Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid ?-Substitution Enzyme That Catalyzes C-C Bond Formation

Article abstract of DOI:10.1021/jacs.0c03535

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze transformations of l-amino acids at α, β, and γpositions. These enzymes are frequently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S,6S)-6-methyl pipecolate 1, from the biosynthetic pathway of citrinadin. The key enzyme CndF is PLP-dependent and catalyzes the synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed this enzyme performs ?-elimination of O-acetyl-l-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C?-C? bond in 3 and complete the ?-substitution reaction. CndF displays promiscuity toward different β-keto carboxylate and esters. With use of an Aspergillus strain expressing CndF and CndE, feeding various alkyl-β-keto esters led to the biosynthesis of 6-substituted l-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

Full text of DOI:10.1021/jacs.0c03535

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Article
Discovery and Biocatalytic Application of a PLP-Dependent Amino
Acid #-Substitution Enzyme that Catalyzes C-C Bond Formation
Mengbin Chen, Chun-Ting Liu, and Yi Tang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c03535 • Publication Date (Web): 20 May 2020
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Discovery and Biocatalytic Application of a PLP-Dependent Amino
Acid -Substitution Enzyme that Catalyzes C-C Bond Formation
Mengbin Chen,1 Chun-Ting Liu,1,3 Yi Tang1,2,*
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1Department of Chemical and Biomolecular Engineering, 2Department of Chemistry and Biochemistry, University of California Los
Angeles, Los Angeles, California 90095, USA. 3Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massa-
chusetts 02139, USA.
Supporting Information Placeholder
ABSTRACT: Pyridoxal phosphate (PLP)-dependent enzymes can catalyze transformations of L-amino acids at ,  and  positions. These
enzymes are frequently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive
biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2S, 6S)-6-methyl pipecolate 1, from the biosynthetic pathway
of citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (S)-2-amino-6-oxoheptanoate 3 that is in equilibrium with
the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1. Biochemical characterization of CndF showed
this enzyme performs -elimination of O-acetyl-L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack
by acetoacetate to form the new C-C bond in 3 and complete the -substitution reaction. CndF displays promiscuity towards different -keto
carboxylate and esters. Using an Aspergillus strain expressing CndF and CndE, feeding various alkyl--keto esters led to the biosynthesis of 6-
substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.
16
INTRODUCTION
Therefore, mining and characterization of new enzymes from sec-
ondary metabolism can be fruitful for discovery of new biocatalysts
for NAA synthesis.
Nature is remarkable in building and using structurally diverse
amino acids.1-2 Nonproteinogeneic amino acids (NAAs), which con-
stitute 96% of the naturally occurring amino acids,1 are frequently
incorporated into small molecules to broaden reactivity and to es-
tablish biologically relevant conformers.3 Peptides that contain
NAAs are less susceptible to proteolysis, thereby increasing the half-
lives during circulation.4-5 The usefulness of NAAs have also been ex-
tensively explored through incorporation into recombinant proteins
expressed from Escherichia coli, yeast, and mammalian cells.6 Be-
cause of these important features, new methods to efficiently synthe-
size NAAs have gained significant attention.7-8
Parallel to organic synthesis, biocatalysis has been applied in the
enantioselective synthesis of NAAs.9 For example, threonine al-
dolases can produce -hydroxy--amino acids with complete stere-
oselectivity at C positions and moderate stereospecificity at C po-
sitions.10 Aminomutases have been used to convert -amino acids to
value-added -amino acids.11 Enzymes discovered from natural
product biosynthetic pathways that catalyze challenging C-H func-
tionalization reactions have been recently used as biocatalysts for
NAA synthesis.12-14 A notable example by Renata and coworkers
showed that GriE, -ketoglutarate (KG)-dependent hydroxylase,
can stereospecifically hydroxylate remote sp3 carbons in a wide range
of -amino acids at preparative scales.13 Chang et al recently identi-
fied an KG-dependent radical halogenase that can halogenate un-
activated sp3 carbons of free amino acids.14 In these examples, the
natural functions of the enzymes are to build NAAs for further mod-
ification or uptake by nonribosomal peptide synthetases (NRPS).15-
Figure 1. Pipecolates are important synthons in medicinal chemistry. Ex-
amples of FDA approved drugs containing N-heterocyclic amino acids.
Pipecolate and derivatives are privileged building blocks fre-
quently used in medicinal chemistry. Pipecolate is present in several
FDA approved therapeutics, such as levobupivacaine and nelfinavir
(Figure 1).4 Pipecolate is also found in natural products as high-
lighted by rapamycin, in which the L-pipecolate is involved in for-
1
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mation of the macrolactam and interactions with FKBPs through hy-
We explored the synthetic utility beyond the native substrate to pro-
duce a suite of 6-alkyl-pipecolates starting from -keto esters.
drogen bonding.17 Altering the L-pipecolate moiety changes the po-
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tency of rapamycin derivatives.18
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Synthetically, pipecolates can be constructed via multiple ap-
proaches, including aza Diels-Alder cycloaddition between an acti-
vated carbodiene and an imine,19 full reduction of a substituted pic-
olinate,20 ring closing metathesis (RCM),21 or cyclization/reduction
of oxo-amino acids generated from sulfinimine-mediated asym-
metric Strecker synthesis.22 The last approach resembles one way
used by Nature to synthesize unsubstituted L-pipecolate: L-lysine is
deaminated to give a cyclic imine, followed by reduction to pipeco-
late.23 In rapamycin biosynthesis, a single cyclodeaminase LCD cat-
alyzes both L-lysine deamination and imine reduction (Scheme
1A).24 In plants, the -amine of L-lysine is first converted to ketone
followed by Schiff base formation between C and N.25 Subsequent
reduction takes place stereospecifically to give L-pipecolate (Scheme
1B). In fungi, flavin-dependent saccharopine oxidase can generate a
C-semialdehyde from saccharopine, which can form a cyclic imine
between N and C (Scheme 1C).26 This mechanism is analogous to
microbial biosynthesis of methylprolines, common building blocks
in natural products. Oxygenases can first catalyze remote oxidation
of aliphatic amino acids such as L-leucine or L-isoleucine, followed
by imine reduction (Scheme 1D).27-28
RESULTS AND DISCUSSIONS
Citrinadin contains a substituted piperidine. Active against mu-
rine leukemia L1210 cells, citrinadin A and B were isolated from ma-
rine Penicillium citrinum (Scheme 2).30-31 Citrinadin A is modified
from citrinadin B with an N,N-dimethylvaline ester.31 Both citrina-
din A and B contain a pentacyclic core structure comprised of spi-
rooxindole and quinolizidine. The complex structures have inspired
numerous total synthesis of citrinadin A and B, and the structurally
related PF1270A–C.32-36 Biosynthetically, citrinadin A is a prenyl-
ated indole alkaloid, which can be morphed from a monoketopiper-
azine that is derived from two amino acid building blocks.37-39 One of
the two amino acid is L-tryptophan as indicated by the oxoindole
moiety, while the other becomes part of the quinolizidine, and is pre-
dicted to be (2S, 6S)-6-methyl pipecolate 1 (Scheme 2). Retrobio-
synthetically, we propose 1 can be derived from the stereospecific
reduction of the Schiff base 2, analogous to the last step in proline
and pipecolate biosynthesis (Scheme 1). 2 is expected to be in equi-
librium with the ring-opened amino acid (S)-2-amino-6-oxohep-
tanoate 3, which should be biosynthesized by dedicated enzymes in
the citrinadin biosynthetic cluster starting from primary metabolites
(Scheme 2B).
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Scheme 2. Citrinadin A and B contain a 6-methyl-piperidine that is
likely derived from 6-methyl-pipecolate1.
As proposed, the biosynthesis of citrinadin A would require a two-
module NRPS that activates and condenses tryptophan and 1, and
at least one dimethylallyl diphosphate transferases (DMAT). The
N,N-dimethylvaline ester group in citrinadin A is likely installed by
an additional single-module NRPS.29 Using the colocalizations of
genes encoding a two-module NRPS and DMAT as requirement, we
located a biosynthetic cluster (renamed cnd) in several sequenced
Penicillium citrinum strains (Figure 2A). Among a handful of P. cit-
rinum strains that were screened, P. citrinum DSM1997 was the only
one that produce citrinadin A under laboratory conditions with a ti-
ter of 5 mg/L when grown on potato dextrose agar (Figure S2A). To
confirm the detected compound is citrinadin A, we performed large-
scale culturing of P. citrinum DSM1997, purified the compound and
verified its structure by NMR analysis (Tables S1-S2, Figures S15-
S16). Next, the cndA gene encoding the NRPS was deleted by ho-
mologous recombination from the genome to generate P. citrinum
cndA (Figure S2). Metabolic analysis of P. citrinum cndA
showed citrinadin A production was abolished (Figure S2), confirm-
ing the cnd cluster is responsible for citrinadin biosynthesis.
Scheme 1. Enzymatic routes to pipecolate and methylated prolines.
Despite their occurrences in drugs such as Nelfinavir, alkyl-substi-
tuted pipecolates are extremely rare in natural products. To our
knowledge, our recent discovery of 5,5-dimethyl-L-pipecolate in fla-
vunoidine is the only example.29 Given the linear aliphatic chain in L-
lysine and the limited side chain lengths of natural aliphatic amino
acids, it is evident that formation of an alkyl-substituted pipecolate
via mechanisms shown in Scheme 1 is not possible. Instead, biosyn-
thesis of alkyl-substituted pipecolates will likely involve C-C bond
formation between two building blocks to reach the required num-
ber of carbons. Uncovering the enzymes that can perform such func-
tion can therefore lead to development of biocatalysts for diverse
pipecolate synthesis. Here, we reported the discovery of a two-en-
zyme pathway, including a PLP-dependent -substitution enzyme
and an imine reductase, to produce (2S, 6S)-6-methyl pipecolate 1.
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Figure 2. Biosynthesis of citrinadin by Penicillium citrinum DSM 1997. (A) The identified cnd gene cluster. The four enzymes that are studied here are
colored. Abbreviations: FMO: flavin-dependent monooxygenase; DMAT: dimethylallyltransferases; MT: methyltransferase; SDR: short-chain dehy-
drogenase/reductase; NRPS: nonribosomal peptide synthetase (A: adenylation; C: condensation; T: thiolation; R: reductase); (B) Proposed early
steps of citrinadin biosynthesis involves the NRPS CndA and (2S,6S)-6-methyl-pipecolate 1.
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1 is a building block of citrinadin. Since 1 is not a known primary
metabolite, we reasoned that dedicated enzymes responsible for its
biosynthesis may be encoded in cnd cluster. Through comparative
analysis of cnd and known gene clusters of other related indole alka-
loids that do not incorporate 1, such as those of notoamide B and
taichuanamide A (Figure S3),37 a set of cnd genes (cndE-cndG ) that
are uniquely present in cnd cluster were identified and highlighted
in Figure 2A. Basic local alignment search tool (BLAST) searches
using CndE as a query revealed that it belongs to short-chain dehy-
drogenase/reductase (SDR) family. Considering that the final steps
by the NRPS and is incorporated into the dipeptide (Figure 2). Both
compounds are proposed to be off-pathway shunt products that are
related to the on-pathway intermediate monoketopiperazine 5. For-
mation of the carboxylate 6 can be a result of nonenzymatic hydrol-
ysis of dipeptidyl-S-PCP from the peptidyl-carrier protein (T2) do-
main of CndA, prior to reductive release as the aldehyde 4 by the R
domain (Figure 2B). A sluggish R domain in the heterologous host
A. nidulans may lead to nonenzymatic hydrolysis of the thioester
bond to offload 6. On the other hand, once released, 4 is in equilib-
rium with the monoketopiperazine 5, in which the amide bond is in
a less favorable cis configuration.46 In the absence of downstream en-
zymes that can process 5 to more advanced intermediates, the alde-
hyde of 4 can be further reduced to the alcohol 7 by endogenous re-
ductases as a detoxification mechanism (Figure 2B).47
in L-proline and L-pipecolate biosynthesis both require reduction of
cyclized imine by imine reductases (Scheme 1), CndE may play a
similar role in biosynthesis of 1.23, 27-28 The remaining two unique en-
zymes encoded in the cluster are CndF, a pyridoxal phosphate
(PLP)-dependent enzyme; and CndG, a putative HMG-CoA ly-
ase.40 The closest known homologue of CndF is PLP-dependent
cystathionine--synthase (CGS), with a sequence identity of 36%.41-
42 In fungi, CGS catalyzes the -elimination of O-acetyl-
L-homoser-
ine 9 to give a vinylglycine ketimine.43 The nucleophilic thiolate of
cysteine can subsequently attack the C atom to form a new C-S
bond.44 We reasoned that CndF may catalyze C-C bond formation
between vinylglycine and a three-carbon precursor in a -substitu-
tion reaction to give (S)-2-amino-6-oxoheptanoate 3 (Scheme 2). If
so, CndF would be the first example of a -substitution, PLP-
dependent enzyme that catalyzes C-C bond formation.
To determine whether 1 is a building block of citrinadins, we het-
erologously expressed CndA in Aspergillus nidulans A1145 EM.45
Expressing cndA alone did not lead to new metabolites, indicating
possible lack of a specific amino acid substrate. Feeding commer-
cially available (±)-cis-6-methyl pipecolate, which contain both (2S,
6S) and (2R, 6R) enantiomers, to the cndA expression strain led to
the production of two new compounds 6 and 7 (Figure 3, vii). NMR
characterization revealed 6 and 7 are dipeptides derived from tryp-
tophan and 6-methyl pipecolate (Tables S5 and S6, Figures S28-
S37). Therefore, the exogenously supplied pipecolate is recognized
Figure 3. Heterologous expression in A. nidulans established that
CndE and CndF are necessary and sufficient for the formation of 1.
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Figure 4. CndF and CndE catalyze tandem reactions to yield 1. (A) acetoacetate 8a is the precursor to 1. (B) In vitro characterization of CndF revealed
that O-acetyl- -homoserine 9 is the preferred amino acid substrate. (C) In vitro reconstitution of activities of CndF and CndE to give 1. (D) Proposed
biosynthetic pathway to 1.
L
Determination of the biosynthetic enzymes responsible for pro-
ducing 1. To identify the enzymes required to biosynthesize 1 and
test the hypothesis that the PLP-dependent CndF catalyzes a C-C
bond forming reaction, we reconstituted the activities of CndE,
CndF and CndG in A. nidulans. Expression of cndF alone or with
cndG did not yield any new product compared to control (Figure 3,
i-iii). Co-expression of cndE and cndF led to formation of a new
product with a m/z =144 ([M+H]+) (Figure 3, iv) that agrees with
the molecular weight of 1. We purified the compound with a titer of
5 mg/L and confirmed the structure to be that of 1 by NMR (Table
S3, Figures S17-S22). NOESY analysis showed that 2-carboxylate
and 6-methyl substituents in 1 are in a cis configuration. Purified 1
was derivatized with Marfey’s reagent,48 and the product profile was
compared with that of derivatized (±)-cis-6-methyl pipecolate (Fig-
ure S4). Derivatized 1 elutes as a single peak, whereas the derivatized
(±)-cis-6-methyl pipecolate elutes as two separate peaks (Figure
S4). This demonstrates that 1 is a single enantiomer. Based on the
total synthesis and stereochemical assignment of the quinolizidine
methyl group in citrinadin to be in S configuration,33-34 we suggest
the C6-methyl group in 1 is also in S configuration. Hence the struc-
ture of 1 is assigned to be (2S, 6S)-6-methyl-pipecolate. This result
shows that cndE and cndF are necessary and sufficient to produce 1.
Expression of cndE and cndF in the presence of cndA led to the pro-
duction of 6 and 7 (Figure 3, vi), which is consistent with the feeding
study with (±)-cis-6-methyl pipecolate.
were made to obtain cDNA of cndG from the native producer, we
found that cndG remained silent under the citrinadin producing
conditions tested in the lab. Suppression of cndG expression may be
due to sufficient cytosolic concentration of acetoacetate through pri-
mary metabolism to support the production of citrinadin.
To verify 1 is derived from acetoacetate 10, we supplied [2,4-13C2]
ethyl acetoacetate 8a to A. nidulans expressing cndE and cndF. We
reasoned the ethyl ester of 8a may be hydrolyzed by an endogenous
esterase to give 10. As a result of feeding, an increase of 2 mu in
MWT of 1 was detected by mass spectrometry (Figure 4A). This in-
dicates both 13C atoms of 8a were incorporated into 1. We purified
this labeled compound and NMR analysis revealed that carbon at-
oms at C5 and the C6-methyl group are isotopically labeled (Figure
4A, Table S4, Figures S23-S27). Based on differences in reactivities
of the carbon atoms, we assigned C5 of 1 to correspond to the more
nucleophilic C atom of 8a, while C6-methyl to correspond to the
terminal C of 8a. C-C bond formation thus occurs between C (C4
in 1) and C (C5 in 1) to forge 3, the precursor of 1 (Figure 4D).
This agrees with our hypothesis that PLP-dependent enzyme CndF
catalyzes C-C bond formation between two smaller precursors.
CndF is a PLP-dependent -substitution enzyme that catalyzes C-
C bond formation. Having established 10 is a precursor of 1, we next
sought to elucidate the mechanism of CndF. Since CndF is proposed
to catalyze a -substitution reaction via a PLP-dependent mecha-
nism, the first substrate must be an amino acid with a good leaving
group at C position. To identify the amino acid substrate of CndF,
we heterologously expressed and purified it from E. coli with 40 M
PLP supplemented in all purification buffers. To confirm that CndF
binds PLP, we used UV-vis spectrophotometer to examine to ab-
sorbance profile. A characteristic absorption was observed at 420 nm
for the purified enzyme, indicating the formation of internal al-
dimine between PLP and a lysine residue in the active site (Figure
S5).49 Extensive dialysis of CndF in the presence of hydroxylamine
eliminated the absorbance peak at 420 nm, indicating the enzyme
has been converted to the apo form (Figure S5).
When cndG encoding a predicted HMG-CoA lyase was coex-
pressed in the strain expressing CndE and CndF, no significant
changes of metabolic profile were observed (Figure 3, v). However,
the clustering of cndG with cndE and cndF suggests a possible role
in increasing the availability of a biosynthetic precursor to 1. CndG
shows >60% sequence identity with fungal HMG-CoA lyases, which
indicates CndG is likely to catalyze the same reaction: the cleavage
of HMG-CoA into acetyl-CoA and acetoacetate 10 (Figure 4D).40
The product 10 could therefore be the three-carbon building block
that is used by CndF in constructing 1. Although extensive efforts
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Scheme 3. Proposed mechanism of CndF in catalyzing the -substitution reactions to yield 3.
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Next, we tested a panel of amino acids, including L-homoserine,
O-acetyl-L-homoserine 9, O-phospho-L-homoserine, O-succinyl-L-
homoserine, L-homocysteine, L-cystathionine, and S-adenosyl-L-
methionine (SAM), as substrates of CndF in the -substitution reac-
tion. In vitro reactions of holo-CndF were carried out in the presence
of 10. To facilitate detection of 2 (in equilibrium with 3) by LC-MS,
we used o-aminobenzaldehyde (oAB) to derivatize the cyclic imine
2 into the dihydroquinazolinium complex that has a distinct absorb-
ance at 440 nm (Figure S6) and a m/z of 245. The assays showed
that 9 displayed the highest activity among all substrates (Figure
4B), and is therefore the most likely natural substrate for CndF. The
only other substrate that supported the formation of 2 was O-suc-
cinyl-L-homoserine.
The proposed mechanism of CndF is shown in Scheme 3. In the
first step of the reaction following formation of the external aldimine
(I) between PLP and 9, PLP serves as an electron sink to delocalize
the negative charge generated by the initial deprotonation event at
C position of 9 to give quinonoid (II). Divalent metal ions are
known to promote electron displacement as a way to stabilize the C
carbanion and accelerate catalysis for PLP-dependent enzymes.53-56
We carried out in vitro enzymatic reactions of CndF in the presence
of different divalent metal ions, and found Co2+ expedites the reac-
tion most (Figure S8). The remaining steps of -elimination and
substitution by CndF are analogous to that of cystathionine -syn-
thase.44 Protonation of II gives the ketimine (III), which can undergo
C proton abstraction and expulsion of the acetyl group to yield the
PLP-bound ketimine form of vinylglycine (IV). Next, the acidic pro-
ton at C position of acetoacetate is abstracted and the resulting eno-
late attacks the electrophilic IV to form an enamine adduct (V).
Reprotonation of C forms the ketimine (VI), followed by qui-
nonoid (VII) formation and C protonation to arrive at the new ex-
ternal aldimine (VIII). Release of the adduct 11 as product regener-
ates the internal enzyme-PLP aldimine. Under physiological condi-
tions where primary amines are abundant, the -carboxylate of 11
can undergo nonenzymatic decarboxylation to arrive at the desired
ketone 3.57-59 While the C-C bond formation step (IV  V) may also
be initiated through the decarboxylation of 10, our data below with
-keto ethyl esters suggest the decarboxylation can take place after
C-C bond formation. Furthermore, although decarboxylation is de-
picted to occur after product release in Scheme 3, we cannot exclude
that from taking place during the later catalytic steps (V  VIII) in
the CndF active site.
CndF is a unique PLP-Dependent enzyme. PLP-dependent en-
zymes are essential in all kingdoms of life to catalyze many reactions
in metabolism, and arguably represent the most versatile biocata-
lysts.42 The PLP cofactor enables chemical transformations on
amino acids that are otherwise difficult to perform, including trans-
amination, decarboxylation, / elimination and substitution, etc.41
Recently, oxidase activities were also identified in PLP-dependent
enzymes.60 The mechanism of CndF reported here is distinct from
known PLP-dependent, -substitution enzymes by catalyzing C-C
bond formation between the vinylglycine ketimine and the nucleo-
philic C in 10. The closet examples to CndF are cystathionine--
synthase and LolC found in the loline biosynthetic pathway, which
catalyze C-S and a proposed C-N bond formation using the vinylgly-
cine ketimine as electrophile, respectively.42, 44, 61 Other known C-C
bond forming PLP-dependent enzymes, such as UstD in ustiloxin B
biosynthesis, can perform -substitutions using an enamine-PLP as
a nucleophile.42, 62 Threonine aldolases and -aminolevulinate syn-
thase, which are PLP-dependent enzymes, catalyze C-C bond for-
mation at -position of glycine using the nucleophilic glycyl-qui-
nonoid.10 The recent discovered bi-functional FlvA is proposed to be
mechanistically similar to CndF, although biochemical validation is
not available.29
Searches of the nonredundant protein database in the National
Center for Biotechnology Information (NCBI) for CndF homo-
logues revealed numerous candidates across filamentous fungi.
None of these homologues have known functions, implicating the
untapped functional diversity of these enzymes. Phylogenetic analy-
sis showed that the closest homologues to CndF are all co-clustered
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with other biosynthetic enzymes (Figure S9-10). One group of these the esterase that is presumably present in A. nidulans. Feeding L-ho-
1
2
3
4
5
6
7
8
clusters are highly conserved across several Aspergillus spp. and all
contain a NRPS-independent siderophore (NIS) synthetase, while
other cluster contains a single-module NRPS (Figure S10). It is
likely that these PLP-dependent enzymes also provide NAAs for
downstream incorporation in a secondary metabolite.
CndE is an imine reductase. To probe the role of the SDR homo-
log CndE in reducing 2 to 1, we carried out combined enzymatic re-
actions in vitro. Purified CndE and CndF (28 M each final concen-
tration) were incubated with 9 (1 mM), 10 (1 mM), NADPH (1
mM), and Co(NO3)2 (2.5 mM) in 50 mM phosphate buffer (pH
6.5) for 1 hour at room temperature. Subsequently, the reaction mix
was derivatized with Fmoc-Cl and analyzed by LC-MS. The Fmoc-
derivatized product eluted at the same time as Fmoc-1(Figure 4C).
Formation of 1 is not detected in the absence of CndE or NADPH.
Together with earlier chiral derivatization result showing that 1 is a
single enantiomer, these data established 2 that can be stereoselec-
tively reduced to 1 with S-configuration at C6 by CndE.
The steady-state kinetics of CndF were then determined in a cou-
pled assay with CndE through the monitoring of NADPH consump-
tion. The KM values of CndF towards 9 and 10 are 0.48 mM and 2.8
mM, respectively (Table 1, Figure S7). The KM of 9 is ~5 fold lower
than the reported value for O-acetyl-homoserine sulfhydrylase,50
which may result in only a limited amount of 9 to be redirected from
primary metabolism for the biosynthesis of 1. The kcat values of CndF
towards 9 and 10 are 0.086 s-1 and 0.064 s-1, respectively (Table 1).
These kcat values are comparable with other PLP-dependent en-
zymes in secondary metabolism.51-52
moserine (10 mM final concentration) and 8a (10 mM final concen-
tration) to E. coli culture after IPTG-induced protein expression led
to the production of 14 at 30 mg/L after 6 hours. The compound
was purified and the structure was solved by NMR to be the enamine
14 shown in Scheme 4 (Table S7, Figures S38-S42). The max of 14
at 286 nm is also consistent with reported enamine absorbance (Fig-
ure S12A).63 Formation of 14 confirms the cyclic Schiff base 13 to be
the product of CndF. On the other hand, coexpression of CndE with
CndF in E. coli did not reduce 13 to the corresponding pipecolate.
This can be attributed to the more stringent substrate specificity of
CndE against bulky, C5-substituted cyclic imines. The production
of 14 also provides additional mechanistic insight of CndF. In pro-
posing a mechanism for CndF using 10 as the nucleophile, we did
not know if decarboxylation is required for enolate formation. Con-
version of 8a to 14 indicates C-C bond formation by CndF does not
require acetoacetate decarboxylation to drive enolate formation.
Therefore, the presence of a general base (B4 in Scheme 3) to ab-
stract C proton in the -keto substrates is required to form the eno-
late nucleophile. Furthermore, we determined steady-state kinetics
of CndF in the presence of 8a (kcat = 0.027 s-1, KM = 1.9 mM) (Table
1). Comparable KM values between 8a and 10 suggests that the ethyl
ester group in 8a does not significantly weaken its binding to CndF.
Turnover number of 8a is 2.4-fold lower than that of 10. This differ-
ence in kcat values is insufficient to favor the -decarboxylation driven
C-C bonding formation during CndF catalysis when 10 is used (Ta-
ble 1, Figure S7).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Steady-state kinetic constants of CndF.
substrate
kcat (s-1)
KM (mM)
kcat/KM
(M-1 s-1)
O-acetyl-L-homoserine 9
acetoacetate 10
0.086 ± 0.014 0.48 ± 0.003 1.8 x 102
0.064 ± 0.012
2.8 ± 0.9
1.9 ± 0.2
22
14
ethyl acetoacetate 8a
0.027 ± 0.002
CndF can synthesize enamines with -keto esters. Having verified
the activity of CndF, we next probed the substrate scope of CndF by
varying the structures of -keto nucleophile (Figures S11-S12). We
chose -keto ethyl esters as substrates because very few -keto acids
are commercially available due to spontaneous -decarboxylation.
Substrates with different sizes at C, as well as those containing cyclic
-ketones are tested in the assay. We used LCMS to monitor appear-
ance of new products, as well as quantifying the consumption of sub-
strates (Figure S11). Using 8a as an example as shown in Scheme 4,
if the ester 8a can be recognized by CndF and can form a C-C bond
with the vinylglycine ketimine IV, then adduct 12 and the cyclized
Schiff base 13 are expected to be products. If the -position of the
ester nucleophile is not substituted as in 8a, the Schiff base 13 is ex-
pected to tautomerize and form the stable enamine 14 (Scheme 4).
When tested in an the in vitro assay with CndF, 8a can indeed be
efficiently converted to a new product with the same MWT as 14,
with nearly complete consumption of 8a (Figure S12A). To produce
sufficient amount of the product for structural determination, we
transformed E. coli with a plasmid harboring homoserine O-acetyl-
transferase (HAT) and CndF. Overexpression of HAT increases the
intracellular levels of 9 for the biotransformation. In addition, 8a
does not undergo hydrolysis readily in E. coli due to the absence of
Scheme 4. CndF can convert ethyl acetoacetate 8a to enamine 14.
We then assayed a panel of commercially available -ketoesters
with CndF in vitro (Figure 5 and Figures S11-12). C-Mono-, di-,
tri-fluoro ethyl acetoacetate can all be converted into new products
based on LCMS analysis, with full consumption of substrates (Fig-
ure S12). Increasing the sizes of C substituent from methyl in 8a to
isopropyl in 8b, tert-butyl in 8c, cyclobutyl in 8d led to decrease in
substrate conversions, while product formation can still be clearly
detected by LCMS (Figure S12). Further increasing the sizes of C
substituent to cyclopentyl in 8e or phenyl abolished conversion by
CndF, indicating the cyclobutyl -keto ethyl ester 8d is at the size
limit of the CndF active site available for the nucleophile. We ob-
served ~50% consumption of the cyclopentanone-containing sub-
strate 8f and emergence of a product with expected MWT, (Figures
S11-S12), which indicates that CndF may also tolerate substitution
at the C position of the nucleophile. Other substrates tested, in-
cluding amide-containing and halogenated esters, were not accepted
by CndF (see list in Figure S11).
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Journal of the American Chemical Society
A microbial platform to produce (2S, 6S)-6-alkyl pipecolate. The
several notable exceptions attributed to the removal of the ethyl es-
ter in cellulo. For -keto esters (8b-d) that can be consumed by
CndF in vitro to give enamine esters, 6-substituted pipecolates 15,
16, and 17 were synthesized, respectively (Tables S8-S10, Figures
S43-S60). While 8e was not transformed by CndF in vitro, we can
detect and purify the corresponding 6-cyclopentane-pipecolate 18
from A. nidulans (Table S11, Figures S61-S66). This suggests that
once the ethyl ester is hydrolyzed to the carboxylate, the active site
of CndF can accommodate the bulky C substituent. However,
larger groups such as the phenyl group still cannot be recognized by
CndF to make 6-phenyl-pipecolate. The formation of 15-18 also
shows the SDR CndE can tolerate substantially larger substituents at
C6 of the cyclic Schiff base in performing imine reduction. The ste-
reochemical configurations of these compounds have all been con-
firmed by NOESY analyses. Our results show there are no endoge-
nous reductases in A. nidulans that can intercept the imine interme-
diates generated by CndF to produce the (2S, 6R)-diastereoisomer.
Therefore, coupling CndF with a promiscuous R-specific imine re-
ductase can potentially lead to the production of (2S, 6R)-6-alkyl
pipecolate.65
In addition to 1, we observed production of enamine 14 in large
amounts from A. nidulans when 8a is supplied (Figure S14A). When
C substituent is bulkier as in 8b or 8c, enamine formation becomes
barely detectable (Figure S14A). We reasoned that the ethyl esters 8
is subjected to competing hydrolysis by esterases and C-C bond for-
mation by CndF in A. nidulans. When C substituent is small as in
8a, the rates of hydrolysis and -substitution are comparable, which
results in the formation of both enamine ester 14 and pipecolate 1.
When C substituent becomes bulkier, the C-C bond formation rate
of ethyl ester is slower than that of ester hydrolysis, leading to the
formation of nearly all 6-alkyl pipecolate products (Figure S14B).
Lastly, bicyclic 19 and 20 can be produced from ethyl 2-oxocyclo-
pentanecarboxylate 8f and ethyl 2-oxocyclohexanecarboxylate 8g,
respectively (Tables S12-14 and Figures S67-S84). Similar to 8e, we
did not observe enamine formation for 8g in vitro, but 20 was de-
tected and isolated in vivo as a result of ethyl ester hydrolysis. Both
8f and 8g were supplied as racemic mixtures due to facile racemiza-
tion at the C position. From biotransformation, we observed a pair
of diastereomers for both 19 and 20 at nearly 1:1 ratio (Figure S13).
8f was converted to octahydro-1H-cyclopenta[b]pyridine-2-carbox-
ylic acid 19cis and 19trans at 1:1 ratio, although we were only able to
structurally characterize 19cis by NMR due to separation difficulties
associated with 19trans. Octahydro-1H-cyclopenta[b]pyridine-6-car-
boxylic acid, an analogue of 19, is a conformationally restricted -
aminobutyric acid (GABA) analogue.66 Considering the noted neu-
roactivity of pipecolates,67 we speculate that 19 could be useful as
conformationally restricted analogues of L-pipecolates.67-68 Similar to
8f, 8g is presumably hydrolyzed by A. nidulans esterase to yield race-
mic -keto acids, which were converted to a pair of cyclic imines at
1:1 ratio by CndF. CndE can reduce the imines to either the cis-aza-
decalin 20cis or trans-azadecalin 20trans, as confirmed by NMR analy-
sis. Previous studies showed that racemic 20 produced by hydro-
genation of quinoline-2-carboxylic acid is a potent inhibitor of Angi-
otensin I converting enzyme.69 Coupling of CndF and CndE offers a
biocatalytic route to produce decahydroquinoline-2-carboxylic ac-
ids. We reason that scrambling of the C5 stereocenter in 19 and 20
may occur as a result of -decarboxylation after the C-C bond for-
mation step, during which formation of an enol intermediate effec-
tively scrambles the previously established C5 stereocenter.
1
2
3
4
5
6
7
8
above data showed that CndF has considerable substrate promiscu-
ity to form adducts between vinylglycine and different nucleophiles.
However, the use of -keto ethyl esters in vitro or in E. coli limits the
utility of the enzyme to make alkyl-pipecolate derivatives, due to the
inability of CndE to reduce the ester product 13 (Scheme 4). The
incorporation of [2,4-13C2]-8a to 1 using A. nidulans as a host
showed that the ethyl esters can be converted to the corresponding
-keto carboxylates in cellulo by endogenous esterase and lipases
(Figure 4A).64 Encouraged by this finding, we explored the use of A.
nidulans expressing CndF and CndE as a biotransformation host to
convert various -keto ethyl esters into 6-alkyl-pipecolates (Figure
5). The esters were added to the A. nidulans culture and after growth
for 4-5 days, formation of 6-alkyl-pipecolates was monitored by
LCMS. New pipecolate compounds were extracted and purified
from culture (Figure S13), and were structurally characterized by
NMR. We were able to obtain several 6-alkyl pipecolates through
microbial fermentation, with yields ranging from 1-5 mg/L.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A. nidulans
overexpressing
CndF & CndE
O
O
O
O
H2
N
COO
R1
R2
R1
OEt
R1
OH
esterase
CndF
CndE
R2
R2
substrate
product
titer (mg/L)
H2
N
O
O
O
Me
COO
5
Me
OEt
OEt
8a
8b
1
Me
H2
N
O
COO
COO
COO
Me
Me
5
3
Me
15
Me
H2
N
Me
Me
O
O
Me
Me
OEt
Me
16
8c
8d
H2
N
O
O
3
1
OEt
OEt
17
O
O
H2
N
COO
COO
8e
18
H2
N
O
O
3
2
OEt
OEt
8f
19
O
O
H2
N
COO
8g
20
Figure 5. In vivo biosynthesis of substituted pipecolates.
The abilities of the different esters to be transformed into pipeco-
late generally match to what was observed in vitro (Figure 5), with
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Steinreiber, J.; Fesko, K.; Reisinger, C.; Schürmann, M.; van Assema,
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9
Abundant genome sequences have provided unprecedented op-
portunities to uncover new enzymatic reactivities from natural prod-
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pathway to synthesize the noncanonical amino acid (2S, 6S)-6-me-
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between O-acetyl-L-homoserine and acetoacetate, catalyzed by a
PLP-dependent enzyme. The second step is stereoselective Schiff
base reduction catalyzed by an imine reductase. The substrate toler-
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a panel of 6-alkyl pipecolate derivatives. In addition, CndF can ac-
cept -keto ethyl esters to generate enamine containing pipecolate
molecules. CndF displays new catalytic mechanism in catalyzing -
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ASSOCIATED CONTENT
Supporting Information.
This material is available free of charge via the Internet at
http://pubs.acs.org. Experimental procedures, chromatograms, and
spectroscopic data.
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AUTHOR INFORMATION
Corresponding Author
Kano, T.; Kumano, T.; Sakamoto, R.; Maruoka, K., Catalytic
*Yi Tang yitang@ucla.edu
Notes
Rutjes, F. P. J. T.; Schoemaker, H. E., Ruthenium-catalyzed ring
The authors declare no conflict of interest.
ACKNOWLEDGMENT
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This work was supported by the NIH 1R35GM118056 to YT. Structural
characterization by NMR is based upon work supported by the National
Science Foundation under equipment grant no. CHE-1048804.We
thank Drs. Y. Hai and W. Cheng for helpful discussions.
Pérez-García, F.; Max Risse, J.; Friehs, K.; Wendisch, V. F.,
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