Discovery, characterization and engineering of ligases for amide synthesis

Article abstract of DOI:10.1038/s41586-021-03447-w

Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-l-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways^1–4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown^1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and l-Ile in plants⁵. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions—Jar1 producing a compound essential for plant development^4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.

Full text of DOI:10.1038/s41586-021-03447-w

Article
Discovery, characterization and engineering
of ligases for amide synthesis
https://doi.org/10.1038/s41586-021-03447-w
Received: 21 August 2020
Michael Winn1, Michael Rowlinson1, Fanghua Wang1,2, Luis Bering1, Daniel Francis1,
1 1
ꢀ✉
Colin Levy & Jason Micklefield
Accepted: 11 March 2021
Coronatine and related bacterial phytotoxins are mimics of the hormone
jasmonyl-L-isoleucine ( JA-Ile), which mediates physiologically important plant
signalling pathways1–4. Coronatine-like phytotoxins disrupt these essential pathways
and have potential in the development of safer, more selective herbicides. Although
the biosynthesis of coronatine has been investigated previously, the nature of the
enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains
unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases
(CfaLs), and resolve their structures. We found that CfaLcan also produce JA-Ile,
despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and
L-Ile in plants5. This suggests that Jar1 and CfaLevolved independently to catalyse
similar reactions—Jar1 producing a compound essential for plant development4,5, and
the bacterial ligases producing analogues toxic to plants. We further demonstrate
how CfaLenzymes can be used to synthesize a diverse array of amides, obviating the
need for protecting groups. Highly selective kinetic resolutions of racemic donor or
acceptor substrates were achieved, afording homochiral products. We also used
structure-guided mutagenesis to engineer improved CfaLvariants. Together, these
results show that CfaLs can deliver a wide range of amides for agrochemical,
pharmaceutical and other applications.
Published online: 19 May 2021
Check for updates
Coronatine 3 (COR) is an important phytotoxin produced by bacterial Supplementary Table 1). Previous attempts to characterize this ligase
plant pathogens; it is composed of the polyketide coronafacic acid 1 have been unsuccessful13. Other plant pathogens possess a COR-like
(CFA), conjugated via an amide bond to coronamic acid 2 (CMA), an unu- biosynthetic gene cluster14,15, including Streptomyces scabies which
sual cyclopropyl amino acid (Fig. 1a)1,2. COR is a structural mimic of JA-Ile has a putative CfaL and CFA biosynthetic genes, but no CMA pathway
(6), a ubiquitous plant hormone that is essential for plant development (Supplementary Fig. 2b and Supplementary Table 1)15. Consequently,
and defence3. The biologically active stereoisomer is (3R,7S)-JA-Ile, but this strain produces predominantly CFA-L-Ile 4, along with smaller
the C7 stereocentre rapidly epimerizes at physiological pH to the more quantities of L-Val and L-allo-Ile adducts, which have also been detected
stable but inactive trans (3R,7R) diastereoisomer, which modulates from P. syringae16–18. As CFA must be coupled to an amino acid to elicit
its activity4. In contrast, COR is configurationally stable, endowing it biological activity, we sought to characterize the key CfaLenzymes to
with increased potency and longevity. The conjugation of JA and L-Ile enable new routes to COR-like phytotoxins as potential herbicides19. We
in plants is catalysed by the adenosine triphosphate (ATP)-dependent were also interested in exploring the relationship between the bacterial
ligase Jar1 (Fig. 1b), a member of the ANL superfamily5. ANL enzymes CfaL and the functionally related plant ligase Jar1.
generate acyl-adenylate (acyl-AMP) intermediates that undergo sub-
As well as being fundamental in nature, amide formation is one of the
stitution with various nucleophiles. For example, acyl-CoA synthetases mostwidelyusedsynthetictransformations. Althoughcouplingacidsand
(ACSs) are common ANLenzymes generating thioesters, which can be amines is relatively simple, it often requires three steps, protect–couple–
coupled to an amine by a secondary N-acyltransferase. Jar1 is an amide deprotect, to install each amide. Stoichiometric quantities of expensive
bond synthetase (ABS), a rarer subclass of the ANLsuperfamily which and deleterious coupling reagents are typically required and purification
accept amine nucleophiles directly without requiring an additional can be problematic20. While some progress has been made in the develop-
partner enzyme (Supplementary Fig. 1)5,6
.
mentofchemocatalyticmethodsforamidesynthesis, thesehavenotbeen
CFA is assembled by a type I polyketide synthase via the cyclization widely adopted21–27. Consequently, there is interest in the development of
of a β-ketothioester intermediate (Fig. 1c)1,7–9. The biosynthesis of CMA enzymaticalternatives6,20,28–31. Inthiswork, wecharacterizeCfaLligasesand
occurs via a nonribosomal peptide synthetase (NRPS)-mediated cryptic demonstrate how they are highly versatile biocatalysts for the synthesis of
chlorination and cyclization (Fig. 1d)10–12. A putative ligase (CfaL) within ubiquitous amides. Additionally, using structure-guided mutagenesis, we
the Pseudomonas syringae COR biosynthetic gene cluster is predicted generateimprovedligasesprovidingmoresustainable, alternativeroutesfor
to couple CFA and CMA to form COR (Fig. 1a, Supplementary Fig. 2a and productionofpharmaceuticals,agrochemicalsandothervaluablematerials.
1Department of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK. 2Present address: School of Food Science and Engineering, South China
✉
University of Technology, Guangzhou, China. e-mail: jason.micklefield@manchester.ac.uk
Nature | Vol 593 | 20 May 2021 | 391

Article
a
O
O
O
CO2H
H
CfaL
H2N
or
CO2H
H
CO2H
+
O
OH
N
H
O
N
H
O
CMA (2)
CFA (1)
or L-Ile
COR (3)
CFA-Ile (4)
P. syringae
S. scabies
b
O
O
O
O
7
Jar1
L-Ile
CO2H
CO2H
3
O
OH
O
OH
O
N
H
O
N
H
(3R,7S)-5
(3R,7R)-5
(3R,7S)-6 Active
(3R,7R)-6 Inactive
Jasmonic acid (JA)
Jasmonyl-isoleucine (JA-Ile)
c
ER
ER
Cfa6
Cfa7
DH KR
DH KR
ACP
KS AT
ACP TE
ACP
AT ACP KS AT
ACP
Cfa1
Cfa1
Cfa2 (DH)
Cfa4 (?)
Cfa9
(TE)
O
O
S
O
S
O
S
O
S
S
CFA (1)
O
O
O
O
CHO
O
O
Cfa1 (ACP),
Cfa3 (KS)
malonyl-CoA
CmaA
CmaD
CmaD
d
O
A
T
T
T
CmaC
CmaT (TE)
CmaE
CmaB
OHC
R
O
S
O
S
O
S
CMA (2)
R = OH
R = SCoA
CfaL or
Cfa5
H2N
H2N
H2N
Cl
Fig. 1 | Biosynthesis of coronatine (COR) and jasmonyl-L-isoleucine (JA-Ile).
a, Bacterial CfaL enzymes are predicted to ligate coronafacic acid (CFA) 1 with
coronamic acid (CMA) 2 or L-amino acids to generate phytotoxins, including
COR 3 and CFA-Ile 4. b, In plants, the enzyme Jar1 ligates jasmonic acid (JA) and
L-isoleucine to produce JA-Ile epimers (3R,7S)-6 and (3R,7S)-6. c, Polyketide
synthase (PKS) assembly of 1 from succinic semialdehyde. PKS consists of
acyl-carrier protein (ACP), acyl-transferase (AT), keto-synthase (KS),
dehydratase (DH), enoyl-reductase (ER), keto-reductase (KR) and thioesterase
(TE) domains. d, Nonribosomal peptide synthetase (NRPS)-mediated
biosynthesis of 2 in P. syringae. NRPS consists of adenylation (A), thiolation (T)
and thioesterase (TE) domains.
adenylation can be measured in isolation (Extended Data Table 1 and
Supplementary Fig. 7). The rate of adenylation (kcat) was greatest with
Characterization of the CfaL family
Overproduction of the CfaL from P. syringae (PsCfaL) in Escherichia
( )-CFA but the aromatic analogue 7 was found to have a lower Michaelis
coli resulted in only trace amounts of active enzyme. However, assays constant, Km. Both (3R,7R)- and (3S,7S)-enantiomers of trans-JA 5 were
demonstrated that PsCfaL catalyses the ATP-dependent coupling of also accepted, albeit at a lower level than CFA or 7, with a preference
L-isoleucine and CFA, obtained from acid hydrolysis of coronatine, towards the natural (3R,7R)-5 stereoisomer. This indicates that, despite
confirming the function of CfaLfor the first time (Supplementary Figs. 3 low amino acid sequence similarity (16%), the bacterial SsCfaLand plant
and 4). The low quantity of PsCfaLavailable prevented full characteriza- Jar1 both catalyse the ligation of JA with L-Ile (Supplementary Fig. 8).
tion, and so alternative CfaLhomologues were explored. In addition to Reactions with deactivated enzyme confirmed that the adenylation of
the putative S. scabies ligase (SsCfaL), other candidates located within 5 and the subsequent reaction with L-Ile are both enzyme-catalysed.
putative COR-like clusters were selected from BLAST analysis (Supple- Samples of trans-JA (Extended Data Table 1) contain a minor amount of
mentary Fig. 2). Of these, PbCfaLfrom Pectobacterium brasiliense and the less stable cis epimer, owing to facile C7-epimerization (Fig. 1b)4. To
AlCfaLfrom Azospirillum lipoferum were chosen for characterization, explore the stereoselectivity of SsCfaLfurther, all four stereoisomers
as they are predicted to be more amenable to crystallization (Supple- of configurationally stable 7-methyl-jasmonic acid were synthesized
mentary Table 2). While P. brasiliense is a well-known plant pathogen32,33
A. lipoferum is a root-dwelling, nitrogen-fixing plant symbiont that is 7-methyl-jasmonic acid were separated and tested as a racemic mixture.
not known to produce coronatine34
However, these were poor substrates for SsCfaL, hence the resolu-
,
(Supplementary Fig. 9). Initially the trans and cis diastereoisomers of
.
SsCfaLwas overproduced in E. coli (Supplementary Fig. 5) and assays tion of all four stereoisomers was not carried out. The selectivity of
with synthetic ( )-CFA19, L-isoleucine and ATP showed the direct forma- SsCfaL was further tested by incubating CFA 1 or ( )-jasmonic acid 5
tion of CFA-L-Ile 4 via a CFA-AMP intermediate (Supplementary Fig. 6), with 21 proteinogenic amino acids, resulting in a wide range of amino
confirming CfaLis an ABS enzyme. In addition, SsCfaLalso accepted the acid conjugates (Supplementary Figs. 10 and 11). Hydrophobic amino
aromatic CFA variant 7 which when coupled to L-Ile forms coronalone, a acids such as L-isoleucine and L-valine were preferred by SsCfaLwhich
simplified synthetic COR analogue with promising herbicidal activity35
.
reflects the COR-like metabolites isolated from S. scabies16. No activity
Given adenylation occurs in the absence of amine substrate, the rate of was seen with D-amino acids, or with primary amines and dipeptides
392 | Nature | Vol 593 | 20 May 2021

a
b
c
(Supplementary Fig. 12). AlCfaL and PbCfaL, expressed from codon
optimized synthetic genes, were seen to be functionally similar to
SsCfaL, although with lower activities (Supplementary Fig. 13).
O
7
P219
O
OH
W220
The structure of a CfaL ligase
L223
Y180
T316
G289
Crystallography trials revealed that only PbCfaL yielded crystals of
sufficient quality for structural studies. A PbCfaLstructure in the ade-
nylation conformation was solved to 2 Å resolution (Fig. 2), which is
consistent with the ANL superfamily. Despite sharing low sequence
identity (<20%), PbCfaLshowed substantial structural similarity (>70%)
to several other ANL ligases from a variety of organisms, including
bacterial benzoate CoA ligases and firefly luciferases (Fig. 2b, Sup-
plementary Table 3). By contrast, PbCfaL shares very little structural
similarity with the catalytically equivalent Jar1 (30%), suggesting that
the two enzymes evolved independently (Supplementary Fig. 14).
Plants are known to possess other ACSs that do share high structural
homology with PbCfaL (Supplementary Table 3). However, Jar1 and
related plant acyl-AMP forming enzymes that conjugate salicylate or
indole-6-acetic acid (IAA) with amino acids appear to have evolved
3.6 Å
G290
S312
P321
G314
G320
Q315
D291
Flexible C-terminal
region
Carboxylic acid
binding site
separately and specifically for plant hormone signalling36
.
The structure of PbCfaL is composed of a large N-terminal domain
(residues 1–403) and a smaller, flexible C-terminal domain (residues
403–516, Fig. 2). As with other members of the ANL superfamily, it
is likely that the C-terminal domain undergoes a large rotation fol-
lowing acyl-adenylate formation to close off the acyl binding pocket
and form the amino acid binding site (closed conformation) (Fig. 2).
Co-crystallography of PbCfaL with 7 revealed that the extremely
solvent-accessible acyl binding pocket lies between the two domains
(Fig. 2, Supplementary Fig. 15). Sequence alignment between the CfaLs
in this study showed a small number of conserved residues (Supple-
mentary Fig. 16), with only W220 likely to make direct contact with 7,
probably aligning the carboxylic acid via π–π stacking, explaining the
higher binding affinity of 7 versus CFA (Fig. 2). Other conserved residues
around 7 probably define the width and depth of the binding pocket.
When aligned with the most structurally similar proteins from the Pro-
tein Data Bank (PDB; Supplementary Fig. 17) there are few conserved
sequences, the most similarity occurring in the ATP binding SSGTTG
motif (residues 168–173)37. Despite many attempts, determination of
a PbCfaL structure in the closed conformation with AMP and amino
acid bound could not be achieved.
N-terminal region
Rotation of McbA
into “closed” state
PbCfaL
McbA (6SQ8)
CfaL substrate scope and engineering
Fig. 2 | Structure of PbCfaL. a, Main image, X-ray crystal structure of PbCfaL
(2 Å) in the ‘open’ or ‘adenylation’ conformation (PDB ID, 7A9I). PbCfaLhas a
large N-terminal region (residues 1–403), shown in blue, and a flexible
C-terminal region (residues 403–516), shown in red. The boxed region and
magnified inset highlight the active site region, which is shown with 7
co-crystallized; labelled residues are conserved between all four CfaLs in this
study. 7 lies 3.6 Å from a conserved tryptophan residue (W220), which probably
helps to align the substrate via π–π stacking interactions. b, PbCfaL
We next explored if the synthetic scope of CfaL enzymes could be
extended towards other amide targets. The CfaLenzymes were found
to possess extremely broad substrate tolerance, accepting a variety of
aryl and heteroaryl carboxylic acids 8–29 as well as aliphatic carbox-
ylic acids 30–46, including several chiral compounds 39–46 (Fig. 3,
Extended Data Table 2). Several acyl-donor substrates possessed
other reactive functionalities, such as electrophilic ketones (11, 35,
43, 45), alkenes (33), as well as nucleophilic alcohol (40, 44) or amine
groups (13, 17, 18, 19, 46), which would require protecting for tradi-
tional coupling chemistries, but do not interfere with the enzymatic
ligation to amino acid acceptor substrates. In addition to proteino-
genic amino acids (Fig. 4a, Extended Data Table 3), CfaLenzymes also
accept a wide range of non-proteinogenic amino acids 47–61, includ-
ing common pharmaceutical building blocks, with a preference for
hydrophobic amino acids (Fig. 4b, c, Extended Data Table 4). Although
polar, particularly charged, amino acids are not well accepted by CfaL,
both L-2,4-diaminobutyrate 47 and L-ornithine 48 can be selectively
acylated at the α-amino group, obviating the need for protection of the
side-chain amino group (Fig. 4c, Supplementary Fig. 17).
superimposed onto McbA (gold; PDB ID, 6SQ8)29, the closest structural
homologue to PbCfaL. In the ‘adenylation’ state, the two structures show high
levels of similarity. In this state the carboxylic acid binding site (shown) is
located between the N-terminal and the flexible C-terminal regions and is
solvent accessible. c, PbCfaL superimposed on McbA in the ‘closed’ state (also
referred to as the ‘thiolation’ state in related ACS enzymes). Like all ANLs, the
C-terminal region of McbA undergoes a large rotation (direction indicated by
dashed red arrow) to lie on top of the carboxylic acid binding site, trapping the
adenylated intermediate before amine attack. The more rigid N-terminal
region does not substantially change conformation. We would expect the
C-terminal region of PbCfaL to undergo a similar rotation during catalysis.
Structural alignment was performed with Chimera (version 1.14) MatchMaker.
In general, SsCfaL and AlCfaL both performed better than PbCfaL
(Figs. 3 and 4, Extended Data Tables 2–4), which was found to be less
Nature | Vol 593 | 20 May 2021 | 393

Article
a
b
O
O
CfaL
H
N
H2N
OH
+
OH
OH
O
O
100
50
0
Aryl
O
O
O
8
9
O
O
O
OH
OH
OH
OH
OH
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
HO
8
9
10
11
12
O
NH2
O
NH2
O
O
O
Cl
OH
OH
OH
OH
OH
N
H
Br
Cl
13
14
15
16
17
NH2
O
F
O
OMe
Cl
O
O
O
Br
OH
OH
OH
OH
OH
H2N
18
19
20
21
22
H
Heteroaryl
O
H
N
O
HO
N
H
HO
O
OH
N
OH
O
H
N
N
O
HO
O
24
25
26
27
23
O
O
Aliphatic
OH
OH
OH
O
N
OH
OH
O
N
N
S
O
O
28
29
30
31
32
O
O
O
OH
O
OH
OH
OH
O
OH
O
F3C
33
34
35
36
37
Chiral aliphatic
O
O
O
O
OH
38
OH
39
OH
OH
OH
40
41
O
OH
O
H3CO
O
O
H
H
OH
OH
OH
OH
O
N
OH
H
O
42
43
44
45
46
O
Fig. 3 | Carboxylic acid substrate scope. a, Diverse structures of carboxylic
acid (donor) substrates assayed with L-Ile and CfaL enzymes. b, Percentage
conversion for ligation of carboxylic acids 8–46 with L-Ile catalysed by CfaL
enzymes (column headings). Assays were carried out with wild-type and
engineered CfaLenzymes (25 μM), carboxylic acids 8–46 (2 mM) and L-Ile
(5 mM). Conversion to amide products was determined by HPLC analysis
following 20 h incubation. Actual conversion values and errors can be found in
Extended Data Table 2.
thermally stable and frequently precipitated during the reaction time- of carboxylic acids and some amino acid substrates (Figs. 3 and 4,
scale (Extended Data Fig. 1). On the basis of our crystallographic studies, Extended Data Tables 2–4). An X-ray crystal structure of this mutant was
we sought to improve the activity and stability of PbCfaL via rational, determined, which revealed no overall structural changes (Extended
structure-guided mutagenesis. Sequence comparison between the Data Fig. 2a). However, the melting temperature (Tm) of PbCfaL(R395G)
four CfaL enzymes (Supplementary Fig. 16) identified few obvious increased by 5ꢀ°C relative to the wild type, suggesting that the replace-
distinctions. However, one noticeable difference was found on the ment of this solvent-accessible, charged R395 is beneficial for stability
flexible hinge-region linking the N- and C-terminal domains (at posi- (Extended Data Fig. 1).
tion 395, Extended Data Fig. 2a). This position is solvent-exposed and
A subsequent double mutant, PbCfaL(R395G/A294P), showed a fur-
likely to be involved in the conformational changes required to shift ther increased Tm and slightly improved activity (Figs. 3 and 4, Extended
between the adenylation (open) and amidation (closed) states of CfaL. Data Table 2–4). The location of this second mutation is within a highly
The large, charged arginine residue that is located in this position of conserved ATP binding loop (G289–L297) that is significantly larger in
PbCfaL is orientated out from the enzyme, while the same position in PbCfaLthan in other related structures, and which may partially occlude
the other CfaLs and ANLs included in the sequence alignment (Sup- the ATP binding site (Extended Data Fig. 2b). The proline found at this
plementary Fig. 16) is occupied by a small and uncharged glycine. A location in SsCfaL, PbCfaLand several other structurally similar ligases
PbCfaL(R395G) mutant showed increased activity against the panel may aid in rotating this loop out of the binding site (Supplementary
394 | Nature | Vol 593 | 20 May 2021

a
b
c
O
O
CfaL
H
H2N
N
OH
+
OH
OH
R
O
R
9
O
100
80
60
40
20
0
O
O
O
L
-a-Ile
L
-Ile
H2N
O
OH
OH
H2N
OH
D
-a-Ile
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
D-Ile
NH2
NH2
NH2
47
49
48
L
-Val
-Leu
-Met
L
O
O
O
OH
L
OH
S
OH
NH2
NH2
NH2
O
52
L-Ala
51
50
L
-Thr
O
O
O
L
-Cys
OH
OH
OH
O
OH
D
-Cys
NH2
NH2
NH2
NH2
55
56
53
54
L
-Ser
O
O
L
-Asn
OH
OH
OH
L
-Gln
-Trp
-Tyr
-Phe
-Arg
NH2
NH2
NH2
57
58
59
L
O
L
O
OH
L
OH
NH2
O
61
NH2
60
L
L
-His
-Lys
-Asp
d
62 (standard)
RP-HPLC
O
L
H2N
OH
63 (standard)
O
L
H
N
O
NH
OH
L
-Glu
SsCfaL 47 + m-methylbenzoate
NH2
62
O
63
Gly
0.50
0.75
1.00
t (min)
1.25
1.50
L
-Pro
Fig. 4 | Amino acid substrate scope. a, Percentage conversion for ligation of
enzymatic reaction (bottom trace) shows selective acylation of the α-amino
group to give amide 62. All assays were carried out with wild-type and
engineered CfaLenzymes (5 μM), carboxylic acid 9 (1 mM) and amino acids
(2 mM). Conversion to amide products was determined by RP-HPLC analysis
following 20 h incubation. Actual conversion values and errors can be found in
Extended Data Tables 3 and 4.
carboxylic acid 9 with acceptor proteinogenic amino acids (rows). b, Percentage
conversion for ligation of 9 with acceptor non-proteinogenic amino acids.
a-Ile = allo-isoleucine. c, Structures of non-proteinogenic amino acids.
d, Reversed-phase (RP)-HPLC trace of ligation product of L-Dab 47 (green) and 9
(m-methylbenzoate, red) catalysed by SsCfaL, compared to HPLC traces of
synthesized standards of the two possible products 62 and 63. Product of the
Fig. 16). Using these two PbCfaLmutants, which no longer precipitate (PolyP) kinase enzyme (CHU)38 and an inexpensive PolyP phosphate
during the reaction, we were able to substantially improve the conver- donor (Extended Data Fig. 3). Although the isolated yield was lower
sions of both the panel of carboxylic acid and amino acid substrates in this case (52%), there is further scope for optimization. While CfaL
(Figs. 3 and 4), demonstrating that minimal structure-guided mutagen- cell lysate shows good activity for up to 12 h, we sought to improve
esis can be used to engineer improved CfaL variants.
enzyme stability/longevity through immobilization of CfaLin the form
of a cross-linked enzyme aggregate (CLEA)39. PbCfaL(R395G/A294P)
CLEAs were shown to retain activity over an extended period of five
days, and could be isolated and recycled in five sequential ligation reac-
Synthetic applications of CfaL
To demonstrate the synthetic utility of CfaL, we sought to establish tions (Extended Data Fig. 4a). Purified PbCfaL(R395G/A294P) was also
preparative-scale ligation reactions. Accordingly, conditions were shown to tolerate several solvents, including MeOH, ethylene glycol and
optimized for the ligation of carboxylic acid 10 and L-Ile (Fig. 5a). Reac- the widely used ‘green’ solvent 2-methylTHF (Extended Data Fig. 4b).
tion of 10 (at 15 mM concentration) with PbCfaL(R395G/A294P) cell free
To further demonstrate the synthetic potential of CfaL, a series of
lysate afforded amide 64 in near quantitative conversion as determined pharmaceutical-relevant scaffolds were prepared in excellent yields
by high-performance liquid chromatography (HPLC). The reaction (Fig. 5a, b and Supplementary Fig. 18). For example, amides 65 and
mixture was subjected to a simple solvent extraction, providing 1.48 g of 67 were prepared in >70% isolated yields at around 100 mg scale. Fur-
crude 64 from 400 ml of reaction mixture, which would be sufficiently thermore, ligations of cinnamic acid 33 and indole carboxylic acids
pure (>92% purity by NMR, Extended Data Fig. 3) for further synthetic 26 (Fig. 3) with cyclopropyl amino acids 56 and L-Leu, respectively
derivatization. Purification of the extract by column chromatography (Fig. 4), produced amides 68 and 69, which are precursors for the
provided 1.37 g of pure 64 in 87% isolated yield (Fig. 5a). To avoid the use manufacture of promising SARS-CoV-2 protease inhibitors, including
of stoichiometric quantities of the expensive co-factor ATP, we repeated PF-07304814 (Pfizer; in phase I clinical trials) (Fig. 5a, b and Supplemen-
the ligation of 10 and L-Ile at the same scale, omitting ATP and instead tary Fig. 18)40,41. Similarly, ligation of the thiazole carboxylic acid 29 and
introducing an ATP recycling system consisting of a polyphosphate O-methyl-L-serine 49 provided amide 70; this is a key component of
Nature | Vol 593 | 20 May 2021 | 395

Article
a
Conversionc (%)
Amide synthesis
b
O
O
Enzyme
65
66
67
68
69
70
CfaL
OH
H
N
H2N
OH
+
OH
SsCfaL
AlCfaL
PbCfaL
PbCfaL(R395G)
PbCfaL(R395G/A294P)
98 0.5
75 1.0
93 3.5
99 0.01
99 0.1
32 0.5
40 2.2
72 5.1
88 6.1
99 4.8
42 7.8
7.5 1.4
8.8 0.4
14 0.2
29 0.7
1.1 0.03 0.9 0.1
10 0.3
10 1.0
3.7 0.2
25 0.9
0.1 0.02
0.8 0.3
0.6 0.1
R
O
R
O
O
21 2.9
8.3 0.4
14 2.1
99
0
11 0.04 23 8.1
N
H
CO2H
64 (1.37 g, 87%)a
PbCfaL (R395G/A294P)
O
H
O
N
SARS-CoV-2,
3CL protease inhibitor
O
N
H
CO2H
H
H
N
CO2H
N
H
CO2H
O
NH
O
O
65 (72%)b
H
N
68 (29%)c
AlCfaL
O
SsCfaL (100 mg scale)
67 (78%)b
N
H
N
H
CO2H
N
H
SsCfaL (100 mg scale)
O
O
66 (99%)c
SsCfaL (100 mg scale)
O
OMe
OMe
NH
O
O
O
P
O
N
H
N
S
N
O
O
H
O–
O–
H
H
O
H
H
N
N
N
CO2H
N
CO2H
O
N
H
N
H
N
O
N
H
S
H
O
O
O
O
O
SARS-CoV-2,
O
Phase II
69 (14%)c
PbCfaL(R395G/A294P)
70 (25%)c
AlCfaL
3CL protease inhibitor
PF-07304814 (Pzer)
multiple myeloma
ONX-0912 (oprozomib)
Enantioselectivity of CfaL, (E)d
Resolution acid (donor)
d
c
RP-HPLC
Enzyme
71
72
73
74
75
76
77
CfaL
O
O
(S)
(S)-71
SsCfaL
AlCfaL
PbCfaL
PbCfaL(R395G)
PbCfaL(R395G/A294P)
1.7
0.4
ND
ND
ND
>200
>200
97
45
30
ND
94
ND
ND
ND
2.9
4.4
1.1
1.0
1.0
2.4
2.7
ND
7.0
5.0
ND
ND
ND
2.4
1.8
1.1
0.4
2.0
1.8
2.4
L-ILe
N
CO2H
OH
H
NSAID prodrug
71 (54 1.3%),b
Ibuprofen
41
d.r. = 93:7 0.1, E = 94c
(R)-71
O
O
4.2 4.4 4.6
t (min)
(S)
Resolution amine (acceptor)
(S)
e
Chiral HPLC
N
CO2H
N
CO2H
H
(S)
H
OH
72 (24 1%),b
(R)
73 (47 2.3%),b
O
d.r. = 79:21 0.2, E = 4.4c
d.r. = 80:20 0.2, E = 7.0c
OH
+
77
AlCfaL
PbCfaL (R395G)
H2N
CO2H
9
57
OMe
O
CfaL
ND
O
O
(S)
N
H
CO2H
(S)-77 product
O
N
CO2H
77
H
N
CO2H
(75 0.9%)b
e.r. = 99:1
E > 200c
O
H
OH
ND
(S)
74 (28 3.5%),b
CO2H
76 (59 7.4%),b
75 (39 2.3%),b
N
H
d.r. = 67:33 7.0, E = 2.4c
d.r. = 63:37 1.4, E = 2.4c
PbCfaL(R395G/A294P)
d.r. = 60:40 1.0, E = 1.7c
AlCfaL
PbCfaL(R395G)
SsCfaL
13 14 15 16 17
t (min)
Fig. 5 | Synthetic potential of CfaL enzymes. a, Amides including
The yield reported is based on 9 which equates to a yield of 33% based on 57
(2 equiv. used). Inset, chiral HPLC analysis of the amide product showing a
single enantiomer, (S)-77. Enantiomeric ratio (e.r.) values determined by chiral
HPLC. aIsolated yield preparative-scale synthesis of 64 with PbCfaL(R395G/
A294P) lysate 10 (15 mM), L-Ile (45 mM) and ATP (36 mM) incubated for 24 h.
bIsolated yields of about 100-mg-scale reactions catalysed by SsCfaLcell lysate
with carboxylic acid (5 mM), amine (15 mM) and ATP (15 mM) following 24 h
incubation. cConversions determined from HPLC peak area ratios, following
assays including CfaLenzymes (25 μM), carboxylic acids (2 mM) and amino acid
(6 mM) incubated for 20 h. dE value was calculated from average d.r. or e.r.
values, as described previously43. Percentage conversions and d.r. values
represent means where n = 3, error denotes s.d.
pharmaceutical scaffolds synthesized by CfaL enzymes. b, Comparison of
percentage conversion for CfaL enzymes in the synthesis of 65–70. c, Kinetic
resolution of racemic carboxylic acids (donor). Absolute configuration and
diastereoisomeric ratios (d.r.) were determined by RP-HPLC using synthetic
standards. Inset, the HPLC chromatogram for (S)-71 formed in the kinetic
resolution of racemic ibuprofen (41) with L-Ile and AlCfaL (E = 94).
d, Comparison of the enantioselectivities of the different enzymes. Values of
E = 15–30 are considered moderate–good, E > 30 are excellent43. For
conversions <30% the calculation of E is unreliable, so the values were not
determined (ND). e, Kinetic resolution of racemic amino acid (acceptor) 57
(2 mM) with acid 9 (1 mM) and AlCfaL (5 μM) (E > 200) following 20 h incubation.
oprozomib, which is in phase II clinical trials for treatment of multiple
The potential of CfaL for use in kinetic resolution of racemic syn-
myeloma42. Probing the limits of potential CfaL-reaction scope, we thetic carboxylic acids was also investigated (Fig. 5c, d). Notably, race-
found that the mutant PbCfaL(R395G/A294P) also allowed the gen- mic ibuprofen could be resolved, with excellent enantioselectivity
eration of amide precursors required for the synthesis of the antiviral (E = 94)43 leading to the biologically active (S)-ibuprofen-L-Ile amide
telaprevir and the anti-cancer agent bortezomib (Extended Data Fig. 5). 71. Amide conjugates of ibuprofen and related NSAIDs with amino
These products were only produced in low quantities, but with further acids have been explored extensively for applications as prodrugs and/
engineering it may be possible to synthesize these precursors at higher or hydrogel-based nanomedicine44,45. Five other racemic acids were
levels. Overall, these reactions (Fig. 5) clearly demonstrate how struc- subjected to kinetic resolution, affording amides 72–76, with modest
turally diverse carboxylic acids can be combined with proteinogenic E values (Fig. 5c, d and Supplementary Fig. 19). In the case of amide 73,
and synthetic amino acids to produce pharmaceutically important the mutant PbCfaL(R395G) was superior to any of the wild-type CfaLs,
compounds.
illustrating how protein engineering could be used to achieve more
396 | Nature | Vol 593 | 20 May 2021

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biosynthesis of the important coronatine family of phytotoxins. BLAST
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398 | Nature | Vol 593 | 20 May 2021

Acknowledgements We thank the BBSRC (grants BB/K002341/1 and BB/N023536/1) and
Syngenta for funding. F.W. was supported by the China Scholarship Council (grant no.
201806155100) and L.B. was funded by the Deutsche Forschungsgemeinschaft (DFG, grant BE
7054/1). The Michael Barber Centre for Collaborative Mass Spectrometry provided access to
MS instrumentation. We also thank J. Vincent and N. Mulholland (Syngenta) for helpful
discussions in the early stages of the project, and N. J. Turner (University of Manchester) for
kindly providing the CHU plasmid. We also thank Diamond Light Source for beamtime access
on i03 and i04-1 (proposal mx17773-56 and 76).
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
Author contributions M.W. and J.M. designed experiments; M.W., M.R., F.W., L.B. and D.F.
carried out the experiments and provided additional experiment design; C.L. performed
crystallographic studies. M.W. and J.M. wrote the manuscript. J.M. led the study.
Nucleotide sequences for the mutants generated as part of this study are
available in Supplementary Information. Other nucleotide sequences
for the enzymes used in this study were obtained from GenBank, and
their accession numbers are provided within the paper or in Supple-
mentary Information. The original materials and data that support
the findings of this study are either available within the paper or are
available from the corresponding author upon reasonable request.
Crystallographic coordinates of wild type and mutant PbCfaL have
been deposited in the Protein Data Bank as 7A9I (wild type) and 7A9J
(R395G mutant).
Competing interests The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material available at
https://doi.org/10.1038/s41586-021-03447-w.
Correspondence and requests for materials should be addressed to J.M.
Peer review information Nature thanks Francesca Paradisi and the other, anonymous,
reviewer(s) for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.

Article
Extended Data Fig. 1 | Melting point determination of CfaL enzymes.
Melting point temperatures (Tm) of the CfaLs in this study, obtained using a
fluorescence-based assay conducted in a Bio-Rad CFX Connect qPCR machine.
Higher Tm indicates improved thermal stability. The Tm is calculated as the
lowest point when plotting the negative derivative of RFU (relative
fluorescence units) as a function of temperature (dT), versus the temperature
(degrees Celsius).

Extended Data Fig. 2 | Rational mutagenesis of PbCfaL. a, Structural
comparison between PbCfaL (left) and the mutant PbCfaL(R395G) (right). R395
(circled) of PbCfaL (PDB ID 7A9I) is in the hinge region between the N-terminal
domain (blue) and the flexible C-terminal domain (red). In PbCfaL(R395G) (PDB
ID 7A9J) this large arginine residue is replaced by a much smaller glycine
(circled) that is found in the other members of the CfaL family and many other
similar ANL ligases. The overall structure of this mutant exhibits no other
substantial structural difference from that of the wild type. b, Overlay of
PbCfaL with three published ATP-dependent ligase structures (in ellipse)
showing the conserved ATP binding location. When superimposed, PbCfaL
(PDB ID 7A9I, blue), McbA (AMP bound, PDB ID 6SQ8, red), GrsA (ATP bound,
PDB ID 1AMU, green) and AuaEII (anthranoyl-AMP bound, PDB ID 4WV3, light
brown) show the conserved location of ATP binding. The corresponding loop in
PbCfaL(inset, arrowed) is larger than in the other structures which may affect
ATP binding. The location of this region within the structure of PbCfaL(grey) is
also shown for reference. Structural alignment was performed using Chimera
(version 1.14) MatchMaker.

Article
Extended Data Fig. 3 | Synthesis and NMR spectra of crude product 64.
a, Preparative-scale synthesis of 64 from 10 and L-Ile catalysed by
PbCfaL(R395G/A294P) (lysate), with either the addition of ATP (87% isolated
yield) or recycling the endogenous ATP present in the lysate using the kinase
(CHU) and polyphosphate (polyP) (52% isolated yield). b, 1H-NMR spectrum of
crude product 64. c, 13C-NMR spectrum of crude product 64.

Extended Data Fig. 4 | Catalysed conversion of 10 and L-Ile to give 64.
a, Catalysed by PbCfaL (R395G/A294P) CLEAs. Reactions (100 mM Tris-HCl,
10 mM MgCl2, 10 mM ATP, 1 mM 10, 3 mM L-isoleucine, 50 ml total volume) were
run for 24 h; the CLEA (cross-linked enzyme aggregate) was then removed,
washed, and reintroduced to an identical reaction. While activity was seen to
reduce over the 5 days, the CLEA still retained high levels of productivity even
after 5 recycles over 5 days, whereas cell lysates generally precipitated and lost
all activity within 12 h. Although CfaL undergoes extensive conformational
changes during catalysis, encapsulating it within CLEAs shows the potential of
immobilization to extend the functional lifespan of the CfaL. More
sophisticated immobilization techniques may have the potential to further
retain activity. Conversion values were calculated from HPLC peak area ratios
of product and starting materials, and represent means where n = 5, error bars
denote s.d. b, Catalysed by purified PbCfaL(R395G/A294P), showing
percentage conversions of 10 and L-Ile in the presence of various solvents and
at different concentrations. Conversion values were calculated from HPLC
peak area ratios of product and starting materials and represent means where
n = 3, error bars denote s.d.

Article
Extended Data Fig. 5 | LCMS analysis and extracted-ion chromatograms
(EICs) of fragments of telaprevir and bortezomib synthesized by
towards anti-cancer agent bortezomib via the synthesis of 112 by CfaL(see
reaction at top). The expected product of the reaction was detected by LCMS
(top trace, expected m/z 270.0884, observed 270.0878 [M-H]−). An additional
peak consistent with an L-Phe dipeptide (113) was also detected (bottom trace,
expected m/z 311.1401). This indicates that L-Phe can function as both acyl
donor and amine acceptor. c, The reaction between carboxylic acid substrate
(9), which is a good substrate for the enzyme, and cyclohexylglycine (58) gives
only the desired product (114, top trace, expected m/z 274.1449, observed
274.1460 [M-H]−). No cyclohexylglycine homodimer (dipeptide 111) was
evident in this case, indicating that homocoupling of 58 only takes place when
carboxylic acid (acyl donor) substrates that are not well accepted by CfaLare
used.
PbCfaL(R395G/A294P). a, Proposed route towards the antiviral agent
telaprevir by CfaL (see reaction at top). The expected product of the reaction,
110, was detected by LCMS (top trace, expected m/z 262.1197, observed
262.1193 [M-H]−). Additional peaks consistent with dipeptide 111, formed from
condensation of two cyclohexylglycines, 58, were also detected (bottom trace,
111a and 111b, expected m/z 295.2027, observed 295.2036 [M-H]−). Although
CfaLare highly selective for L-amino acid substrates, the appearance of two
products of the same mass suggests formation of diastereomers, which may be
due to a lack of enantioselectivity in the adenylation step forming the acyl
donor when racemic cyclohexylglycine (58) is used. This indicates that 58 can
function as both a carboxylic acid and an amine donor. b, Proposed route

Extended Data Table 1 | Table of kinetic parameters for adenylation reactions
These reactions were catalysed by SsCfaL and used substrates CFA (1), aromatic variant 7, enantiomers of trans-jasmonic acid 5 ((3R,7R)-5
and (3S,7S)-5) and racemic ( )-5. The JA samples also contain a small amount of cis-epimer. Michaelis–Menten plots can be found in Sup-
plementary Fig. 7. Values represent means, error denotes s.d., with n ≥ 3.

Article
Extended Data Table 2 | Percentage yield of ligation reactions of carboxylic acids
Table shows yields of ligation of carboxylic acids (8–46) with l-Ile to give the corresponding amides (64, 71–76, 78–109) catalysed by CfaLs. Assays were
carried out with wild-type and engineered CfaL enzymes (25 μM), carboxylic acids (8–46) (2 mM) and l-Ile (5 mM). Conversions were almost all calculated
by HPLC or liquid chromatography mass spectrometry (LCMS) using standard curves of synthesized standards following 20 h incubation (see footnote a
for the sole exception). Values represent means, error denotes s.d., with n = 3.
ND, not detected.
aYield of compound 85 was calculated using HPLC peak area ratios of product and starting material.

Extended Data Table 3 | Ligation of 9 to a range of proteinogenic amino acids
Table shows yields of ligation of carboxylic acid 9 to a range of proteinogenic amino acids catalysed by CfaLs. Assays were carried out with wild-type
and engineered CfaL enzymes (5 μM), carboxylic acid 9 (1 mM) and amino acids (2 mM). Conversion of 9 to amide products was calculated using HPLC
peak area ratios of product and starting material following 20 h of incubation. Low level of activity with d-cysteine possibly occurs via native chemical
ligation mechanisms. Values represent means, error denotes s.d., with n = 3. ND, not detected.

Article
Extended Data Table 4 | Ligation of 9 to a range of non-proteinogenic amino acids
Table shows yields of ligation of carboxylic acid 9 to a range of non-proteinogenic amino acids catalysed by CfaLs. Assays were carried out with
wild-type and engineered CfaL enzymes (5 μM), carboxylic acid 9 (1 mM) and amino acids (2 mM). Conversions of 9 to amide products were calculated
using HPLC peak area ratios of product and starting material following 20 h of incubation. Values represent means, error denotes s.d., with n = 3. ND,
not detected.