isolation of 7 (Figure S15).
To demonstrate PhnH is the sole enzyme required for the hy-
droalkoxylation reaction, PhnH was purified to homogeneity from
yeast (Figure S6). A combined assay of PhnB, PhnF and PhnH in the
presence of 4 led to formation of 7 as the predominant product (Fig-
ure 2D, iii). Direct incubation of 6a and 6b with PhnH led to con-
version to 7 (Figure 2D, iv-vii), with steady state kinetic parameters
of kcat = (2.9 ± 0.3) × 103 min−1, Km = 216 ± 19 μM, and kcat/Km = 13
± 1.4 min−1 μM−1 (Figure S34B). Compared to the uncatalyzed re-
action rate in DMSO (k = 0.0086 min−1, Figure S34A), PhnH af-
forded a > 3 × 105-fold rate enhancement. Addition of 1 mM EDTA
has no effect on the reaction rate (Figure S12), indicating no metal-
cofactor is required. The optimum pH for the enzyme is 7.5 as meas-
ured by the pH dependence assay, while nearly all activities were
abolished when the pH is lowered to 6.0 (Figure S34C).
The stereochemical outcome of the hydroalkoxylation reaction
catalyzed by PhnH is consistent with the isolation of 2’R-configured
herqueinone 2 from P. herquei.21 The spontaneous, nonstereoselec-
tive formation of 7* from 6 can also account for the isolation of the
trace amounts 2’S-configured isoherqueinone.22 Identification of
PhnH function also allowed us to complete the biosynthetic path-
way of 2. By sequentially adding remaining uncharacterized genes
to the yeast host that produces 7, we identified the O-methyltrans-
ferase PhnC to be responsible for converting 7 to deoxyherqueinone
10 (Figure S4, vi).23 The last C6 hydroxylation step that converts 10
to 2 was confirmed to be catalyzed by the FMO PhnG (Figure S4,
vii). Hence, six enzymes are required to synthesize 2, another exam-
ple of the efficient biosynthetic pathways found in fungi.
We propose that PhnH catalyzes the hydroalkoxylation reac-
tion by favoring binding of the substrate 6a in a near-attack confor-
mation, thus aligning the lone electron pair of the deprotonated phe-
noxide anion in 6a’ (Figure 3A) with the Bürgi-Dunitz trajectory at
the Si-face of the C2’-C1’ double bond. Concerted proton transfer
from a nearby acid leads to protonation at C1’ and yields product
2’R-7. Due to the extended aromatic system of 6, the C7-phenol in
6a is much more acidic with a pKa of 6.9 (obtained from ACE JChem
pKa predictor,24 Figure S34D), which indicates that no general base
is required to generate the phenoxide anion of 6a’ at physiological
pH (Figure 3A). Our DFT calculations show that acid catalysis likely
occurs after the C7-OH is deprotonated, with an energy barrier of
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21.8 kcal/mol using acetic acid as a general acid model (Figures 3B
and S36A). Those reaction mechanisms that are not initiated by the
deprotonation of C7-OH have been ruled out based on calculations,
due to either the high-energy barrier or unstable intermediates (Fig-
ures S36 and S37).
(2) (a) Thomas, C. M.; Hothersall, J.; Willis, C. L.; Simpson, T. J. Nat. Rev.
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Proteins belonging to the DUF3237 superfamily are widely dis-
tributed in Nature, from bacteria to fungi, with many PhnH homo-
logs embedded in potential natural product gene clusters (Figure
S41). PhnH displays high sequence identity and homology to many
members of the family (Figure S39). We generated a homology
model of PhnH by using the I-TASSER server.25 The model shows
an overall -barrel fold, typically adopted by the other members in
the DUF3237 superfamily (Figure 3C). The model reveals a puta-
tive active site: a hydrophobic pocket with a Glu (E104) residue po-
sitioned at the bottom of the solvent-exposed cavity. Mutation at
this residue compromised the enzymatic activity by ~60%. This in-
dicates E104, which may be deprotonated under assay conditions, is
not a general acid, but may be involved in proper positioning a water
molecule (probably in its hydronium form) as a specific acid during
the protonation reaction step. We also performed single-residue
mutations at other conserved acidic positions based on sequence
alignment. Different mutations led to varying degrees of attenuation
in enzyme activity and expression levels, although no single muta-
tion completely abolished PhnH activity (Table S8). Lastly, we syn-
thesized 2-allyl resorcinol as a simplified substrate to test the prom-
iscuity of PhnH. No rate acceleration of the hydroalkoxylation ad-
duct was observed, which may be due to lack of recognition of the
much smaller substrate by the enzyme, and/or due to the signifi-
cantly higher pKa of the phenol group.
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In conclusion, PhnH is the first functionally characterized
member of protein in the DUF3237 family. The discovery of PhnH
complements other enzymatic strategies to synthesize cyclic ethers
in natural products.1a,26 Therefore PhnH provides an enzymatic var-
iant of asymmetric hydroalkoxylation of olefins, an important trans-
formation in synthetic chemistry.
ASSOCIATED CONTENT
Supporting Information
Experimental details, spectroscopic and computational data. This mate-
rial is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
(20) Neill, K. G.; Raistrick, H. Biochem. J. 1957, 65, 166.
(21) (a) Tansakul, C.; Rukachaisirikul, V.; Maha, A.; Kongprapan, T.;
Phongpaichit, S.; Hutadilok-Towatana, N.; Borwornwiriyapan, K.;
Sakayaroj, J. Nat. Prod. Res. 2014, 28, 1718. (b) Quick, A.; Thomas, R.;
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(24) ACE/JChem, ACE and JChem acidity and basicity calculator. 2016,
https://epoch.uky.edu/ace/public/pKa.jsp.
(25) Zhang, Y. BMC Bioinformatics 2008, 9, 40.
(26) Tang, M. C.; Zou, Y.; Watanabe, K.; Walsh, C. T.; Tang. Y. Chem.
Rev. 2017, ASAP. doi: 10.1021/acs.chemrev.6b00478.
Corresponding Author
yitang@ucla.edu, houk@chem.ucla.edu, neilgarg@chem.ucla.edu
ACKNOWLEDGMENTS
This work was supported by the NIH 1DP1GM106413 and
1R35GM118056 to YT; and the NSF CHE-1361104 to K.N.H.; and the
Chemistry–Biology Interface training program (J.S.B., NRSA
5T32GM008496-20). M.G.-B. thanks the Ramón Areces Foundation
for a Postdoctoral Fellowship.
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment