Frontiers and opportunities in chemoenzymatic synthesis

Article abstract of DOI:10.1021/jo101124n

Natural product biosynthetic pathways have evolved enzymes with myriad activities that represent an expansive array of chemical transformations for constructing secondary metabolites. Recently, harnessing the biosynthetic potential of these enzymes through chemoenzymatic synthesis has provided a powerful tool that often rivals the most sophisticated methodologies in modern synthetic chemistry and provides new opportunities for accessing chemical diversity. Herein, we describe our research efforts with enzymes from a broad collection of biosynthetic systems, highlighting recent progress in this exciting field.

Full text of DOI:10.1021/jo101124n

pubs.acs.org/joc
Frontiers and Opportunities in Chemoenzymatic Synthesis
Jonathan D. Mortison and David H. Sherman*
Life Sciences Institute, Department of Chemistry and Department of Medicinal Chemistry,
210 Washtenaw Avenue, University of Michigan, Ann Arbor, Michigan 48109
davidhs@umich.edu
Received June 24, 2010
Natural product biosynthetic pathways have evolved enzymes with myriad activities that represent an
expansive array of chemical transformations for constructing secondary metabolites. Recently, harnes-
sing the biosynthetic potential of these enzymes through chemoenzymatic synthesis has provided a
powerful tool that often rivals the most sophisticated methodologies in modern synthetic chemistry and
provides new opportunities for accessing chemical diversity. Herein, we describe our research efforts with
enzymes from a broad collection of biosynthetic systems, highlighting recent progress in this exciting field.
Introduction
chemoenzymatic approaches to generate natural products
have emerged, particularly through the manipulation of
enzymes from biosynthetic pathways of secondary meta-
bolites. These strategies leverage the unique selectivity and
catalytic power of these enzymes, which are responsible for
producing countless natural products in marine and terres-
trial microorganisms. These methods may utilize a large
assembly line of enzymes to produce a full natural product
scaffold such as 6-deoxyerythronolide B (6-DEB) (Figure 1a)
or a single enzyme to effect a difficult chemical transforma-
tion, such as the selective P450-catalyzed oxidation of a C-H
bond (Figure 1b). In this Perspective, we present some of
the most recent advances in chemoenzymatic synthesis and
identify current trends and opportunities in this growing
field. Particular attention will be centered on chemoenzy-
matic oxidation (e.g., hydroxylation and epoxidation) and
chain termination (e.g., macrocyclization) strategies.
Polyketides, nonribosomal peptides, and their hybrids con-
stitute large classes of structurally diverse natural products
that possess a wealth of pharmacological activities including
antimicrobial, antimycotic, antiparasitic, antitumor, and
immunosuppressive, making them extremely valuable lead
compounds in drug discovery and development. In fact, over
two-thirds of newly introduced drugs worldwide in the
past two decades have been natural products or derivatives
thereof.1 However, clinical development of many of these
promising candidate drugs is challenged by difficulties in
large-scale compound production, as natural product iso-
lates are often low-yielding and their structural complexities
typically make total synthesis a limited option. Furthermore,
difficulty in generating analogue libraries of a parent struc-
ture limits the effectiveness of SAR studies to modulate the
desired properties of the lead compound. Thus, in order to
efficiently identify next-generation drugs from natural pro-
duct scaffolds, it has become imperative to explore new methods
for rapid generation of structurally diverse compound
libraries.
Polyketide Synthases and Nonribosomal Peptide Synthetases
Many enzymes utilized in chemoenzymatic synthesis come
from natural product biosynthetic pathways. The building
of polyketide and nonribosomal peptide natural products
occurs on large megaenzyme complexes called polyketide
synthases2 (PKS) and nonribosomal peptide synthetases3
To address this issue, modern efforts to expand access
to chemical diversity have increasingly employed multi-
disciplinary tools and strategies. From these tools, promising
DOI: 10.1021/jo101124n
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Published on Web 09/30/2010
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FIGURE 1. Illustration of (a) the DEBS PKS responsible for biosynthesis of the erythromcycin aglycon 6-deoxyerythronolide B (6-DEB) and
(b) in vitro chemoenzymatic oxidation of 6-DEB to erythronolide B by the EryF P450 hydroxylase: KS = ketosynthase, AT = acyltransferase,
ACP = acyl carrier protein, KR = ketoreductase, DH = dehydratase, ER = enoyl reductase, TE = thioesterase. * denotes an inactive domain.
(NRPS), respectively. In type I PKS systems, a modular assembly
of enzymatic domains links simple malonyl-CoA derivatives
via sequential decarboxylative Claisen condensations to
build complex polyketide chains (Figure 2a). Modules from
a type I PKS are responsible for a single elongation step in
building the growing polyketide chain, and a minimal mod-
ule consists of three core domains: a ketosynthase (KS), an
acyltransferase (AT), and an acyl carrier protein (ACP).
During elongation, the AT domain selects an appropriate
acyl-CoA extender unit and loads it onto the ACP. The KS
domain then catalyzes decarboxylation of the extender unit
to generate a transient enolate nucleophile that reacts in a
Claisen condensation with the growing polyketide chain to
give an extended β-keto acyl-ACP intermediate. Following
extension, the intermediate can then undergo various levels
of processing by reductive enzyme domains (i.e., ketoreductases,
dehydratases, and enoyl reductases) to introduce functional
and stereochemical diversity to the polyketide backbone.
The architecture of these PKS systems is such that the
collinear arrangement of the modules encodes the chemical
structure of the corresponding polyketide product. As a result,
rational engineering of these PKS enzymes provides an
exciting avenue for design of new polyketide compounds.
In NRPS systems, enzymes are again organized into modules
containing a similar trio of core domains: an adenylation
domain (A), a condensation domain (C), and a peptidyl
carrier protein (PCP) (Figure 2b). The A domain selects an
amino acid monomer, activates the carboxyl group as an
O-AMP ester, and loads it onto the PCP. The C domain then
catalyzes formation of the new peptide bond between the
PCP-loaded monomer and the growing peptidyl inter-
mediate. Similar to PKS systems, the collinear nature of the
NRPS modules leads to products with predictable amino acid
sequences. While many of these biosynthetic systems are
composed exclusively of PKS or NRPS modules, some contain
hybrid PKS/NRPS modules, generating products that contain
both polyketide and peptidyl units in their structures.
Following PKS and NRPS extension and processing,
the intermediates are often converted to macrocyclic ring
scaffolds via a terminal cyclization domain or off-loaded as
linear structures through alternative chain termination
events.4,5 The final bioactive compounds are then obtained
by further tailoring of these scaffolds by enzymes such as
P450 hydroxylases and glycosyl transferases.
Macrocyclization
In their biologically relevant form, many PKS and NRPS-
derived natural products contain macrocyclic rings that
equal or exceed eight atoms, making them challenging
targets for synthetic chemists. In generating macrocyclic
lactones6 and lactams by synthetic methods, common hurdles
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FIGURE 2. Illustration of (a) PKS mechanism: (1) AT loading of the ACP with a methylmalonate extender unit, (2) loading of the KS with the
pendant polyketide intermediate from the upstream ACP domain, and (3) KS-catalyzed decarboxylative Claisen condensation of the extender
unit with the polyketide intermediate. Illustration of (b) NRPS mechanism: (1) loading of the AT activated O-AMP amino acid onto the PCP,
(2) C domain-catalyzed condensation of the PCP-loaded amino acid with the upstream PCP-bound peptidyl intermediate.
P450 Hydroxylation and Epoxidation
Another set of exceptional challenges for synthetic organic
chemists are the selective hydroxylation and epoxidation of
unactivated C-H bonds and olefins, respectively, by traditional
methodologies. Mild oxidation of organic compounds, however,
can be accomplished biocatalytically by the P450 superfamily of
enzymes, which are heme-containing proteins that couple with a
reductase partner protein and ferredoxin cofactor to activate
molecular oxygen using NADPH/NADH.7 Specifically, P450
FIGURE 3. Illustration of TE-mediated macrocyclization: (1)
enzymes from natural product biosynthetic pathways are cap-
able of effecting many difficult oxidative transformations on
natural product scaffolds in both a regio- and stereospecific
manner, making them attractive biocatalysts for chemoenzy-
matic synthesis.
transfer of final intermediate to TE active site serine and (2)
regioselective intramolecular cleavage of the acyl enzyme intermedi-
ate by an internal nucleophile.
include the need for orthogonal protecting group strategies,
complex conformational effects (entropic and enthalpic factors),
regioselectivity, and competing intermolecular dimerizations/
oligomerizations. These factors often lead to extended synthetic
routes and decreased yields of target compounds.
Cryptophycin
The cryptophycins are a class of hybrid PKS/NRPS natural
products isolated from a pair of related lichen-derived cyano-
bacterial symbionts, Nostoc sp. ATCC 53789 and GSV 224.8,9
These compounds are exceptionally potent tubulin-depolymerizing
agents and are not active substrates of P-glycoprotein (P-gp) or
multiple drug resistance-associated protein (MRP),10 making
them viable chemotherapeutic alternatives against cancers that
overexpress both of these transporters and are resistant to the
vinca alkaloids and paclitaxel. Recently, synthetic crytophycin
analogue LY355703 (cryptophycin-52) was discontinued as a
candidate in phase II clinical trials due to dose-limiting toxicity
resulting in peripheral neuropathy.11 Nevertheless, these nat-
ural products still garner significant interest in their develop-
ment as anticancer drugs,12 with current efforts aimed toward
generating cryptophycins that eliminate these undesired effects.
In PKS and NRPS systems, cyclization is often catalyzed
by a discrete terminal thioesterase (TE) domain, containing a
characteristic serine, histidine, aspartate catalytic triad found in
the homologous serine hydrolases. During this process, the
final biosynthetic intermediate from the last PKS or NRPS
elongation module is passed to the TE active site serine residue
to form an O-acyl enzyme intermediate, followed by cleavage of
the acyl enzyme by regiospecific nucleophilic attack of an
internal hydroxyl or amino group to produce the macrocyclic
lactone or lactam (Figure 3). Due to the unique ability of these
TE enzymes to efficiently catalyze macrocyclization, many
efforts have explored their potential as biocatalysts when
excised from their native PKS or NRPS multifunctional protein
context.
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FIGURE 4. Illustration of the Crp PKS/NRPS responsible for biosynthesis of the cryptophycin peptolide core. Epoxidation by CrpE and
chlorination by CrpH generates cryptophycin 1: C = condensation domain, A = adenylation domain, PCP = peptidyl carrier protein, MT =
methyltransferase, E = epimerase.
Recent access to the cryptophycin (Crp) biosynthetic gene
cluster has spurred development of specific enzymes from
this pathway as biocatalysts in the generation of new crypto-
phycins with improved pharmacological profiles. The pepto-
lide core of the most abundant cryptophycin analogue,
cryptophycin 1, consists of a PKS-derived phenyloctenoic acid
(unit A) and three NRPS-derived amino acids: 3-chloro-
O-methyl-D-tyrosine (unit B), methyl β-alanine (unit C), and
L-leucic acid (unit D) (Figure 4). However, there is significant
diversity within this class of compounds, which contains more
than 25 analogues that incorporate many substitutional varia-
tions on the core scaffold. This diversity suggests a high degree
of flexibility in the enzymes of the Crp biosynthetic machinery,
a hypothesis that was further borne out in precursor-directed
biosynthesis studies, where a significant number of unnatural
subunits were incorporated into new cryptophycin analogues
by the Crp enzymes.13
In generating synthetic Crp analogues, efficient macro-
cyclization and epoxidation represent two challenging issues
FIGURE 5. Chemoenzymatic macrocyclization of (a) solution-phase
and (b) solid-phase linear cryptophycin intermediates by Crp TE in vitro.
TE and instead primarily hydrolyzed to the seco-acid. This
indicated that a terminal aryl group is critical for efficient
that can be addressed by chemoenzymatic methods. In a recently
described approach, the Crp TE was excised and hetero-
macrocyclization.
In a subsequent study, it was shown that this methodology
logously overexpressed as a recombinant enzyme for cyclization
of linear Crp precursors.14 NAC thioester-activated seco-
could be expanded to solid-phase bound cryptophycin precur-
sors.15 Several seco-cryptophycin analogues were synthesized as
acyl sulfonamides on safety-catch PEGA resin, which were
activated with iodoacetonitrile and subsequently subjected to
macrocyclization reactions with Crp TE (Figure 5b). Here, Crp
TE was not only capable of catalyzing cyclization of these
substrates directly from the activated solid support but also
cryptophycins were chemically synthesized, incorporating three
different Unit C moieties, and utilized to interrogate the in vitro
activity and substrate specificity of Crp TE (Figure 5a). Robust
macrocylization was observed in each case, suggesting changes
to the β-alanine site are well tolerated by the thioesterase.
Signficantly, a linear cryptophycin precursor lacking the phenyl
ring on the styryl moiety of unit A was poorly cyclized by Crp
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tolerated changes to the unit B tyrosyl ring as well as a switch
from an ester to an amide linkage between units C and D. These
results clearly demonstrate the value of the cryptophycin thio-
esterase as a versatile biocatalyst for the synthesis of novel
cryptophycins from linear and resin-bound precursors.
The cryptophycin β-epoxide group confers a 100-fold
increase in compound potency, thus representing a key
functional group in this class of natural products.16 Intro-
duction of the functional group can only be accomplished
with modest diastereoselectivity by chemical methods, and
separation of the resulting mixture of R/β diastereomers is
cumbersome.17,18 Recently, the engineered P450 epoxidase
CrpE identified from the cryptophycin biosynthetic gene
cluster has been shown to be a viable alternative to synthetic
strategies. In those studies, CrpE was expressed as a recombi-
nant protein containing an N-terminal maltose-binding pro-
tein (MBP) tag and incubated with a small library of desepoxy
cryptophycin substrates in vitro19 (Figure 6). The resulting
β-epoxy cryptophycins were generated efficiently as single
diastereomers, though linear cryptophycins were not epoxidized,
indicating that cyclization is a prerequisite for CrpE activity. At
thesametime,theepoxidasealsodemonstratedtolerancetoward
functional changes on units B and C. Finally, it was also shown
in vitro that Crp TE and CrpE can be used in tandem to cyclize
and epoxidize linear cryptophycins in a single reaction.13 This
has the potential to greatly streamline the chemoenzymatic
synthesis of new cryptophycins by accomplishing the two most
difficult steps in an efficient and economical manner.
Pikromycin and Erythromycin
Macrolides are a large family of antibiotics that bind to the
50S ribosomal subunit of pathogenic bacteria and block
peptidyl transferase activity for amino acid chain elongation.20
The representative member of this family, erythromycin, has
been in clinical use for nearly 60 years to combat a variety of
common infections and has served as a template for the produc-
tion of new antibiotics (e.g., ketolides) in this class.21,22 Despite
the success of the macrolides, however, there is a critical need for
rapid access to new antibiotics due to the evolution of macrolide-
resistant bacteria, pathogens that pose a growing threat to
human health.23 Currently, the complex structural and func-
tional features of macrolides make them particularly difficult
targets for synthetic efforts, and semisynthesis has been the only
FIGURE 6. In vitro chemoenzymatic (a) epoxidation of crypto-
phycin intermediates by the CrpE epoxidase and (b) tandem epoxda-
tion and macrocyclizaiton of linear Crp intermediates by CrpE and
Crp TE.
FIGURE 7. Illustration of the Pik PKS responsible for the biosynthesis of the methymycin and pikromycin aglycons 10-deoxymethynolide
(10-Dml) and narbonolide (Nbl), respectively.
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FIGURE 9. Chemoenzymatic synthesis of macrolactones with PikAIII
and PikAIV modules, utilizing native chain-elongation intermediates.
FIGURE 8. Probing molecular specificity in PikAIII and PikAIV
modules with diastereomeric diketide substrates to give triketide
lactones and tetraketides. Only syn diketides were accepted by either
module with a strong preference for the (2S,3R) diketide. PikAIII
only accepted the (2S,3R) diastereomer.
reliable but limited avenue for next generation macrolides such
as the azalides24 and ketolides.21 As a result, focused study has
been centered on engineered biosystems for the chemoenzymatic
synthesis of new macrolide scaffolds.
The erythromycin25,26 (DEBS, Figure 1a, vide supra) and
pikromycin27 (Pik, Figure 7) PKSs are hallmarks for poly-
ketide natural product biosynthetic systems, producing the
antibiotic aglycons 6-deoxyerythronolide B and narbonolide,
respectively. The modular architecture of these two enzyme
systems makes them prime candidates for rational pathway
engineering, through which specific and predictable changes
can be introduced to the core structures of their putative
polyketide products.28-32A wealth of studies have been per-
formed on both the Pik and DEBS PKSs, exploiting engineered
whole modules as well as individual enzymes such as isolated
thioesterase domains and P450 monooxygenases for chemo-
enzymatic synthesis.
Utilization of complete PKS modules for chemoenzymatic
synthesis presents a significant challenge due to the size
(often greater than 150 kDa) and complexity of the enzymes
involved. However, the reward for overcoming this challenge is
great, as efficient generation of hybrid PKSs through combina-
torial reorganization of PKS modules would lend access to
extraordinary chemical diversity. Toward this end, modules 5
(PikAIII) and 6 (PikAIV) from the Pik PKS present an excellent
experimental system, since both modules are natively expressed
as monomodular enzymes. Initial studies in this system exploited
the facile functional expression and purification of recombinant
PikAIII, PikAIII-TE (module 5 with a C-terminal thioesterase
fusion), and PikAIV, which were then incubated with short
diketide model substrates to generate tri- and tetraketide lactone
products in vitro33,34 (Figure 8). These studies not only demon-
strated the versatility of recombinant PKS modules but also laid
a foundational understanding of their substrate specificity and
catalytic efficiency. During subsequent work, synthetic full-
length native penta- and hexaketide chain elongation inter-
mediates for PikAIII and PikAIV were used for the in vitro
chemoenzymatic synthesis of the complete 12- and 14-membered
ring aglycons 10-deoxymethynolide (10-Dml) and narbonolide
(Nbl)35 (Figure 9). Interestingly, comparative steady-state
kinetic analysis of the loading, extension, processing, and
FIGURE 10. Comparative analysis of molecular specificity in the
Pik and DEBS PKS: (a) Ery5-TE and PikAIII-TE accept, elongate,
and cyclize their native pentaketide substrates but do not tolerate
substrates from the reciprocal system; (b) Ery6 shows remarkable
flexibility in processing the non-native DEBS pentaketide and Pik
hexaketide substrates.
cyclization events in these modules revealed a significant
difference between rates when diketide substrates were used
versus native chain elongation intermediates. An inherent
preference for native pentaketide and hexaketide substrates
(presumably controlled through key molecular recognition
elements) has been demonstrated by the ability of PikAIII and
PikAIV modules to process them 2-3 orders of magnitude
more efficiently than model diketides.
Similar in vitro studies have been conducted in the DEBS
system, where individual engineered DEBS modules 5 (Ery5)
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FIGURE 11. (a) In vitro chemoenzymatic synthesis of 10-deoxymethy-
nolide with Pik TE. (b) Reduction at C7 of the hexaketide substrate
abolishes TE-mediated cyclization.
and 6 (Ery6) were incubated with short-chain diketide sub-
strates to give triketide lactone products. As in the Pik
system, it was also revealed that each of the final DEBS
modules has an inherent substrate specificity profile.36-38 At
the same time, though, it was demonstrated that the DEBS
modules were nearly 3 orders of magnitude more efficient
than their Pik counterparts in processing these diketides.
Recent experiments in the DEBS PKS with synthetic native-
chain elongation intermediates confirmed these findings and
laid out a more detailed understanding of their molecular
specificity parameters, as well as a comparative analysis with
the analogous Pik modules. In these studies, both Pik module
5 (PikAIII) and DEBS module 5 (Ery5) were shown to
exhibit a fairly high specificity, as evidenced by the fact that
the synthetic pentaketide substrate from each system was not
tolerated by the corresponding reciprocal module39 (Figure10a).
This was surprising as the Pik and DEBS pentaketide
substrates are structurally similar, only differing at the
C6-C7 positions. Nevertheless, these changes were enough
to preclude efficient enzymatic processing of the penta-
ketides by the noncognate PKS modules. On the other hand,
Pik module 6 (PikAIV) and DEBS module 6 (Ery6) displayed
a remarkable flexibility toward noncognate substrates, with
Ery6 showing a particularly relaxed specificity. Ery6 was
capable of accepting, extending, processing, and cyclizing
both the non-native synthetic DEBS pentaketide substrate
and the Pik hexaketide substrate (Figure 10b), which not
only have differing chain lengths of 10 and 12 carbons,
respectively, but contain variant proximal and distal func-
tional group substitution patterns. This relaxed specificity
profile indicates that module 6 of both Pik and DEBS may be
excellent candidates for future PKS engineering efforts.
Due to the pivotal role thioesterase domains play in natural
product biosynthesis, understanding the specificity of the Pik
and DEBS TEs is important for their applications both in vitro
and in vivo. As a result, both TEs have been the subject of
experiments to explore their chemoenzymatic potential. When
the Pik TE was excised from its native context, it was shown to
catalyze macrocyclization of the synthetic pikromycin hexa-
ketide SNAC substrate, exclusively forming 10-Dml40 (Figure 11).
However, when the R,β-unsaturated ketone of the substrate
was reduced at C7 to the corresponding allylic alcohol, cycliza-
tion by Pik TE was abrogated. Based on this result, we reasoned
that the rigid enone structure of the Pik hexaketide provides a
favorable entropic contribution necessary for TE-mediated
cyclization. This was further supported by structural biology
studies where the Pik TE was cocrystallized with covalently
bound affinity labels mimicking a portion of the pikromycin
heptaketide chain elongation intermediate (PDB ID 1HFJ).41
˚
FIGURE 12. Pik TE crystal structure (1.8 A) (a) active site with a
bound affinity label mimicking the Pik cyclization intermediate. An
ordered water network on right side of the channel forms a “hydro-
philic barrier”, inducing a curl into the cyclization intermediate;
(b) electrostatic surface representation of the Pik TE substrate channel.
FIGURE 13. Probing specificity of the DEBS TE from in vitro
reactions of Ery5-TE with synthetic DEBS pentaketide SNAC.
Formation of 12-membered ring macrolactone is attenuated upon
exclusion of NADPH, giving C3-unreduced hexaketide seco-acid as
the major product.
Here, it was shown that cyclization appears to be primarily a
structure-driven process with Pik TE, as minimal contacts were
seen between the affinity label and the TE active site residues. A
water network on one side of the thioesterase channel appeared
to form a “hydrophilic barrier” that directed curling of the
linear intermediate toward cyclization (Figure 12). Also, mo-
lecular modeling of the enone structure for the Pik hexa- and
heptaketide cyclization intermediates showed a favorable dis-
position of the appropriate internal hydroxyl group as a nucleo-
phile for macrocyclization.
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FIGURE 14. Hydroxylation patterns of YC-17 and narbomycin by
the PikC P450.
FIGURE 15. Demonstration of substrate flexibility in the PikC
P450 hydroxylase on desosamine-anchored unsubstituted cyclo-
alkanes. Carbons distal to the desosamine were hydroxylated
(shown in red).
FIGURE 16. Illustration of (a) chain termination in the final CurM
module, generating the terminal olefin in curacin A. (b) The
sulfotransferase (ST) in CurM preferentially adds a sulfate to the
(R)-hydroxyl of a synthetic curacin mimic, followed by sequential
hydrolysis and decarboxylation by the Cur TE to give the terminal
alkene.
Until recently, despite the availability of solved crystal
structures for DEBS TE,42,43 little information had been gained
about its ability to catalyze macrocyclization. Recent in vitro
work, however, has provided fresh insights into key mechan-
istic requirements for macrolactonization with DEBS TE.
When Ery5-TE was incubated with its native DEBS penta-
ketide substrate, an unnatural 12-membered ring macrolactone
was formed, demonstrating a similar flexibility to Pik TE for
generating varying ring sizes.39 However, reactions with DEBS
pentaketide substrate that excluded NADPH cofactor (thus
eliminating β-keto reduction by the KR domain) resulted in
drastically reduced levels of cyclized product and instead DEBS
TE-catalyzed hydrolysis to a linear hexaketide intermediate
(Figure 13). Here, the oxidation state at the β-position of the
pendant hexaketide cyclization intermediate was critical, as a
change from a β-hydroxy to a β-keto group resulted in attenua-
tion of TE-mediated macrocyclization, thus indicating a key
enzyme-substrate active site hydrogen bond interaction at this
position. This hypothesis is consistent with a previous study
involving modeling of 6-DEB into the active site of the DEBS
TE crystal structure, which suggested that the 3-position
hydroxyl does participate in a hydrogen-bond with Asn-180
and the backbone carbonyl of Tyr-171.42 This contrasts with
the above-described substrate binding mode for the Pik TE,
which is capable of efficiently cyclizing both a hexaketide
containing a β-hydroxy and a heptaketide containing a β-keto
group to give 10-Dml and Nbl, respectively.35
FIGURE 17. In vitro assay of tautomycetin (Tmc) TE activity.
(a) TMC TE preferentially hydrolyzes synthetic Tmc SNAC sub-
strate mimics containing an (R)-hydroxyl at the β-position.
(b) Decarboxylation and dehydration of the resulting seco-acid
are catalyzed by unidentified enzymes to give the terminal olefin.
12-membered ring macrolide YC-17 (10-Dml glycoslyated
with desosamine on the C-3 hydroxyl) as well as both the C12
and C14 positions of the 14-membered ring macrolide
narbomycin (narbonolide glycosylated with desosamine on
the C-5 hydroxyl)44-46 (Figure 14). Here, it was determined
that substrate anchoring of the macrolactones by the deso-
samine sugar in the PikC active site was vital for activity and
selectivity.47 Also, the alternative hydroxylation patterns
seen in these in vitro studies were identified natively in vivo
as minor Pik metabolites neomethymycin/novamethymycin
and neopikromycin/novapikromycin.46,48 The only required
substitution for hydroxylation by PikC was the desosamine
sugar appendage on the macrocycle. Also, the cocrystal
structures between PikC and substrate revealed that due
to the desosamine anchor the most distal carbons on the
Finally, the PikC P450 hydroxylase from pikromycin
biosynthesis represents an attractive candidate for chemoen-
zymatic synthesis since it exhibits broad substrate selectivity.
In its native form, PikC installs the difficult quaternary
C10 and C12 hydroxyls in the methymycin and pikromycin
metabolites, respectively.44 In a series of in vitro studies with
recombinant PikC, the P450 was shown to be extraordinarily
flexible, hydroxylating both the C10 and C12 positions of the
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macrocycles are positioned close to the enzyme Fe center,
and are subsequently hydroxylated (Figure 15). This detailed
understanding of PikC function has recently motivated an
effort to explore its ability to catalyze remote C-H bond
activation for hydroxylation of a series of desosaminylated
hydrocarbon rings of varying size. Remarkably, PikC showed a
facile ability to selectively hydroxylate these compounds that
also exhibited surprising antibiotic activity.49
The ability of recombinantthioesterases to chemoenzymatically
generate macrocyclic natural product scaffolds holds outstand-
ing promise for medicinal chemistry, providing a robust and
environmentally friendly alternative to difficult chemical
macrocyclization strategies. The examples presented in this
Perspective are representative of the broad utility of TEs as
biocatalysts. Salient examples have also been demonstrated
with thioesterases in other PKS/NRPS systems such as
tyrocidine52-56 and epothilone,57,58 further supporting this
general premise. In order to leverage the catalytic power and
specificity of recombinant TEs, however, future studies must
first aim at detailed understanding of their many underlying
structural details and catalytic limitations. It can be envisioned
that through structure-based protein engineering efforts,
rational development of TEs with expanded substrate tolerance
and increased catalytic efficiencies will be within reach.
At the same time, P450 hydroxylases and epoxidases are
well positioned to address the challenging task of selectively
oxidizing C-H bonds and alkenes in a stereo- and regio-
specific manner. Both the CrpE epoxidase and PikC hydroxy-
lase are powerful examples of the utility of P450s for post-PKS/
NRPS tailoring of natural product scaffolds. Nonetheless,
development of P450 enzymes for chemoenzymatic synthesis
includes significant challenges. For example, P450s require
reductase enzyme partners and ferredoxin cofactors for activity
and may not operate efficiently with in vivo partners during
heterologous expression. For in vitro work, this requires the use
of an exogenous reductase/ferredoxin partner, such as com-
mercially available spinach ferredoxin reductase.19,44 This
strategy, however, is not economical for large-scale chemo-
enzymatic synthesis and does not necessarily reflect general
reductase compatibility of other natural product pathway
P450s. In the case of PikC, this hurdle was overcome by making
a PikC-RhFRED fusion to generate a self-sufficient reductase-
coupled P450.45 General applicability of this strategy was
demonstrated in the same study, where a similar fusion was
made with one of the erythromycin P450s, EryF, to provide a
self-sufficient enzyme. Finally, substrate specificity require-
ments of P450 enzymes, such as the desosamine anchoring
mechanism of PikC,47,49 must be considered in order to success-
fully apply them for biocatalytic conversion of new substrates.
Nevertheless, despite these challenges, the opportunity afforded
by biosynthetic P450s remains an exciting prospect for future
work.
Working in larger systems, the manipulation of complete
PKS modules with multiple enzymatic domains represents a
distinctly challenging goal in chemoenzymatic synthesis but
one that can pay extraordinary dividends. By harnessing the
biosynthetic potential of full modules, it can be envisioned
that an array of polyketide scaffolds containing myriad
stereochemical and functional permutations could be
accomplished in combinatorial fashion. In this Perspective,
the examples from the Pik and DEBS PKSs are a testament to
the utility of intact modules for chemoenzymatic synthesis
both in vitro and in vivo. At the same time, current work in
other systems such as epothilone57,58 and rapamycin59 con-
tinues to expand our access to new PKS modules with unique
substrate specificities and catalytic capabilities. Thus, while
current understandingof PKS modulesinmany systemsisstill
insufficient or incomplete for practical metabolic engineering
efforts, the most recent studies using native chain elongation
intermediates from Pik and DEBS35,39 have been particularly
Curacin and Tautomycetin
With the ongoing discovery of new natural products and
their putative biosynthetic pathways, the biochemical tool-
box for chemoenzymatic synthesis continues to expand. This
has resulted in increasing levels of chemical diversity and the
uncovering of enzymes that can perform unique chemical
transformations. As has been discussed earlier, macrocycli-
zation by a PKS or NRPS terminal thioesterase domain is
often a final step in forming bioactive natural products;
however, some natural product pathways have evolved to
terminate in unusual ways. This is the case with the natural
products curacin A50 (Cur) and tautomycetin51 (Tmc), where
distinctive chain termination events give rise to linear pro-
ducts with rare terminal olefin groups. In both cases, a series
of hydrolysis, decarboxylation, and dehydration reactions
appears to be required for formation of this functionality,
though the mechanisms for accomplishing this are divergent
in the two systems.
For the Cur and Tmc systems, terminal thioesterases
hydrolyze their respective intermediate metabolites from
upstream ACPs to give linear seco-acids. In curacin bio-
synthesis, this TE-mediated cleavage is preceded by an
unusual embedded sulfotransferase (ST) enzyme in the final
CurM module that transfers a sulfonate to the β-hydroxy
group of the ACP-tethered intermediate4 (Figure 16). Fol-
lowing sulfonation, the TE domain catalyzes hydrolysis
of the intermediate phosphopantetheinyl thioester with
concomitant decarboxylation and elimination of sulfate to
give the terminal olefin. By contrast, in tautomycetin bio-
synthesis the linear seco-acid formed by TE hydrolysis of
the final intermediate is unactivated for elimination at the
β-hydroxy position. Here, hydrolysis occurs in a stereospecific
manner favoring the (R) hydroxyl group at the β-position. At
the same time, unlike in Cur biosynthesis, decarboxylation/
elimination is uncoupled from the thioesterase activity.5
Rather, it appears that committed decarboxylase/dehydratase
enzymes are responsible for decarboxylation and elimination of
water to give the final product (Figure 17). Currently, two
putative decarboxylases and a dehydratase, encoded by tmcJ,
tmcK, and tmcM are candidate enzymes for catalyzing these
reactions.
Future Outlook
Realization of the promise of chemoenzymatic synthesis is
rooted in a thorough understanding of the fundamental
mechanistic underpinnings of the enzymes of interest. Through
a synergistic merging of synthetic chemistry with enzymology
and structural biology, detailed knowledge can be obtained for
natural product biosynthetic systems, thus paving the way for
their rational application toward generating new compounds of
biological interest.
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Mortison and Sherman
JOCPerspective
crucial in guiding detailed biochemical evaluations of under-
lying mechanisms in those two systems. This suggests that
future studies must continue to employ native or near-native
substrates and their analogues to rigorously probe inherent
molecular specificities of modular PKS domains for accepting,
elongating, processing, and cyclizing substrates.
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Acknowledgment. The generous support of NIH (NIGMS
and NCI) through grants to DHS and the Hans W. Vahlteich
Professorship at the University of Michigan has made this work
possible. The cover art photography was graciously provided
by Pam Schultz.
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