Elucidation of a carboxylate O-methyltransferase NcmP in nocamycin biosynthetic pathway

Article abstract of DOI:10.1016/j.bmcl.2017.08.010

Nocamycins belong to the tetramic acid family natural products and show potent antimicrobial activity. Recently, the biosynthetic gene cluster of nocamycin was identified from the rare actinomycete Saccharothrix syringae and an S-adenosylmethionine (SAM) dependent methyltransferase gene NcmP was found to be located within the gene cluster. In this report, the methyltransferase gene NcmP was disrupted and a new nocamycin intermediate nocamycin E was isolated from the mutant strain. Meanwhile, NcmP was heterologously expressed in Escherichia coli BL21 (DE3) and biochemically characterized as a carboxylate O-methyltransferase in nocamycin biosynthetic pathway. Compared to nocamycin I, nocamycin E showed inferior antibacterial activity, indicating the methyl group is essential to antibacterial activity.

Full text of DOI:10.1016/j.bmcl.2017.08.010

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Elucidation of a carboxylate O-methyltransferase NcmP in nocamycin
biosynthetic pathway
b
a
Xuhua Mo a, , Chun Gui , Qingji Wang
⇑
a Shandong Key Laboratory of Applied Mycology, School of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China
b CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China
Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Rd., Guangzhou 510301, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nocamycins belong to the tetramic acid family natural products and show potent antimicrobial activity.
Recently, the biosynthetic gene cluster of nocamycin was identified from the rare actinomycete
Saccharothrix syringae and an S-adenosylmethionine (SAM) dependent methyltransferase gene NcmP
was found to be located within the gene cluster. In this report, the methyltransferase gene NcmP was dis-
rupted and a new nocamycin intermediate nocamycin E was isolated from the mutant strain. Meanwhile,
NcmP was heterologously expressed in Escherichia coli BL21 (DE3) and biochemically characterized as a
carboxylate O-methyltransferase in nocamycin biosynthetic pathway. Compared to nocamycin I, noca-
mycin E showed inferior antibacterial activity, indicating the methyl group is essential to antibacterial
activity.
Received 3 July 2017
Revised 30 July 2017
Accepted 6 August 2017
Available online xxxx
Keywords:
Nocamycins
Methyltransferase
Post-tailoring modification
Enzyme reaction
Antibacterial activity
Ó 2017 Published by Elsevier Ltd.
The tetramic acid class natural products nocamycin I (1) and II
(2) (Fig. 1) were isolated from the rare actinomycete Saccharothrix
syringae NRRL B-16468 and they showed potent inhibitory activity
toward Gram-positive and Gram-negative bacteria, especially
toward some anaerobic bacteria.1–7 Due to the complex structure,
the total synthesis of nocamycin has not been achieved until now
and the diversification of nocamycin by chemical synthesis was
limited. Thus, biosynthetic pathway engineering provides an alter-
native approach to generate nocamycin analogs by manipulating
biosynthetic gene cluster. Recently, we have identified the noca-
mycin biosynthetic gene cluster that contained 21 open reading
frames (ORFs),8 including five type I polyketide synthases genes
(NcmAI, NcmAII, NcmAIII, NcmAIV and NcmAV), a non-ribosomal
peptide synthetase gene (NcmB), a Dieckmann cyclase gene NcmC,
a short chain dehydrogenase gene (NcmD), two cytochrome P450
oxidase genes (NcmO and NcmG), a glycoside dehydratase gene
(NcmE), a SAM dependent methyltransferase gene NcmP, and five
regulators genes (NcmN, NcmJ, NcmK, NcmI and NcmM). The back-
bone of nocamycin is assembled by PKS-NRPS and the tailoring
enzymes are employed to further modify the structure to yield
the nocamycin I. In our previous report, we have isolated two
new nocamycin derivatives from the cytochrome P450 gene NcmG
disruption mutant strain, which provide hints to the nocamycin
biosynthetic pathway.8 In this work, we report (i) isolation of a
new intermediate nocamycin E from the methyltransferase gene
NcmP disruption mutant strain; (ii) biochemically characterization
of NcmP as a carboxylate O-methyltransferase in nocamycin
biosynthesis pathway; (iii) antibacterial activity evaluation of
nocamycin E towards a panel of bacteria.
Bioinformatics analysis revealed that NcmP is the only methyl-
transferase gene located within the nocamycin biosynthetic gene
cluster, which is accord to the hypothetical nocamyicn biosyn-
thetic pathway. Sequences alignments revealed that NcmP belongs
to SAM dependent methyltransferase family and it shares the three
common conserved motifs distributed in SAM dependent methyl-
transferase family (see Supporting information, Fig. S1).9,10 NcmP
shows identity to NokK (44% identity) involved in K-252a pathway
from Nonomuraea longicatena,11 NivG (43% identity) involved in
nivetetracyclates pathway from Streptomyces sp. Ls2151,12 and
AknG (41% identity) involved in aclacinomycins pathway from
Streptomyces galilaeus.13 Given that all NcmP homologues are pro-
posed to catalyze methyl esterification reaction, NcmP is most
likely to be the candidate involved in the methyl esterification in
nocamycin biosynthetic pathway.
To determine the function of NcmP, we inactivate the NcmP by
replacing partial internal NcmP with aac(3)IV gene cassette through
k-RED recombination technology to generate the double cross
mutant strain S. syringae MoS-1004 (see SI). The double-crossover
mutant was selected by apramycin resistant kanamycin sensitive
⇑
Corresponding author.
E-mail address: xhmo2013@163.com (X. Mo).
http://dx.doi.org/10.1016/j.bmcl.2017.08.010
0960-894X/Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Mo X., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.010

2
X. Mo et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
lated from the
D
NcmG mutant strain,8 we assumed that the methyl
esterification in nocamycin pathway is likely to be a post-tailoring
process. Next, S. syringae MoS-1004 was cultured with other nine
media (see SI). All the fermentation broths were analyzed by HPLC.
Results of HPLC revealed that a new more polar peak with charac-
teristic UV absorption to that of nocamycin was detected in four
different media (Fig. 2).
To determine the structure of the newly produced nocamycin
derivative, 6 L scale fermentation media (1% starch, 1% yeast
extract, 0.3% corn flour, 2% glucose, 0.1% beef extract, 0.05%
K2HPO4, 0.05% MgSO4ꢀ7H2O, 0.2% CaCO3, pH 7.0) was prepared to
culture S. syringae MoS-1004. Ultimately, by using a series of chem-
ical isolation methods, compound 3 (about 10 mg) was purified
and subjected to high resolution mass spectrum (HR-MS) and
NMR analyses.
Compound 3 was isolated as a yellowish amorphous solid. Its
molecular formula was determined as C25H31NO9 on the basis of
ion peaks at m/z 490.2068 [M + H]+, m/z 512.1901 [M + Na]+, and
m/z 1001.3903 [2 M + Na]+ by HR-ESI-MS analysis (Fig. S2), 14
mass units less than that of nocamycin I, corresponding to a
demethylation derivate of nocamycin I. Comparisons of the 1H
and 13C NMR spectroscopic data indicate that 3 showed similar
structure to that of nocamycin I, except that one methoxy proton
Fig. 1. The structures of nocamycin I (1) and nocamycin II (2).
phenotype and the genotype was then verified by PCR (Fig. 2). The
mutant strain S. syringae MoS-1004 was firstly cultured by using
the fermentation medium contained 1% soybean, 3% glycerol,
8
0.2% NaCl, 0.5% mycose, 0.2% CaCO3. After seven days fermenta-
tion, the broth was extracted by organic solvent and analyzed by
HPLC. HPLC results revealed that the mutant strain S. syringae
MoS-1004 (DNcmP) lost the capability to produce nocamycin I
and nocamycin II. To our surprise, no other nocamycin derivatives
have been detected. Based on the intermediates nocamycin III iso-
Fig. 2. Construction of
DNcmP gene replacement mutant S. syringae MoS-1004 and HPLC analysis of the metabolite profile. (A) Depiction of NcmP inactivation. The mutant
MoS-1004 was generated by replacing 648-bp internal NcmP fragment with the oriT and acc3(IV) fragment in pMoS-1004, resulting from a double-crossover recombination
event. (B) PCR analysis of the double-crossover mutant. W: S. syringae wild type (827 bp); M: mutant strain S. syringae MoS-1004 (1527 bp); Marker: DNA molecular ladder.
(C) HPLC analysis of the metabolite profiles. 1 represents nocamycin I, 2 represents nocamycin II, 3 represents the newly produced nocamycin derivative. (D) The UV
absorption of compound 3 and 1.
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X. Mo et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
1H and 13C NMR spectroscopic data for nocamycin E (3) and nocamycin I (1) in CD3OD.
nocamycin E (3)
nocamycin I (1)
position
dC type
dH mult. (J in Hz)
position
dC type
dH mult. (J in Hz)
1
2
3
4
5
6
176.2,
117.5
150.6
135.9
145.9
36.2
1
2
3
4
5
6
176.2,
117.2
150.6
135.9
145.3
36.2
7.19, d (15.0)
7.58, d (15.4)
7.22, d (15.6)
7.54, d (15.6)
6.26, d (9.7)
2.94, m
6.12, d (9.8)
2.94, m
7
8
80.1
36.5
3.57, d (10.7)
2.01, m
7
8
80.1
36.5
3.58, d (11.4)
2.02, m
9
80.5
4.08, s
9
80.2
4.09, s
10
11
12
13
14
15
16
17
18
19
20
21
209.7
52.0
83.2
107.8
64.4
74.3
20.3
12.4
17.1
11.9
10
11
12
13
14
15
16
17
18
19
20
21
22
10
209.7
52.1
83.2
107.8
64.4
74.4
20.5
12.3
17.1
11.9
2.60, s
2.63, s
2.90, d (8.5)
4.50, m
1.26, d (5.8)
1.90, s
1.08, d (6.6)
0.85, m
1.41, s
2.90, d (17.7)
4.53, m
1.27, d (6.0)
1.92, s
1.06, d (6.8)
0.85, d (6.8)
1.42, s
21.8
172.1
21.8
172.1
52.5
3.81, s
3.82, s
10
20
30
40
50
Not observed
Not observed
Not observed
52.2
20
Not observed
Not observed
Not observed
52.4
30
40
3.81, s
50
2 mM SAM in 50 mM Tris-Cl (pH 8.0) buffer. Control experiment
with boiled NcmP protein (100 °C, 15 min) was also set. The
enzyme reaction was performed on 30 °C for 1 h. Then, the reaction
product were extracted by 200
lL ethyl acetate and the organic
phase was evaporated to dryness and dissolved in 20
lL methanol
for HPLC analysis. Comparing with the control set, NcmP can cat-
alyze the conversion from compound 3 to compound 1 completely.
The structure of enzymatically converted compound 1 was deter-
mined by comparison with standard nocamycin I and LC-HR-MS
analyses (Fig. 4).
Based on in vivo gene disruption and in vitro biochemical evi-
dences, NcmP was identified as a carboxylate O-methyltransferase.
For the production of nocamycin I and nocamycin II in wild type S.
syringae, we propose that NcmP can accept both compound 3 and
compound 4 as substrates to yield nocamycin I and nocamycin II,
respectively (Fig. 5).
Fig. 3. The structure and selected key HMBC correlations (arrows) for compound 3.
The antibacterial activities of nocamycin E and nocamycin I
toward Bacillus subtilis (CCTCC AB2013121), Bacillus thuringiensis
(CCTCC AB2013119), Micrococcus luteus (CCTCC AB2013120), Sta-
phylococcus aureus ATCC 29213, and Enterococcus faecalis
ATCC29212 were evaluated and the results were shown in Table 2.
Both nocamycin E and nocamycin I show no inhibitory activity
against B. subtilis at the concentration of >128 mg/mL. Notably,
nocamycin E displays much less potent antibacterial activity than
that of nocamycin I, although only a methyl group disparity
between nocamycin I and nocamycin E. This observation high-
lighted the importance of the carboxylate O-methyl group for the
antibacterial property of nocamycin.
In summary, we construct the methyltransferase gene NcmP
disruption strain through k-RED recombination technology and
isolate a new nocamycin intermediate nocamycin E from the
mutant strain by varying culture condition. NcmP has been bio-
chemically characterized as a carboxylate O-methyltransferase in
nocamycin biosynthetic pathway. The bioactivity assays indicated
the importance of the methyl group at C-21 for antibacterial prop-
erty of nocamycin, and nocamycin E with no carboxylate O-methyl
group showed inferior antibacterial activity.
signal at C-21was absent (Table 1, Figs. S3–S7). Key HMBC correla-
tions from H-14 to C-21 and from H-15 to C-21 confirmed the loca-
tion of the –COOH group at C-21. Inspections of other signals were
fully assigned on the basis of 2D NMR analyses. Hence, the struc-
ture of 3 was determined as 21-demethyl-nocamycin I and it was
designated as nocamycin E (Fig. 3).
Based on the structure of compound 3, we proposed that NcmP
is responsible for the methyl esterification in nocamycin biosyn-
thetic pathway. To further fully identify the function of NcmP
in vitro, the NcmP gene was amplified from cosmid p5-A-9 and
cloned into a pET-28a (+) vector for heterologously overexpression
as a soluble N-terminal His6-tagged protein in E. coli BL21 (DE3).
The recombinant protein was purified as homogeneity by using
Ni-NTA affinity chromatography. SDS-PAGE analysis revealed the
purified protein has a molecular weight of 29.8 KDa, which was
consistent with the calculated molecular weight for N-His6-tagged
NcmP (Fig. 4). Then, the enzyme reaction activity was tested
in vitro. Enzymatic reactions were performed on 100 mL volume
system consisted of 1 mM compound 3, 5 mM enzyme NcmP,
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X. Mo et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 4. Biochemical characterization of NcmP in vitro. (A) SDS-PAGE analysis of recombinant NcmP. (B) HPLC analyses of in vitro NcmP assays with nocamycin E (3). (I): an
assay of NcmP with 3; (II) the nocamycin I (1) standard; (III) a control assay of 3 with boiled NcmP. (C): LC-HR-MS analysis of enzymatically converted compound 1. The
molecular of 1 is C26H33NO9 (m/z for [M + Na]+ 526.2048, m/z for [2 M + Na]+ 1029.4194), which is identical to authentic nocamycin I (1).
Fig. 5. Proposed biosynthetic pathway catalyzed by NcmP.
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X. Mo et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Table 2
Antibacterial activity (MIC in mg/mL) of nocamycin I (1) and nocamycin E (3).
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Acknowledgments
Research in our laboratory was supported by Grants from the
National Natural Science Foundation of China (No. 31300063), Nat-
ural Science Foundation of Shandong Province (No. ZR2013CL020),
Research Foundation for Advanced Talents of Qingdao Agricultural
University (No. 631301) and Open Research Foundation of CAS Key
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08.
010.
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