C-S bond cleavage by a polyketide synthase domain

Article abstract of DOI:10.1073/pnas.1508437112

Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2- dithiolane moiety is essential for LNM's antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using L-cysteine and L-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing L-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.

Full text of DOI:10.1073/pnas.1508437112

C-S bond cleavage by a polyketide synthase domain
Ming Maa,1, Jeremy R. Lohmana,1, Tao Liub,1, and Ben Shena,b,c,d,2
aDepartment of Chemistry, The Scripps Research Institute, Jupiter, FL 33458; bDivision of Pharmaceutical Sciences, University of Wisconsin-Madison,
Madison, WI 53705; cDepartment of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458; and dNatural Products Library Initiative at
The Scripps Research Institute, The Scripps Research Institute, Jupiter, FL 33458
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved July 15, 2015 (received for review April 29, 2015)
Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featur-
ing an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to
a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-
dithiolane moiety is essential for LNM’s antitumor activity, by virtue
of its ability to generate an episulfonium ion intermediate capable
of alkylating DNA. We have previously cloned and sequenced the
lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and
in vitro characterizations of the LNM biosynthetic machinery have
since established that: (i) the 18-membered macrolactam backbone
is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl
branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF,
and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the
nascent product of the LNM hybrid nonribosomal peptide synthe-
tase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS).
Sulfur incorporation at C-3 of LNM E1, however, has not been
addressed. Here we report that: (i) the bioinformatics analysis reveals
a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine
lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the
LnmJ-SH domain catalyzes C-S bond cleavage by using L-cysteine and
L-cysteine S-modified analogs as substrates through a PLP-dependent
β-elimination reaction, establishing L-cysteine as the origin of sulfur at
C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence ho-
mology with any other enzymes catalyzing C-S bond cleavage, rep-
resents a new family of PKS domains that expands the chemistry and
enzymology of PKSs and might be exploited to incorporate sulfur
into polyketide natural products by PKS engineering.
peptide-polyketide macrolactam backbone is synthesized by a hy-
brid NRPS-acyltransferase (AT)-less type I polyketide synthase
(PKS), consisting of LnmQ (adenylation protein), LnmP (peptide
carrier protein), LnmI (a hybrid NRPS-AT-less type I PKS), LnmJ
(PKS), and LnmG (AT) (21, 23, 24); (ii) the alkyl branch at C-3 of
LNM is installed by a novel pathway for β-alkylation in polyketide
biosynthesis, featuring LnmK (AT/decarboxylase), LnmL (ACP),
LnmM (hydroxymethylglutaryl-CoA synthase), and LnmF (enoyl-
CoA hydratase) that act on the growing polyketide intermediate
tethered to the ACP domain of PKS module-8 of LnmJ (25–27);
and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is
the nascent product of the LNM hybrid NRPS-AT-less type I PKS
(28) (Fig. 1). Although sulfur incorporation into the 1,3-dioxo-1,2-
dithiolane moiety of LNM has not been addressed, the structures
of biosynthetic intermediates and shunt metabolites accumulated
in the SB3029 (ΔlnmK), SB3030 (ΔlnmL), and SB3031 (ΔlnmM)
mutant strains suggest that the β-alkyl branch at C-3 is installed
before the sulfur incorporation, within the PKS module-8 of LnmJ
(26), and the structure of LNM E1 further suggests that one sulfur
at C-3 is introduced after the β-alkyl branch formation, but before
the linear polyketide is released from the PKS module-8 of LnmJ by
the thioesterase (TE)-catalyzed cyclization (28). Therefore, the re-
gion of approximately 850 aa between the ACP and TE domains in
PKS module-8 of LnmJ represent potential candidates responsible
for the sulfur incorporation at C-3 of LNM E1 and LNM (Fig. 1).
We here report that: (i) the bioinformatics analysis reveals a
PLP-dependent domain, we termed cysteine lyase (SH) domain
(LnmJ-SH), together with a domain of unknown function (DUF),
located between the ACP and TE domains in PKS module-8 of
LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by
cysteine metabolism enzyme mechanism genome mining
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pathway engineering sulfur metabolism
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ulfur is found in many primary metabolites, such as thiamin,
biotin, molybdenum cofactors, lipoic acid, iron-sulfur clus-
S
Significance
ters, and nucleosides including 2-thiocytidine, 4-thiouridine, and
2-methylthio-N6-isopentenyl adenosine (1). There is a wealth of
information on how sulfur is incorporated into these primary
metabolites, the key step of which is catalyzed by a desulfurase to
produce a protein-bound cysteine persulfide by using cysteine as
a substrate (2–5). Sulfur is also found in many secondary me-
tabolites (interchangeably referred to as natural products here).
The major groups of sulfur-containing natural products are
peptides produced by ribosome or nonribosomal peptide syn-
thetases (NRPSs). Sulfur incorporation into these natural prod-
ucts from intact cysteine or methionine is well characterized.
However, sulfur incorporation into other natural products remains
poorly understood. Only a few cases featured by C-S bond for-
mation or C-S bond cleavage steps were reported to date, and
most of the mechanisms require further investigation (6–15).
Leinamycin (LNM) is a sulfur-containing antitumor antibiotic
produced by Streptomyces atroolivaceus S-140 (16). The structure
of LNM is characterized by an unusual 1,3-dioxo-1,2-dithiolane
moiety that is spiro-fused to a thiazole-containing 18-membered
lactam ring (Fig. 1). The 1,3-dioxo-1,2-dithiolane moiety is essen-
tial for LNM’s antitumor activity by virtue of its ability to alkylate
the N7 position of the deoxyguanosine of DNA through an epis-
ulfonium ion intermediate (17–19). We have previously cloned and
sequenced the lnm gene cluster from S. atroolivaceus S-140 (20–
22). In vivo and in vitro characterizations of the LNM biosynthetic
machinery have since established that: (i) the 18-membered hybrid
Sulfur incorporation into natural products remains poorly un-
derstood except for those derived from intact cysteine or me-
thionine. Leinamycin (LNM) is a sulfur-containing antitumor
antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety.
A pyridoxal phosphate-dependent domain, termed cysteine ly-
ase (SH) domain, is identified within the LNM polyketide syn-
thase (PKS) module-8 of LnmJ. The LnmJ-SH domain catalyzes
C-S bond cleavage by using L-cysteine and L-cysteine S-modified
analogs as substrates, shares no sequence homology with any
other enzymes catalyzing C-S bond cleavage, and represents a
new family of PKS domains. This study establishes L-cysteine as
the origin of the C-3 sulfur of LNM, expands the chemistry and
enzymology of PKS, and sets the stage to incorporate sulfur into
polyketide natural products by PKS engineering.
Author contributions: B.S. designed research; M.M., J.R.L., and T.L. performed research;
M.M., J.R.L., T.L., and B.S. analyzed data; and M.M., J.R.L., and B.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The amino acid sequences reported in this paper have been deposited in
the NCBI database. For a list of accession numbers, see SI Appendix.
1M.M., J.R.L., and T.L. contributed equally to this work.
2To whom correspondence should be addressed. Email: shenb@scripps.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1508437112/-/DCSupplemental.
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Fig. 1. Proposed biosynthetic pathway for LNM featuring (i) the LnmQPIJ hybrid NRPS-AT–less type I PKS with the discrete LnmG AT loading the malonyl
CoA extender units to all six PKS modules (21, 23, 24), (ii) the LnmKLMF enzymes catalyzing introduction of the β-alkyl branch at C-3 (25-27), (iii) the DUF
and SH domains of PKS module-8 of LnmJ catalyzing sulfur incorporation into C-3 of LNM from L-cysteine characterized in this study, and (iv) LNM E1 as the
nascent product of the LNM hybrid NRPS-AT-lee type I PKS (28), setting the stage to investigate the tailoring steps that convert LNM E1 to LNM. Color
coding indicates the moieties installed by NRPS (blue), PKS (red), β-alkyl branch (green), and other tailing enzyme (black), and the green ovals denote AT
docking domains. A, adenylation domain; AT, acyltransferase; Cy, condensation/cyclization; DH, dehydratase; DUF, domain of unknown function; KR,
ketoreductase; KS, ketosynthase; MT, methyltransferase; Ox, oxidation; PCP, peptidyl carrier protein; SAM, S-adenosylmethionine; SH, PLP-dependent
cysteine lyase domain; TE, thioesterase.
using L-cysteine and L-cysteine S-modified analogs as substrates
through a PLP-dependent β-elimination reaction, establishing The region between the DUF and TE, we now termed the SH
L-cysteine as the origin of sulfur at C-3 of LNM and LNM E1; and
domain, shares similarity with the family of aromatic amino acid
acyltransferase superfamily [InterProScan (30); E value 2.2 × 10−6].
(iii) the LnmJ-SH domain, sharing no sequence homology with β-eliminating lyases (ABLs) (residues 6689–6964, PFAM E value
any other enzymes catalyzing C-S bond cleavage in natural prod- 2.3 × 10−48). BLAST searches of the LnmJ-SH domain revealed
homology to tyrosine phenol lyases (TPL) and tryptophan indole
lyases (TIL), canonical members of the ABL family (Fig. 2). TPL
and TIL are PLP-dependent enzymes that catalyze Cβ-Cγ bond
cleavage of tyrosine and tryptophan to phenol and indole, re-
spectively (31) (SI Appendix, Fig. S1). Sequence alignments and
molecular modeling of the selected ABLs and LnmJ-SH domain
indicate conservation of all residues involved in PLP and amino
acid substrate carboxylate binding (SI Appendix, Fig. S2).
BLAST searches also revealed homologs of LnmJ-SH (E value
between 7.0 × 10−66 and 2.0 × 10−107) within PKSs from various
organisms, none of which, however, had previously been function-
ally annotated. The SH domain is always located immediately
C-terminal to a DUF (Figs. 2 and 3A and SI Appendix, Fig. S3).
Organisms other than S. atroolivaceus S-140 that possess PKS
modules with DUF-SH domain architecture include Actino-
uct biosynthesis, represents a new family of PKS domains that
expands the chemistry and enzymology of PKSs.
Results
Bioinformatics Analysis of the PKS Module-8 of LnmJ Revealing Novel
Domains. Inspired by the proposal that sulfur incorporation at
C-3 takes place after β-alkyl branch formation and before release
of the mature hybrid peptide-polyketide intermediate from the
ACP domain by TE-catalyzed cyclization to afford LNM E1, we
reexamined the PKS module-8 of LnmJ. We previously noted
the atypical architecture of PKS module-8 of LnmJ but were
unable to assign function to the unknown domains (21, 22, 28).
Close examination of PKS module-8 of LnmJ revealed two ad-
ditional domains between ACP and TE, previously not known
to be associated with PKSs (Fig. 1). C-terminal to the ACP is a
DUF [DUF2156 as annotated by PFAM (29), residues 6299– kineospora sp. EG49, Aquimarina muelleri, Burkholderia pseudomallei
6563, E value 2.9 × 10−18], with weak homology to the acyl-CoA
(various subspecies), Catenulispora acidiphilia DSM 44928, Fulvivirga
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Fig. 2. The phylogenetic analysis for LnmJ-SH in comparison with selected members of ABLs and selected enzymes catalyzing C-S bond cleavage from natural
product biosynthetic pathways. LnmJ-SH forms a distinct clade with functionally uncharacterized homologs, as exemplified by CaJ-SH and MaJ-SH, from
various organisms (cyan). Selected enzymes catalyzing C-S bond cleavage from both primary metabolism, as exemplified by the cysteine desulfurases, cysteine
desulfhydrases, and cysteine S-conjugate β-lyases, and natural product biosynthetic pathways, as exemplified by cysteine sulfoxide lyases, methionine γ-lyases,
and SUR1, fall into another distinct clade (purple). Members of the ABLs form the third clade (orange) lying between the two clades mentioned above. See SI
Appendix for accession numbers of the selected enzymes or domains used in this phylogenetic analysis.
imtechensis, Kordia algicida OT-1, Lysobacter sp. ATCC 53042,
Micromonospora aurantiaca ATCC 27029, Micromonospora sp. L5,
Pseudoqulbenkiania ferrooxidans, Saccharothrix espanaensis, Salinis-
pora arenicola, Sporocytophaga myxococcoides, and Streptomyces
leeuwenhoekii (Fig. 2). None of these organisms, however, is known
to produce LNM or analogs.
no product formation was observed from the negative controls
using boiled LnmJ-SH (SI Appendix, Fig. S21). Compound 16 was
purified from repeated reactions and analyzed by NMR and high-
resolution electrospray mass spectrometry (HR-ESI-MS) (SI
Appendix). The HR-ESI-MS analysis of 16 afforded the [M + H]+
ion at m/z 236.0776 (calculated [M + H]+ ion for C9H17NO2S2
at m/z 236.0778) and [M + Na]+ ion at m/z 258.0596 (calculated
[M + Na]+ ion for C9H17NO2S2 at m/z 258.0598) (SI Appendix,
The PLP-Dependent C-S Bond Cleavage Activity of LnmJ-SH Domain.
We successfully cloned and expressed the lnmJ-SH fragment in
Escherichia coli and purified the LnmJ-SH domain to homoge-
neity (Fig. 3B). The purified LnmJ-SH was yellow in color; dis-
played UV absorption peak at 420 nm, indicative of bound PLP;
and eluted as a homotetramer upon size-exclusion chromatogra-
phy (SI Appendix). Given the sequence homology of LnmJ-SH to
TPL and TIL (SI Appendix, Fig. S2), we tested the cleavage ac-
tivities of LnmJ-SH with L-tyrosine, L-tryptophan, D- and L-cyste-
ine, and L-cysteine S-modified analogs (Fig. 3 A and C and SI
Appendix, Fig. S1). LnmJ-SH was active with L-cysteine and all of
the L-cysteine S-modified analogs tested, but not with L-tyrosine,
L-tryptophan, and D-cysteine, as monitored by production of
pyruvate [as methylquinoxalinol after derivatization by o-phenyl-
enediamine (OPD); SI Appendix, Fig. S4]. The reactions with
L-cysteine (1), L-cystine (2), and S-phenyl-L-cysteine (4) were fur-
ther verified by detection of hydrogen sulfide (SI Appendix, Fig.
S5), thiocysteine (SI Appendix, Fig. S6), and thiophenol (SI Ap-
pendix, Fig. S7), respectively.
1
Fig. S22). The H NMR spectrum of 16 gave the resonances at-
tributed to two coupled methylene groups at δH 3.36 (t, J = 6.7 Hz,
H-3) and δH 3.04 (t, J = 6.7 Hz, H-4), one methylene group at
δH 2.94 (s, H-6), one methyl group at δH 1.94 (s, H-1) and two
overlapped methyl groups at δH 1.49 (s, H-8 and H-9). The two
coupled methylene groups at δH 3.36 (t, J = 6.7 Hz, H-3) and δH
3.04 (t, J = 6.7 Hz, H-4), and the methyl group at δH 1.94 (s, H-1),
together with the resonances at δC 21.1 (C-1), δC 172.0 (C-2), δC
38.7 (C-3), and δC 28.0 (C-4) in the 13C NMR spectrum, suggested
there was a S-substituted N-acetylcysteamine moiety in 16. The
N-acetylcysteamine moiety was confirmed by the correlations of
H-1/C-2, H-3/C-2, H-3/C-4, and H-4/C-3 in the HMBC spectrum.
The HMBC spectrum also showed the correlations of H-4/C-5,
H-6/C-5, H-8 (9)/C-6, H-8 (9)/C-7, and H-6/C-8 (9), establishing
the thioester linkage between an N-acetylcysteamine and a
3-substituted isovaleric acid. The substitution at C-3 of the iso-
valeric acid was established as a thiol group by the molecular
formula of C9H17NO2S2. Thus, 16 was unambiguously identified
as the C-S bond cleavage product of 9 (Fig. 3D and SI Appendix,
Figs. S23–S27). The identity of 17 as the C-S bond cleavage
product of 10 was similarly established (Fig. 3D), based primarily
on the HR-ESI-MS analysis that afforded the [M + H]+ ion at m/z
395.1670 (calculated [M + H]+ ion for C16H30N2O5S2 at m/z
395.1673) and [M + Na]+ ion at m/z 417.1490 (calculated [M + Na]+
ion for C16H30N2O5S2 at m/z 417.1493) (SI Appendix, Fig. S28).
LnmJ-SH Catalyzing C-S Bond Cleavage of Cysteine-Adduct Polyketide
Substrate Mimics. To determine whether LnmJ-SH acts on the
ACP-tethered polyketide intermediate (Fig. 1), we next synthe-
sized the N-acetylcysteamine and pantetheine thioesters of the
L-cysteine-polyketide adduct, 9 and 10, as substrate mimics (Fig.
3D and SI Appendix, Figs. S8–S20). Upon incubation with LnmJ-
SH, 9 and 10 afforded products 16 and 17, respectively, whereas
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activities, with kcat/Km values of 1.6 0.3 and 1.5 0.3 mM-1min−1
,
Kinetic Analysis of LnmJ-SH with L-Cysteine and L-Cysteine S-Modified
Analogs as Substrates. Optimization of the LnmJ-SH catalyzed C-S
bond cleavage reaction revealed maximal activity in 0.1 mM PLP,
20 mM KCl, and a pH of 8.5 when buffered with Tris·HCl (SI
Appendix, Fig. S29). The pseudo-first–order kinetic parameters of
LnmJ-SH toward L-cysteine (1) and L-cysteine S-modified analogs,
including L-cystine (2), S-t-butylmercapto-L-cysteine (3), S-phenyl-
L-cysteine (4), S-benzyl-L-cysteine (5), S-t-butyl-L-cysteine (6),
S-ethyl-L-cysteine (7), S-methyl-L-cysteine (8), and the polyketide
intermediate mimics 9 and 10 (Fig. 3), were determined by
monitoring pyruvate production (Table 1 and SI Appendix, Fig.
S4). The Km or kcat values for all substrates, except 1, 9, and 10,
could not be determined because of their low solubility at higher
concentrations in the reaction buffer. However, their kcat/Km val-
ues can be obtained based on the data obtained in the low con-
centration range. The Km and kcat values for 9 and 10 were
determined at the pH of 7.2 because of significant nonenzymatic
reverse β-addition of 9 and 10, resulting in the loss of L-cysteine, at
pH 8.5 (SI Appendix, Fig. S21). The Km and kcat values for 1 and 4
at pH of 7.2 were also determined for comparison (Table 1).
Among all of the substrates tested, 3 and 4 exhibited the highest
respectively. The catalytic efficiencies of LnmJ-SH toward 1 and 4
decreased as the pH was lowered from 8.5 to 7.2 (Table 1). At pH
of 7.2, the kcat/Km values for 1, 4, 9, and 10 ranged from (23 5) ×
10−3 to 0.38 0.01 mM-1·min−1, indicating substrate tolerance of
LnmJ-SH (SI Appendix, Fig. S30).
PLP-Dependent C-S Bond Cleavage Activities of the LnmJ-SH Domain
Homologs. To examine whether other LnmJ-SH homologs could
catalyze the similar C-S bond cleavage, we overproduced in E. coli
and purified two selected LnmJ-SH homologs, CaJ-SH from
C. acidiphilia DSM 44928 and MaJ-SH from M. aurantiaca ATCC
27029, to homogeneity (Fig. 3). Although not known as LNM
or analog producers, bioinformatics analysis revealed that
both strains contain a biosynthetic gene cluster homologous to the
lnm gene clusters (SI Appendix, Fig. S3). The purified CaJ-SH and
MaJ-SH were both yellow in color, indicative of bound PLP, and
catalyzed C-S bond cleavage by using all of the substrates tested
for LnmJ-SH (Fig. 3 and Table 1). Both enzymes also exhibited
the highest catalytic efficiency toward 4. Compared with the same
substrate, the catalytic efficiencies for the three enzymes in gen-
eral increase noticeably (∼40-fold) in the order of LnmJ-SH <
CaJ-SH < MaJ-SH (Table 1). However, for 9 and 10, the three SH
domains exhibited similar kcat/Km values ranging from (23 5) ×
10−3 to (51 8) × 10−3 mM-1·min−1 (i.e., ∼twofold). Among the
three SH domains, MaJ-SH appeared to be most sensitive to pH,
displaying a sharp decrease in kcat, as exemplified for 1 from 106
6 min−1 at pH 8.5 to 6.7 0.2 min−1 at pH 7.2 (∼16-fold). The Km
and kcat values of MaJ-SH for 4 could be determined at pH 7.2,
benefited from the reduced kcat value at the lower pH (Table 1
and SI Appendix, Fig. S30).
Discussion
LnmJ-SH and Homologs Are Distinct from All Sulfur-Incorporating
Enzymes Known to Date. Sulfur-containing metabolites are well
known from both primary and secondary metabolism (1–15). The
best-characterized sulfur-incorporating enzymes from primary
metabolism include the following: (i) the group I and II cysteine
desulfurases that use L-cysteine as the substrate and catalyze the
production of a protein-bound persulfide and alanine, as exem-
plified by NifS, IscC, and CsdB (1–3); (ii) the D-cysteine desulf-
hydrases that use D-cysteine as the substrate and catalyze the
β-elimination reaction to produce hydrogen sulfide, ammonia, and
pyruvate (32); and (iii) the cysteine S-conjugate β-lyases that use
L-cysteine-S-conjugates as the substrates and catalyze the β-elim-
ination reaction to produce a thiol, ammonia, and pyruvate (33).
They are all PLP-dependent enzymes that share significant
sequence homology and fall into a large clade upon phylogenetic
analysis (Fig. 2).
Sulfur incorporation into secondary metabolites is less well
characterized except for those peptide natural products derived
from intact incorporation of cysteine and methionine by either
ribosomal or NRPS mechanism. Recent literature has witnessed
a flurry of reports on natural product biosynthesis featuring C-S
bond formation or C-S bond cleavage steps (6–15). Thus, as sum-
marized in SI Appendix, Fig. S31, in the biosynthesis of lincomycin A,
LmbT catalyzes the C-S bond formation between GDP-D-α-
D-lincosamide and ergothioneine (EGT), and LmbV then catalyzes
the sulfur exchange between the resultant EGT S-conjugate and
mycothiol (MSH) to generate the new C-S bond (6). In the bio-
synthesis of BE-7585A, the C-S bond formation in the 2-thioglucose
moiety is catalyzed by BexX with the sulfur provided by sulfur
carrier protein and activating protein involved in primary metabolite
biosynthesis (7). The C-S bond formation in ergothioneine and
ovothiol biosynthesis is catalyzed by EgtB using γ-glutamylcysteine
as the sulfur donor and by OvoA using cysteine as the sulfur donor,
respectively (8). The C-S bond formation in gliotoxin biosynthesis is
catalyzed by GliG using glutathione as the sulfur donor, and the
Fig. 3. LnmJ-SH, CaJ-SH, and MaJ-SH as a new family of PKS domains that
catalyzes C-S bond cleavage. (A) Domain organization and architectural
conservation of the PKS modules featuring the DUF and SH domains
reported in this study. (B) SDS/PAGE (12%) of the purified SH domains
(calculated molecular mass, including the extra amino acids from expres-
sion vector, in daltons): LnmJ-SH (49,609.6), CaJ-SH (49,699.9), and MaJ-SH
(48,539.8). (C) LnmJ-SH, CaJ-SH, and MaJ-SH catalyze C-S bond cleavage
using L-cysteine (1) and L-cysteine S-modified analogs (2–8) as substrates.
(D) LnmJ-SH, CaJ-SH, and MaJ-SH catalyze C-S bond cleavage using
N-acetylcysteamine and pantetheine thioester of the L-cysteine-polyketide
adducts 9 and 10 as substrate mimics of the ACP-tethered growing polyketide
intermediate in LNM biosynthesis, affording the corresponding products 16
and 17 featuring a thiol moiety at C-3 (Fig. 1).
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Table 1. Pseudo-first–order kinetic parameters of LnmJ-SH, CaJ-SH, and MaJ-SH toward selected substrates
LnmJ-SH Ca-SH
Substrates* Km, mM kcat, min−1 kcat/Km, mM−1·min−1 Km, mM kcat, min−1 kcat/Km, mM−1·min−1 Km, mM kcat, min−1 kcat/Km, mM−1·min−1
Ma-SH
1
2
3
4
35
5
2.4 0.1
0.07 0.01
0.22 0.01
1.6 0.3
40
5
6.0 0.3
0.15 0.02
1.12 0.07
2.15 0.02
42
5
106
6
2.5 0.3
13
27
68
1
3
3
1.5 0.3
4.53 0.07
5
6
7
8
0.20 0.01
0.26 0.02
0.63 0.07
0.381 0.001
0.137 0.002
(48 1) × 10−3
1.1 0.1
(23 1) × 10−3
(14 1) × 10−3
(6.1 0.2) × 10−3
(63 1) × 10−3
0.38 0.01
(69 3) × 10−3
0.416 0.002
0.22 0.01
1†
4†
9†
10†
13
2
0.82 0.04
18
4
5.2 0.4
0.29 0.01
6.3 0.8 6.7 0.2
1.9 0.2 8.7 0.3
0.93 0.02
4.6 0.5
32
30
6
3
0.75 0.06
0.72 0.03
(23 5) × 10−3
(24 3) × 10−3
42
23
6
2
2.2 0.1
0.66 0.02
(51 8) × 10−3
(29 3) × 10−3
33
23
5
4
1.24 0.07
1.07 0.06
(38 6) × 10−3
(47 9) × 10−3
*Assays were carried out in 0.2 mM PLP, 20 mM KCl, 100 mM Tris·HCl, pH 8.5 (SI Appendix).
†Assays were carried out in 0.2 mM PLP, 20 mM KCl, 100 mM Tris·HCl, pH 7.2 (SI Appendix).
subsequent C-S bond cleavage is catalyzed by a pyridoxal phosphate
at significantly reduced catalytic efficiency (Vmax/Km) of 4% (33) and
(PLP)-dependent enzyme, GliI (9). The C-S bond formation in 1% (34), respectively, relative to their natural substrates tyrosine
sulfoquinovosyl diacylglycerol biosynthesis is catalyzed by SQD1
using sulfite as the sulfur donor (10). The C-S bond formation in
grixazone biosynthesis is proposed by a nonenzymatic reaction be-
tween the quinone imine intermediate and N-acetylcysteine (11).
The C-S bond cleavage in glucosinolate biosynthesis in plants is
catalyzed by SUR1, which belongs to the PLP-dependent cysteine
S-conjugate β-lyases (12). The C-S bond cleavage in the biosynthesis
and tryptophan. In contrast, LnmJ-SH, CaJ-SH, and MaJ-SH
showed no activity toward tyrosine and tryptophan under all
conditions examined. The LnmJ-SH family of enzymes therefore
apparently has evolved to be L-cysteine and L-cysteine-S-conju-
gates specific. Interestingly, substrate promiscuity toward L-cyste-
ine S-modified analogs is also known for ABLs (35, 36). For
example, when S-(o-nitrophenyl)-L-cysteine (SOPC) was tested as
of volatile sulfur containing natural products in plants is catalyzed an alternative substrate, both TPL and TIL exhibited higher cat-
by cysteine sulfoxide lyases, as exemplified by alliinases in the genus
Allium (13, 14). Finally, CalE6 from the calicheamicin biosynthetic
gene cluster in Micromonospora echinospora was found to catalyze
C-S bond cleavage in vitro, using methionine as the substrate, to
produce methanethiol, although no evidence was provided to cor-
relate this reaction to sulfur incorporation in calicheamicin bio-
synthesis (15). While the emerging sulfur-incorporating enzymes
from natural product biosynthetic pathways are very diverse, those
that catalyze C-S bond cleavage are all PLP-dependent enzymes,
including SUR1, cysteine sulfoxide lyases, CalE6, and GliI, and fall
into the same clade as the cysteine desulfurases, cysteine desulfhy-
drases, and cysteine S-conjugate β-lyases from primary metabolism
(Fig. 2).
Remarkably, LnmJ-SH shares no sequence homology with any of
the sulfur-incorporating enzymes known to date from either pri-
mary or secondary metabolisms. Instead, LnmJ-SH forms a distinct
clade, including CaJ-SH, MaJ-SH, and many other homologs from
various organisms, none of which was functionally annotated pre-
viously (Fig. 2). Characterization of LnmJ-SH and selected homo-
logs CaJ-SH and MaJ-SH in this study therefore unveils a new
family of PKS domains that catalyze C-S bond cleavage, using
L-cysteine and L-cysteine-S-conjugates as substrates (Fig. 3).
alytic efficiency toward SOPC than their native substrates, pre-
sumably due to o-nitrothiophenol being a better leaving group (35,
36). These results are reminiscent of the findings from the current
study for the SH domains, which exhibited the highest catalytic
efficiency toward S-phenyl-L-cysteine (4) among all of the sub-
strates tested (Table 1). Regardless of the observed substrate
promiscuity, the LnmJ-SH family of new PKS domains catalyzes
C-S bond cleavage and prefers amino acid substrates bearing
minimally a thio-side chain and L-amino group (Figs. 1 and 3).
Using the sequence alignment and structures of TPL and TIL
(PDB ID codes 2VFL, 2TPL, 2EZ1, 2EZ2, and 1AX4), we gen-
erated a structural model of LnmJ-SH (SI Appendix, Fig. S32). TPL
and TIL are catalytic as dimers, which are stabilized by the binding
of two potassium cations, as such, the active site of each monomer
is closed off from solvent by dimerization (31, 37–39). These di-
mers form higher-order tetramers in solution and crystal struc-
tures. The residues responsible for potassium binding and
dimerization are mutated in LnmJ-SH (S6874[K], W6677[G],
A6694[E], A6696[Y], corresponding TPL residue in brackets), al-
though LnmJ-SH retains potassium dependence and a tetrameric
quaternary structure. A few regions of LnmJ-SH, likely responsible
for alteration of function, could be predicted. The loop between
H6994 and P7000 in LnmJ-SH is missing 11 residues, and the
C-terminal β-α-β-motif, responsible for covering the active site and
substrate specificity, is likely truncated in LnmJ-SH starting at
residue L7038. In addition, the region between R6764 and R6774
in LnmJ-SH is lacking three residues. Together, the truncations
and differences in oligomeric interfaces likely allow LnmJ-SH to
accommodate a large substrate such as the growing peptide-poly-
ketide intermediate proposed for LNM biosynthesis (Fig. 1).
LnmJ-SH Representing a New Family of PKS Domains That Catalyze
C-S Bond Cleavage. We have unambiguously established that the
LnmJ-SH family of PKS domains catalyzes C-S bond cleavage, using
L-cysteine and L-cysteine-S-conjugates as substrates (Fig. 3). These
findings expand the chemistry and enzymology of PKSs, and the
LnmJ-SH family of PKS domains might be exploited to incorporate
sulfur into polyketide natural products by PKS engineering.
It should be noted that the LnmJ-SH family of PKS domains
shares significant sequence homology with the ABL family of en-
zymes. All of the residues required for PLP and amino acid substrate
A Proposal for C-3 Sulfur Incorporation in LNM Biosynthesis. In the
proposed biosynthetic pathway for LNM, after introduction of
binding are conserved (SI Appendix, Fig. S3), but the two families the β-alkyl branch at C-3, there is a requirement for β-addition of
fall into two distinct clades phylogenetically (Fig. 2). Members of the a sulfur nucleophile at the C-3 carbon of the α,β-unsaturated
ABL family enzymes, such as TPL and TIL, are known to use cys-
teine as an alternative substrate, catalyzing C-S bond cleavage, albeit
linear polyketide intermediate (Fig. 1). While the characteriza-
tion of LnmJ-SH in this study establishes that the sulfur at C-3 of
Ma et al.
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LNM is ultimately derived from L-cysteine, we have not accounted combination of both (Fig. 1). However, in this proposed pathway,
for how the C-S bond is formed by the β-addition. Recently,
β-additions, with carbon nucleophiles (i.e., Michael addition), to
generate an alkyl branch in the growing polyketide chain have
been observed (40–42). On the basis of the absolutely conserved
KS-ACP-DUF-SH-TE domain architecture of the PKS module-8
of LnmJ and its homologs (Figs. 1 and 3 and SI Appendix, Fig. S3),
we now propose that a β-addition of L-cysteine, with a sulfur nu-
cleophile, to the α,β-unsaturated polyketide intermediate to afford
the cysteine adduct, and LnmJ-DUF serves as the candidate re-
sponsible for the β-addition (Fig. 1). This proposal is supported
by the ability of LnmJ-SH, CaJ-SH, and MaJ-SH to catalyze C-S
bond cleavage of the L-cysteine S-modified analogs, especially 9
and 10, mimics of the proposed biosynthetic intermediate for
LNM E1 (Figs. 1 and 3) with similar kcat/Km values (Table 1). The
observed Km values of LnmJ-SH, CaJ-SH, and MaJ-SH with all
tested substrates (1–10) are between 1.9 0.2 and 42 6 mM
(Table 1), which are too high to be relevant under the physio-
logical reactions. These high Km values may result from the
structural difference between the native substrates of the SH do-
mains and the substrate mimics tested, the fact that the SH do-
mains were studied in isolation (i.e., in truncation lacking the
context of other domains within the native PKS modules), or a
the LnmJ-SH domain would not need tight substrate binding as
the L-cysteine-adduct intermediate would be tethered to the cog-
nate ACP, overcoming a need for low Km values. These hypoth-
eses would also be consistent with the prediction that the active
site of LnmJ-SH is likely more open to solvent than the other
ABL family members in the structural modeling (SI Appendix,
Fig. S32). Further studies including structural elucidation of
LnmJ-SH and functional characterization of LnmJ-DUF are
clearly warranted to define the precise course of events leading
to sulfur incorporation at C-3 in LNM biosynthesis.
Materials and Methods
Materials, methods, and detailed experimental procedures are provided in SI
Appendix. Included in SI Appendix are cloning and expression of LnmJ-SH,
CaJ-SH, and MaJ-SH; detection of pyruvate, hydrogen sulfide, thiocysteine
and thiophenol; chemical synthesis of compound 9 and 10; detection of
compound 16 and 17; optimization of LnmJ-SH reaction; and 1H and 13C NMR
spectra and HR-ESI-MS spectra for compound 9–11 and 15–17.
ACKNOWLEDGMENTS. We thank Kyowa Hakko Kogyo Co. Ltd for the wild-
type S. atroolivaceus S-140 strain and the NMR Core facility at the Scripps
Research Institute in obtaining 1H and 13C NMR data. This work was sup-
ported in part by NIH Grant CA106150.
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