Identification and characterization of a bacterial cytochrome P450 monooxygenase catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis

Article abstract of DOI:10.1074/jbc.M117.791269

Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by Streptomyces atratus ATCC 14046. Its structure contains three non-proteinogenic amino acids, N-dimethylallyltryptophan, trans-2-crotylglycine, and 3-nitro-tyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of S. atratus and identified the rufomycin biosynthetic gene cluster (ruf cluster), consisting of 20 ORFs (rufA–rufT). We found a putative heptamodular nonribosomal peptide synthetase encoded by rufT, a putative tryptophan N-dimethylallyltransferase encoded by rufP, and a putative trimodular type I polyketide synthase encoded by rufEF. Moreover, the ruf cluster contains an apparent operon harboring putative cytochrome P450 (rufO) and nitric oxide synthase (rufN) genes. A similar operon, txtDE, is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of rufO abolished rufomycin production by S. atratus, which was restored when 3NTyr was added to the culture medium of the dis-ruptant. Recombinant RufO protein exhibited Tyr 3-nitra-tion activity both in vitro and in vivo. Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of 0.1 M. These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an in silico analysis of the ruf cluster, we propose a rufomycin biosynthetic pathway in S. atratus.

Full text of DOI:10.1074/jbc.M117.791269

JBC Papers in Press. Published on August 3, 2017 as Manuscript M117.791269
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.791269
Tyrosine nitration in rufomycin biosynthesis
Identification and Characterization of a Bacterial Cytochrome P450 Monooxygenase Catalyzing
the 3-Nitration of Tyrosine in Rufomycin Biosynthesis
1
,2
1,2
3
1,2
Hiroya Tomita , Yohei Katsuyama , Hiromichi Minami , and Yasuo Ohnishi
1
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of
2
Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; JST, CREST, 7, Gobancho, Chiyoda-ku, Tokyo 102-0076,
3
Japan; Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308,
Suematsu,ꢀNonoichi, Ishikawa 921-8836, Japan.
Running title: Tyrosine nitration in rufomycin biosynthesis
To whom correspondence should be addressed:
Prof. Yasuo Ohnishi, Department of Biotechnology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan, Telephone:
(
+81)-3-5841-5123, FAX: (+81)-3-5841-8021, E-mail: ayasuo@mail.ecc.u-tokyo.ac.jp
Prof. Yohei Katsuyama, Department of Biotechnology, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan, Telephone:
(
+81)-3-5841-5124, FAX: (+81)-3-5841-8021, E-mail: aykatsu@mail.ecc.u-tokyo.ac.jp
Keywords: Cytochrome P450, nonribosomal peptide, rufomycin, 3-nitrotyrosine, Streptomyces
ABSTRACT
Rufomycin is a circular heptapeptide
reported in the 1960s, its biosynthesis, including
3-NTyr generation, remains unclear. To elucidate
the rufomycin biosynthetic pathway, we
assembled a draft genome sequence of S. atratus
and identified the rufomycin biosynthetic gene
cluster (ruf cluster), consisting of 20 ORFs (rufA–
with anti-mycobacterial activity and is produced
by Streptomyces atratus ATCC 14046. Its
structure contains three non-proteinogenic amino
acids,
trans-2-crotylglycine, and 3-nitrotyrosine (3NTyr). rufT). We found
Although the rufomycin structure was already nonribosomal peptide synthetase encoded by rufT,
N-dimethylallyltryptophan,
a
putative heptamodular
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Tyrosine nitration in rufomycin biosynthesis
a putative tryptophan N-dimethylallyltransferase
by rufP, and a putative trimodular type I
polyketide synthase by rufEF. Moreover, the ruf
cluster contains an apparent operon harboring
putative cytochrome P450 (rufO) and nitric oxide
synthase (rufN) genes. A similar operon, txtDE, is
responsible for the formation of 4-nitrotryptophan
in thaxtomin biosynthesis; the cytochrome P450
TxtE catalyzes the 4-nitration of Trp. Therefore,
we hypothesized that RufO should catalyze the
Tyr 3-nitration. Disruption of rufO abolished
rufomycin production by S. atratus, which was
restored when 3NTyr was added to the culture
medium of the disruptant. Recombinant RufO
protein exhibited Tyr 3-nitration activity both in
vitro and in vivo. Spectroscopic analysis further
revealed that RufO recognizes Tyr as the substrate
with a dissociation constant of ~ 0.1 µM. These
results indicate that RufO is an unprecedented
cytochrome P450 that catalyzes Tyr nitration.
Taken together with the results of an in silico
analysis of the ruf cluster, we propose a
rufomycin biosynthetic pathway in S. atratus.
biochemical point of view, these compounds
display diverse chemical structures, and many
studies have focused on their biosynthetic
mechanisms.
The nitro group is not an abundant
functional group in natural products, but it has
been found in some important bioactive
compounds including chloramphenicol, aureothin,
and thaxtomin (1,2). Although the mechanism for
generating the nitro group in natural products is
not fully understood, two possible nitration
mechanisms have been reported. One is the
oxidation reaction of an amino group found in
chloramphenicol, aureothin, and pyrrolnitrin
biosynthesis. In the former two cases, the p-amino
groups
of
p-aminophenylalanine
and
p-aminobenzoic acid are oxidized to p-nitro
groups using molecular oxygen as a substrate by
the non-heme di-iron-containing monooxygenases
CmlI (3,4) and AurF (5–7), respectively. In
pyrrolnitrin biosynthesis, PrnD, which contains a
Rieske iron-sulfur cluster, catalyzes the oxidation
of aminopyrrolnitrin to complete the biosynthetic
pathway (8). Recently, CreD, a flavin-dependent
monooxygenase that is involved in cremeomycin
biosynthesis, was reported to catalyze nitro group
formation by oxidizing the amino group of Asp
(9).
Natural products have been found in a
wide range of organisms including plants, fungi,
microorganisms, and mammals. Because these
compounds
activities
exhibit
various
to
physiological
medical and
applicable
The other mechanism is direct nitration
pharmaceutical products, animal drugs, and
agricultural chemicals, they have been used to
facilitate and improve human life. From a
catalyzed by TxtE, a bacterial cytochrome P450
involved
in
thaxtomin
biosynthesis
in
plant-pathogenic Streptomyces strains including
2

Tyrosine nitration in rufomycin biosynthesis
Streptomyces scabies (10–12). TxtE uses nitric
mechanisms for generating trans-2-crotylglycine
oxide (NO) supplied by a nitric oxide synthase
and
3NTyr
are
unknown.
Although
(
NOS) encoded by txtD as its substrate, and it
catalyzes the selective nitration of Trp to generate
-nitrotryptophan. The txtE and txtD genes are
2-crotylglycine is an intermediate in the
degradation pathway of aromatic rings in
Pseudomonas putida (30), this pathway is
unlikely to be used for 2-crotylglycine synthesis
in rufomycin biosynthesis, and no enzymes for
3NTyr formation have been reported. Therefore,
elucidating the rufomycin biosynthetic pathway
will shed light on novel enzymes involved in the
biosynthesis of 2-crotylglycine and 3NTyr.
Here we report the identification and in
4
located next to each other, and they form a
putative operon in the thaxtomin biosynthetic
gene cluster (txt cluster) (13).
In addition to these compounds,
pyrrolomycins were reported to possess a nitro
group in the pyrrole ring (14–18), but the nitration
mechanism has not been elucidated in detail
(
19,20). NOS is not considered to be involved in
silico characterization of the rufomycin
biosynthetic gene cluster (ruf cluster). We also
report genetic and biochemical analyses of a
cytochrome P450 enzyme, RufO, which catalyzes
the 3-nitration of Tyr. According to these analyses,
we propose a probable rufomycin biosynthetic
pathway.
this mechanism, because NOS inhibitors do not
affect the nitration of pyrrole (20). Instead,
putative nitrate reductases are encoded by its
biosynthetic gene cluster, suggesting that NO is
synthesized by the reduction of nitrate (19).
Rufomycin (21,22), also known as
ilamycin (23–27), is a circular heptapeptide with
anti-mycobacterial activity, which was isolated
from Streptomyces atratus ATCC 14046.
Although its chemical structure was reported in
the 1960s (26,27), its biosynthetic mechanisms are
RESULTS
Draft genome analysis of S. atratus to identify
the ruf cluster.
To elucidate the mechanisms of
rufomycin biosynthesis and 3NTyr generation,
identifying the rufomycin biosynthetic gene
cluster is indispensable. Therefore, we first
determined a draft genome sequence of S. atratus
with the HiSeq 2000 system (Illumina) using a
paired-end sequencing strategy. Then we searched
the draft genome sequence for the rufomycin
biosynthetic gene cluster in silico. Based on the
unclear.
Rufomycin
contains
amino
three
non-proteinogenic
acids,
N-dimethylallyltryptophan, trans-2-crotylglycine,
and 3-nitrotyrosine (3NTyr). The formation of
N-dimethylallyltryptophan
was
found
in
cyclomarin biosynthesis, in which the tryptophan
N-dimethylallyltransferase CymD catalyzes the
N-prenylation of free Trp (28,29). In contrast, the
3

Tyrosine nitration in rufomycin biosynthesis
rufomycin structure, we assumed that the
rufomycin biosynthetic gene cluster should
heptamodular NRPS, the domain component of
which was consistent with the chemical structure
of rufomycin (Fig. 1B). The substrate of each
adenylation (A) domain was predicted by NRPS
predictor 2 (Table S1) (31,32). Although most of
the predictions were not consistent with the
structure of rufomycin, the A domain of the
loading module was predicted to recognize
N-dimethylallyltryptophan with high reliability.
This result indicates that peptide assembly is
initiated from N-dimethylallyltryptophan. The
location of two methyltransferase (MT) domains
at modules 1 and 4 also supports this notion.
Modules 1 and 4 correspond to the second and
fifth amino acid residues, respectively, in the
include
a
nonribosomal peptide synthetase
(
NRPS) gene(s) for peptide assembly,
a
tryptophan N-dimethylallyltransferase gene for the
synthesis of N-dimethylallyltryptophan, and a
gene(s) for the nitro group synthesis. As expected,
we found a gene cluster that satisfied all these
requirements (see below) and named it the ruf
cluster. The nucleotide sequences of partially
sequenced regions were determined by primer
walking to complete the sequence of the ruf
cluster (Fig. 1A and Table 1). Twenty ORFs
(
rufA–T) compose the ruf cluster, and four of
them encode cytochrome P450 enzymes. One of
the four cytochrome P450 genes (rufO) forms a
putative operon with an NOS gene (rufN), and
these two genes were expected to be responsible
for the nitro group synthesis. An NRPS gene
rufomycin
assembly
pathway.
When
N-dimethylallyltryptophan is considered to be the
first amino acid residue, the second and fifth
amino acid residues are both N-methylleucine,
which agree with the presence of MT domains at
modules 1 and 4. Meanwhile, the A domain of
module 2 was predicted to incorporate Tyr. This
module corresponds to the third amino acid
residue of rufomycin, i.e., 3NTyr, assuming
N-dimethylallyltryptophan is the first amino acid
residue. If this prediction is correct, Tyr should be
loaded on the thiolation (T) domain of module 2,
and 3-nitration should occur during or after
peptide assembly. However, because there is no
report of A domains recognizing 3NTyr, it is
impossible to predict 3NTyr as a substrate of any
A domains. Therefore, it is also possible that
(
rufT) and a tryptophan N-dimethylallyltransferase
gene (rufP) were also found in the ruf cluster.
In silico analysis of the ruf cluster.
Here we describe the results of our in
silico
analysis
of
several
for
important
rufomycin
enzyme-encoding
biosynthesis.
genes
(
i) NRPS
Because rufomycin harbors three
non-proteinogenic amino acids, we assumed that
it should be synthesized by an NRPS. In the ruf
cluster, we found a large gene (rufT) encoding a
4

Tyrosine nitration in rufomycin biosynthesis
3
NTy, instead of Tyr, is incorporated to the
module 2 consists of KS, AT, DH, enoylreductase
(ER), KR, and ACP domains. RufF is a TE
domain. Via such a domain organization, the
RufEF PKS is expected to synthesize 4-hexenoic
acid (Fig. 2A). In general, the cis-trans
stereochemistry of a double bond formed by a DH
domain can be predicted by analyzing the KR
domain of the same module. KR domains can be
classified into A, B, and C types based on the
presence or absence of the conserved xxD motif
and Trp residue, and the stereochemistry of their
products can be predicted according to this
classification (34). The KR domain of module 1
belongs to the B type, suggesting that the trans
C=C bond is synthesized by module 1 (Fig. 2C).
Thus, we speculate that RufEF produces
trans-4-hexenoic acid, which is a compatible
precursor for trans-2-crotylglycine biosynthesis.
Hydroxylation and oxidation of the C2 position of
NRPS.
(
ii) Tryptophan N-dimethylallyltransferase
In cyclomarin biosynthesis, CymD
catalyzes the N-dimethylallylation of free Trp
28,29). In the ruf cluster, rufP encodes a CymD
(
homolog (44% identity). We speculate that
N-dimethylallyltryptophan is synthesized by
RufP.
(
iii) Type I polyketide synthase (PKS) for the
synthesis of trans-2-crotylglycine
PKS genes (rufE and rufF) were found
in the ruf cluster. RufE consists of three modules
loading module and modules 1 and 2) and RufF
is a thioesterase (TE) (Fig. 2A), and the RufEF
PKS is most likely responsible for
trans-2-crotylglycine biosynthesis. The loading
module contains ketosynthase (KS),
acyltransferase (AT), and acyl carrier protein
ACP) domains. While KS domains generally
(
trans-4-hexenoic
acid
can
form
trans-2-oxo-4-hexenoic acid, and transamination
of the keto group can form trans-2-crotylglycine
(Fig. 2A). We speculate that these reactions are
catalyzed by a cytochrome P450 enzyme (RufC,
see below) and an aminotransferase (RufI).
(
catalyze decarboxylative Claisen condensation for
Q
C-C bond formation, a group of KSs called KS ,
in which the Cys residue for substrate binding is
substituted by Gln, catalyze only decarboxylation
(
33). The KS domain of the loading module is
(iv) NOS and cytochrome P450 for the 3-nitration
of Tyr
Q
classified as KS (Fig. 2B), suggesting that it
catalyzes
only
the
decarboxylation
of
In the thaxtomin biosynthetic gene
cluster, txtD and txtE (encoding NOS and
cytochrome P450, respectively) form a putative
operon. We also found a putative operon
malonyl-CoA to provide the acetyl starter unit.
Module 1 consists of KS, AT, dehydratase (DH),
ketoreductase (KR), and ACP domains, and
5

Tyrosine nitration in rufomycin biosynthesis
consisting of NOS, P450, and tryptophan
N-dimethylallyltransferase genes (rufN, rufO, and
rufP, respectively) in the ruf cluster. RufO and
RufN show 26% and 58% amino acid sequence
identities with TxtE and TxtD, respectively.
Although the sequence identity between RufO and
TxtE was not high, we assumed that RufO and
RufN should be responsible for the nitration of
Tyr.
other natural product biosynthetic pathways.
Similarly, we predict the functions of RufM and
RufS. Among the characterized enzymes, RufM
and RufS show the highest identity with
CYP107Z4 (46%) and TbtJ1 (45%), respectively.
CYP107Z4 and TbtJ1 catalyze the hydroxylation
of avermectin to produce 4’’-oxo-avermectin (35)
and the hydroxylation of Phe or Leu of the
thiopeptide thiomuracin (36,37), respectively.
This result implies that RufS should catalyze the
hydroxylation of Leu; therefore, RufM is
considered to play the remaining role, which is
epoxidation of the dimethylallyl group.
(
v) Cytochrome P450 monooxygenases
The cytochrome P450 enzymes except for
RufO encoded in the ruf cluster (RufC, RufM, and
RufS) may catalyze occasional oxidative
modification reactions, such as the epoxidation of
N-dimethylallyltryptophan and the hydroxylation
and oxidation of Leu, to synthesize other
rufomycin derivatives. Although this biosynthetic
process is hypothetical, we predict the roles of
three P450 monooxygenases. Using a BLAST
search with the primary sequence of RufC as
query, we discovered several cytochrome P450
monooxygenases (~50% identity with RufC)
Gene disruption of rufO.
To confirm the involvement of the ruf
cluster in rufomycin biosynthesis and to examine
the contribution of the candidate P450 gene (rufO)
to 3NTyr synthesis, we disrupted rufO using the
CRISPR/Cas9 system (38). A disruption plasmid
was designed so that the rufO coding region
should be cut by Cas9 and removed during the
repair of the region, guided by the recombination
whose genes form an operon with PKS genes (Fig. template cassette introduced into the plasmid.
S1A). Interestingly, these operons also encode an
aminotransferase. In addition, the domain
organization of the PKS discovered from
Streptomyces aidingensis strain CGMCC 4.5739
is identical to that of RufE. Thus, RufC is most
likely to be involved in trans-2-crotylglycine
biosynthesis and this trans-2-crotylglycine
biosynthesis pathway seems to be distributed to
After multiple trials, we isolated the rufO deletion
strain (ΔrufO) (Fig. S2).
We cultivated the wild-type and ΔrufO
strains and compared their metabolic profiles in
three nutrient-rich media, Tryptic Soy Broth
(
(
TSB), International Streptomyces Project 2
ISP2), and modified G-2 (MG2). After extracting
metabolites with n-butanol, the organic phase was
6

Tyrosine nitration in rufomycin biosynthesis
analyzed by LC-MS.
In the wild-type strain cultivated in
should be strict and only 3NTyr can be accepted
by RufT as the third amino acid residue in the
peptide assembly line. Thus, nitration seems to
occur prior to peptide assembly.
MG2, we clearly detected the production of four
major compounds whose m/z values were
identical to those of rufomycin derivatives
reported previously (Fig. 3 and Table S2) (39).
This indicates that these compounds are the
rufomycin derivatives. Three of the four
congeners (compounds 1, 2, and 3 in Fig. 3) were
also produced in ISP2 medium, whereas the
rufomycin derivatives were hardly produced in
TSB (data now shown). In contrast, in the ΔrufO
strain, we did not detect any rufomycin
derivatives or shunt products in any of the media.
However, when 3NTyr was added to the culture
medium of the ΔrufO strain, rufomycin
production was fully recovered (Fig. 3). From
these results, we conclude that the ruf cluster is
responsible for rufomycin biosynthesis. In
addition, the recovery of rufomycin production in
the ΔrufO mutant denied the possibility that the
mutant lost heptapeptide production because of
the unexpected inactivation of rufP caused by the
possible polar effect of the rufO deletion; rufP
appears to form an operon, rufNOP, with rufN and
rufO. Inactivation of rufP must inhibit the peptide
synthesis, because rufP presumably encodes a
tryptophan N-dimethylallyltransferase essential
UV-Vis spectroscopic study of RufO.
To investigate the function of RufO in
vitro, the recombinant RufO protein with an 8x
His-tag at its C-terminus was produced in
Escherichia coli and purified by nickel chelate
affinity chromatography (Fig. 4). The color of the
obtained solution ranged from yellow to red,
which is typical of cytochrome P450 solutions.
The purified RufO protein was subjected to
carbon monoxide (CO)-binding and NO-binding
spectral analyses (40). Bubbling of CO into the
RufO solution resulted in the Soret band shifting
from ~ 420 nm to ~ 450 nm (Fig. 5A). In the case
of NO, a shift to ~ 440 nm was observed (Fig. 5B).
These physicochemical properties are consistent
with those of typical P450 enzymes. All these
results indicate that the recombinant RufO was
purified in an active form.
Substrate-binding analysis of RufO.
The substrate specificity of RufO was
examined by investigating substrate-binding
spectra (41). We used five compounds: Tyr, Phe,
Trp, 4-hydroxyphenylpuruvate (the proximate
for
producing
the
first
amino
acid
precursor
-aminophenylalanine. The addition of Tyr
resulted in the typical type I binding spectra, a
of
Tyr
biosynthesis),
and
(
N-dimethylallyltryptophan) of the rufomycin
4
peptide. We think that the substrate specificity of
the A domain in module 2 of the RufT NRPS
7

Tyrosine nitration in rufomycin biosynthesis
Soret band shift from ~ 420 nm to ~ 390 nm (41),
suggesting that Tyr is recognized as a substrate
also
examined
whether
Phe,
Trp,
and
4-hydroxyphenylpyruvate
(
Fig. 5C). The dissociation constant (K
d
) was
4-aminophenylalanine could be used as a substrate
of RufO in this in vitro reaction. Consistent with
the results of the substrate-binding spectrum
analysis described above, these four compounds
were not recognized as a substrate of RufO and no
nitrated products were detected (data not shown).
estimated to be ~ 0.1 µM (Fig. 5D). In contrast,
the other four compounds did not give apparent
binding spectra, suggesting that they are not
recognized as substrates, or that their binding
affinity is too low to be detected.
Examination of the enzyme activity of
recombinant RufO protein.
Production of 3-(N-acetyl)aminotyrosine by a
recombinant E. coli strain expressing rufO and
camAB.
To investigate the ability of RufO to
nitrate Tyr, we conducted an in vitro analysis
using the purified RufO protein. Because bacterial
cytochrome P450s require a ferredoxin and a
ferredoxin reductase as the redox system,
To further confirm the Tyr-nitration
activity of RufO, we attempted to produce 3NTyr
by a recombinant E. coli strain. We used a
Tyr-overproducing strain AN219 (43) as the host
to express rufO and camAB. The recombinant
strain was cultivated and expression of rufO and
camAB was induced, and then DEANO was added
to the culture. After a further 24-h incubation, the
culture supernatant was analyzed with LC-MS. As
a result, 3-(N-acetyl)aminotyrosine was detected,
indicating that Tyr was converted to 3NTyr by
RufO in this strain, because we confirmed that the
nitro group of 3NTyr was efficiently reduced and
N-acetylated to yield 3-(N-acetyl)aminotyrosine
by the E. coli BL21(DE3) resting cells (Fig. S3).
putidaredoxin
(CamB)
and
putidaredoxin
reductase (CamA), which are involved in camphor
biosynthesis in P. putida (42), were also prepared
as reported previously (Fig. 4). RufO was
incubated with Tyr and diethylamine NONOate
(
DEANO), a chemical NO generator, in the
presence of CamA, CamB, and NADH. After
incubating the mixtures, the reactions were
quenched with HCl and then subjected to an
LC-MS analysis. As a result, the formation of
3
NTyr was clearly detected, indicating that RufO
catalyzes the nitration reaction of free Tyr (Fig. 6). In E. coli, two major nitroreductases, NfsA and
However, regardless of repeated trial and error,
we could not increase the amount of 3NTyr
produced in this in vitro reaction, which hampered
the kinetic analysis of RufO (see discussion). We
NfsB, were reported to have wide substrate
specificity (44), and therefore they may reduce
3NTyr. E. coli also has an arylamine
N-acetyltransferase (45,46), which seems to be
8

Tyrosine nitration in rufomycin biosynthesis
responsible for the acetylation of 3-aminotyrosine.
Taken together with the results of all other
experiments in this study, we conclude that RufO
catalyzes 3-nitration of free Tyr.
N-dimethylallyltryptophan as the first amino acid
to synthesize the heptacyclic peptide rufomycin B.
During the peptide elongation, two N-methyl
groups are introduced by the two MT domains
located in modules 1 and 4. Of course, further
experiments are required to confirm this proposed
biosynthetic pathway.
DISCUSSION
In this study, we identified the
Regarding the proposed biosynthetic
pathway for trans-2-crotylglycine, a similar
pathway has been predicted for the biosynthesis of
(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine,
which is a building block of cyclosporin A (47–
49). In this predicted pathway, the polyketide
rufomycin biosynthetic gene cluster (ruf cluster)
in S. atratus. Rufomycin harbors a 3NTyr moiety
in its structure, and we demonstrated that 3NTyr
is synthesized from Tyr by a cytochrome P450
enzyme, RufO.
Based on the bioinformatic analysis of
intermediate is hydroxylated by
a
P450
the ruf cluster and genetic and biochemical studies
of RufO, we propose the biosynthetic pathway of
rufomycin as follows (Fig. 7). In the first step,
three non-proteinogenic amino acids are
synthesized individually. (i) Trp is converted to
N-dimethylallyltryptophan by RufP. (ii) RufE and
RufF, which form a trimodular type I PKS,
produce trans-4-hexenoic acid. A cytochrome
P450 monooxygenase RufC introduces a keto
group into the C2 position of trans-4-hexenoic
acid to generate trans-2-oxo-4-hexenoic acid.
Finally, a transamination reaction catalyzed by
monooxygenase and further oxidized by a
dehydrogenase. However, the ruf cluster contains
no putative dehydrogenase gene. Therefore, we
speculate that the P450 monooxygenase RufC
should catalyze two sequential oxidations
(hydroxylation and dehydrogenation) to generate
the α-keto group, similar to how CYP170A1
catalyzes the two-step oxidation of epi-isozizaene
to produce albaflavenone in Streptomyces
coelicolor A3(2) (50).
During rufomycin biosynthesis, the
timing of Tyr nitration is of great interest. In the
biosynthetic pathway of natural products, many
P450 enzymes that catalyze post-assembly
reactions (called tailoring reactions) have been
reported. In addition, 3NTyr is non-proteinogenic,
and it may be toxic to cells. Therefore, nitration of
the Tyr residue after peptide assembly was
RufI
results
in
trans-2-crotylglycine
(
trans-2-amino-4-hexenoic acid). (iii) RufO
catalyzes the 3-nitration of Tyr, with NO
produced by RufN, to form 3NTyr. In the second
step, the heptamodular NRPS RufT assembles
seven
amino
acids
using
9

Tyrosine nitration in rufomycin biosynthesis
regarded as reasonable. There was also a
possibility that the nitration of Tyr occurs during
peptide assembly. For example, Tyr or some
intermediate peptides attached to a T domain
could be the substrate of the nitration enzyme.
However, all our experimental data indicate that
The catalytic mechanism of the nitration catalyzed
by TxtE was proposed by Barry et al. as follows
(10). After substrate binding and heme reduction,
TxtE combines NO with molecular oxygen to
generate a ferric peroxynitrite intermediate, which
then undergoes either homolytic cleavage to yield
3
NTyr is produced from free Tyr and incorporated
2
NO and an Fe(IV)=O species (compound II), or
into the peptide as a substrate of the RufT NRPS.
First, the ΔrufO mutant produced neither
rufomycin nor its denitrated derivatives, and it
restored rufomycin production following the
exogenous addition of 3NTyr. Second,
recombinant RufO catalyzed the nitration of Tyr
in vitro. Third, 3-(N-acetyl)aminotyrosine, the
reduced and N-acetylated derivative of 3NTyr,
was produced by the recombinant E. coli cells
expressing rufO and camAB. From these three
results, we conclude that Tyr nitration by RufO
occurs prior to peptide assembly. It should be
noted that no rufomycin derivatives lacking the
nitro group on Tyr have been found, although
various patterns of modification have been
observed within rufomycin (39). This also
strongly supports our conclusions that 3NTyr is
produced from free Tyr by RufO and that only
heterolytic cleavage initiated by protonation to
+
yield NO
2
and an Fe(III)-OH species. In the
former case, the nitration of enzyme-bound Trp
should occur via NO addition and hydrogen atom
2
abstraction by compound II, resulting in the
formation of an Fe(III)-OH species. In the latter
case, it should occur via electrophilic aromatic
substitution. In both cases, protonation of the
Fe(III)-OH species is required for the regeneration
2
of Fe(III)-OH to restore the resting state of the
enzyme. Crystal structure analyses of TxtE and an
investigation of substrate analogs indicated the
substrate recognition mechanisms of TxtE and the
substrate properties required for catalysis (11,12).
In addition, a recent study discovered that a single
mutation of His176 in the F/G loop of TxtE
completely shifts the enzyme’s regioselectivity
from the C4 to the C5 position of Trp (51). The
F/G loop is a flexible loop connecting the F and G
helices, and it is involved in the open-to-closed
transitions of the substrate-binding pocket; His176
also plays a key role in gating these transitions.
However, despite these studies, detailed molecular
mechanisms of Trp nitration, including the
formation of a ferric peroxynitrite intermediate, by
3
NTyr is incorporated into the peptide assembly
by the RufT NRPS because of high substrate
specificity of the A domain in module 2.
Many cytochrome P450 enzymes have
been studied extensively, but only TxtE and its
close homologs were reported to catalyze the
direct nitration of aromatic compounds (10–12).
1
0

Tyrosine nitration in rufomycin biosynthesis
TxtE remain to be elucidated. Moreover, the
reason why TxtE does not show conventional
monooxygenase activity toward Trp is of great
interest. RufO is the first cytochrome P450
revealed to catalyze the nitration of Tyr, and
therefore, it will provide new clues to solve the
aforementioned issues of nitrating cytochrome
P450 enzymes. However, difficulty in the analysis
of these nitrating cytochrome P450 enzymes,
TxtE and RufO, seems to be ascribed to their low
enzymatic activities. No kinetic analysis has been
reported for TxtE and we also failed to show
enough RufO activity for the kinetic analysis in
vitro. Therefore, some improvement of the
enzyme assay system may be required for further
analysis. A fusion protein of TxtE with redox
partner proteins was reported to enhance the
catalytic efficiency (52).
and mutagenesis studies of RufO, will provide
important insights into the mechanisms of the
nitration of aromatic compounds, which will
extend our knowledge of cytochrome P450
enzymes.
EXPRERIMENTAL PROCEDURES
Strains, media and culture conditions.
S. atratus ATCC 14046 was obtained
from the NITE Biological Resource Center
(NBRC), Japan, and was cultivated in Mannitol
Soya Flour (MS), TSB, ISP2 and modified G-2
(MG2) media. G-2 is a medium optimized for
rufomycin production (36). MS medium consists
of mannitol (2%) and soya flour (2%). TSB
medium consists of tryptic soy broth (3%). ISP2
medium consists of yeast extract (0.4%), malt
extract (1%), and glucose (0.4%) (pH 7.2). MG2
medium consists of potato dextrin (5%), glucose
(3%), glycerol (1%), molasses (1%), soluble
starch (0.5%), soybean meal (0.5%), corn steep
When we performed a BLAST search using
the primary sequence of RufO as a query, we
could not find any close homologs with more than
5
0% amino acid sequence identity in the database.
liquor (0.5%), MgSO
(0.3%), Fe(NH (SO
and MnSO (0.01%). For solid culture, agar
4
•7H
2
O (0.05%), CaCO
3
However, we found some uncharacterized RufO
homologs whose genes are likely to form an
operon with an NOS homolog gene (Fig. S1B).
Interestingly, these operons encoding RufO and
NOS homologs are located adjacent to NRPS
genes. This observation suggests that these RufO
homologs should function as aromatic amino
acid-nitrating enzymes and be utilized by
respective NRPS systems. Studies of these RufO
homologs, as well as further structural analyses
4
)
2
4
)
2
(0.01%), ZnCl
2
(0.01%),
4
powder (2%) was added to solidify the medium. E.
coli strains JM109 and ET12567/pUZ8002, used
for plasmid construction and conjugation,
respectively, were cultivated at 37°C in
Luria-Bertani (LB) medium with or without
-
1
-1
ampicillin (100 mg l ) and apramycin (50 mg l ).
E. coli strains BL21(DE3) and BLR(DE3), used
for heterologous gene expression, were cultivated
1
1

Tyrosine nitration in rufomycin biosynthesis
at various temperature in LB medium or terrific
broth (TB). Trace element solution consists of 50
disrupted were amplified with the primer pairs
DrufO5'-F/R and DrufO3'-F/R (Table S3),
mM FeCl
MnCl •4H
•6H
•6H
3
•6H
O, 10 mM ZnSO
O, mM CuCl •2H
O, 2 mM NaMoO •2H
and mM BO
2
O, 20 mM CaCl
2
•2H
•7H
O,
O, 2 mM
Specific
2
O, 10 mM
O, 2 mM
mM
respectively.
After
digestion
of
2
2
4
2
pCRISPomyces-2/ΔrufO-PAM with XbaI, both
the amplified fragments were simultaneously
introduced into the plasmid using In-Fusion
reaction to yield the gene disruption plasmid
pCRISPomyces-2/ΔrufO.
CoCl
2
2
2
2
2
2
NiSO
4
2
4
2
Na
2
SeO
3
,
2
H
3
3
.
modifications of media are described in respective
sections below. Unless mentioned otherwise, all
chemicals were purchased from Tokyo Chemical
Industry (Tokyo, Japan), Wako Pure Chemicals
Disruption of rufO.
Transformation of S. atratus was
carried out by a conjugation method with E. coli
ET12567/pUZ8002 (53). The E. coli cells
(
Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).
Enzymes and plasmids.
pCRISPomyces-2 (38) was purchased
harboring
cultivated in LB medium until the OD600 reached
0.5. The cells were harvested by centrifugation
5,000 g, for 15 min, at room temperature),
pCRISPomyces-2/ΔrufO
were
from Addgene (Cambridge, MA, USA). BbsI was
purchased from New England Biolabs (Ipswich,
MA, USA). Other restriction enzymes and
In-Fusion enzymes were purchased from Takara
Bio (Shiga, Japan)
~
(
washed twice with fresh LB medium and
resuspended in fresh LB medium. Spores of S.
atratus were harvested from the culture on ISP2
solid medium incubated for 7 days at 30°C. The
spores were suspended in TSB medium (0.5 ml)
and incubated at 50°C for 10 min. The spores and
E. coli cells were mixed and inoculated on MS
Construction of plasmids for rufO disruption.
A sequence containing a protospacer
and a protospacer adjacent motif (PAM) was
chosen in the reverse complementary sequence of
rufO. Primers containing this sequence, PAM-F/R,
2
solid medium containing MgCl (10 mM). After
incubation for 16 h at 30°C, the culture was
overlaid with sterilized water (1 ml) containing
nalidixic acid (0.75 mg) and apramycin (0.75 mg).
After a further cultivation for several days, a
transformant colony was picked up and inoculated
onto ISP2 solid medium. Then, the transformant
was repeatedly cultivated on ISP2 solid medium
(
Table S3) were annealed with each other, and the
product was introduced into the BbsI site of
pCRISPomyces-2,
resulting
in
pCRISPomyces-2/ΔrufO-PAM.
The
approximately 1-kb fragments of the 5'- and
'-flanking regions of the target sequence to be
3
1
2

Tyrosine nitration in rufomycin biosynthesis
without apramycin until the cells lost apramycin
resistance. Gene disruption was confirmed by
PCR using the primer pair DrufO-F/R (Table S3
and Fig. S2).
end of rufO. pET16b was applied to inverse PCR
using the primer pair 16b-inv-F/R (Table S3), and
the original N-terminal ten-histidine tag-coding
sequence was removed during the PCR. The
resultant two fragments were applied to the
In-Fusion reaction, and the plasmid obtained
(pET16b-rufO) was introduced into E. coli
BLR(DE3) after confirming the absence of
unintended mutations. The transformant was
cultivated at 37°C in TB with 5-aminolevulinic
Comparison of metabolic profiles and
rufomycin production.
S. atratus cells were cultivated in TSB,
ISP2, and MG2 media at 28°C for 7 days.
n-Butanol saturated with water was added to the
medium at a ratio of 1 : 1 (v/v). The organic layer
was concentrated in vacuo, and the residual
materials were dissolved in methanol and
subjected to an LC-electron spray ionization MS
-
1
-1
acid (80 mg l ), Fe(NH
4 2 4 2
) (SO ) (40 mg l ), trace
-
1
element solution (200 µl l ), and ampicillin (100
-
1
mg l ) until the OD
6
00
reached ~ 0.5. After
cooling the medium to room temperature,
isopropyl 1-thio-β-D-galactopyranoside (IPTG)
was added to a final concentration of 0.1 mM to
induce gene expression, and cells were cultivated
for a further 20 h at 15°C. Then, cells were
harvested, resuspended in lysis buffer (20 mM
Tris-HCl (pH 8.0) containing 200 mM NaCl and
(
LC-ESIMS) analysis in an 1100 series
spectrometer (Agilent Technologies) coupled to
high-capacity Trap Plus system (Bruker
Daltonics) equipped with a COSMOCORE 2.6
C
18 column (2.1 ID × 150 mm; Nacalai Tesque,
Kyoto, Japan). The compounds were separated
with a linear gradient of water and acetonitrile
containing formic acid (0.1%) at a flow rate of 0.4
1
0% (v/v) glycerol), and disrupted by sonication.
After centrifugation (20,000 g, 20 min, 4°C),
His60 Ni Superflow Resin was added to the
soluble cell extracts and mixed gently at 4°C for
-
1
ml min .
Production
and
purification
of
the
3
0 min. After the His-tagged proteins were eluted
recombinant RufO, CamA, and CamB
proteins.
with lysis buffer containing 0.5 M imidazole, the
buffer was exchanged for 25 mM Tris-HCl (pH
The coding region of the rufO gene
was first amplified from the genomic DNA of S.
atratus by PCR using the primer pair rufO-F/R
8
0
.0) containing 0.1 mM dithiothreitol (DTT) and
.1 mM ethylenediaminetetraacetic acid (EDTA)
using
Biosciences).
The pET28b-camA and pET28b-camB
a
PD-10 column (GE Healthcare
(
Table S3). During the PCR, an eight-histidine
tag-coding sequence was incorporated into the 3'
1
3

Tyrosine nitration in rufomycin biosynthesis
plasmids (54) were individually introduced into E.
coli BL21(DE3). The transformants were
cultivated at 37°C in LB medium with kanamycin
monoxide (CO) gas was bubbled into cuvette A
for 2-3 min. UV-Vis spectra of both the cuvettes
were measured, and the CO difference spectrum
was obtained by the subtraction of the spectrum of
cuvette B from that of cuvette A.
-
1
(
50 mg ml ) until the OD600 reached ~ 0.5. FeCl
3
was added to the culture medium for the camA
expression at a final concentration of 0.1 mM.
After cooling the medium to room temperature,
IPTG was added to a final concentration of 0.1
mM to induce expression, and cells were
cultivated for a further 12 h at 28°C. Cells were
harvested, resuspended in lysis buffer and
disrupted by sonication. After centrifugation
The RufO Fe(III)-NO complex was
investigated as follows. The protein solution (5
µM RufO in 25 mM Tris-HCl (pH 8.0) containing
0.1 mM DTT and 0.1 mM EDTA) was prepared.
Diethylamine
NONOate
(DEANO)
(Sigma-Aldrich) solution was prepared by
dissolving DEANO into the same buffer. The
protein solution was divided into two cuvettes, A
and B. An equivalent volume of the DEANO
solution was added to cuvette A, whereas the
buffer was added to cuvette B. UV-Vis spectra of
both the cuvette were measured, and the NO
difference spectrum was obtained by the
subtraction of the spectrum of cuvette B from that
of cuvette A.
(
20,000 g, 20 min, 4°C), His60 Ni Superflow
Resin was added to the soluble cell extracts and
mixed gently at 4°C for 30 min. After the
His-tagged proteins were eluted with lysis buffer
containing 0.5 M imidazole, the buffer was
exchanged for 25 mM Tris-HCl (pH 8.0)
containing 0.1 mM DTT and 0.1 mM EDTA using
a PD-10 column. The protein concentrations were
determined using NanoDrop (Thermo Fisher
Sceintific) with the millimolar coefficient of 29.1
for RufO, 38.8 for CamA, and 10.3 for CamB.
Substrate-binding analysis.
The protein solution (3.4 – 5 µM RufO
in 25 mM Tris-HCl (pH 8.0) containing 0.1 mM
DTT and 0.1 mM EDTA) was prepared, and
divided into two cuvettes, A and B. The potential
substrate was added to cuvette A (0.1-5 µM), and
the same volume of the buffer was added to
cuvette B. The solutions were mixed well. After 2
min, UV-Vis spectra of both the cuvettes were
measured. The spectrum of cuvette B was
subtracted from that of cuvette A, resulting in the
UV-Vis spectroscopic analysis.
The RufO Fe(II)-CO complex was
investigated as follows. The protein solution (5
µM RufO in 25 mM Tris-HCl (pH 8.0) containing
0
.1 mM DTT and 0.1 mM EDTA) was prepared,
and sodium hydrosulfite (Sigma-Aldrich, St.
Louis, MO, USA) was added. The solution was
divided into two cuvettes, A and B, and carbon
1
4

Tyrosine nitration in rufomycin biosynthesis
difference spectrum. For determination of the
dissociation constant (K ) with Tyr, the difference
The camA and camB genes were
introduced into pCDFDuet-1. The coding region
of camA was amplified from pET28b-camA by
PCR using the primer pair camA-F/R (Table S3).
pCDFDuet-1 was applied to inverse PCR using
the primer pair pCDFinv-F/R (Table S3). The
resultant two amplified fragments were applied to
d
in absorbance of each spectrum at 386 nm and 422
nm was calculated in triplicate, and the average
was plotted against the substrate concentration.
d
The K value was calculated by fitting the data
with a hyperbolic curve.
the
In-Fusion
reaction,
resulting
in
In vitro analysis using the recombinant
proteins.
pCDFDuet-1-camA. The coding region of camB
was amplified from pET28b-camB by PCR using
the primer pair camB-Nde-F/R (Table S3). This
fragment and NdeI-digested pCDFDuet-1-camA
were applied to the In-Fusion reaction, resulting in
pCDFDuet-1-camAB. After confirming the
In vitro enzymatic reaction mixtures
contained 3.4 µM recombinant RufO, 1.6 µM
recombinant CamA, 3.2 µM recombinant CamB,
1
2
mM Tyr, 1 mM NADH, and 1 mM DEANO in
5 mM Tris-HCl (pH 8.0) containing 0.1 mM
absence
of
unintended
mutations,
pCDFDuet-1-camAB and pET16b-rufO were
introduced together into the Tyr-overproducing E.
coli strain AN219. The transformant was
cultivated at 37°C in TB with 5-aminolevulinic
DTT and 0.1 mM EDTA. When the substrate
specificity of RufO was examined, Tyr was
replaced with Phe, Trp, 4-hydroxyphenylpyruvate,
or 4-aminophenylalanine. The reactions were
carried out at 28°C for 3 h with reciprocal shaking
at 1,500 rpm. After addition of HCl (0.15 M) to
the mixtures to solubilize Tyr and 3NTyr and
denature proteins, the mixtures were centrifuged
-
1
-1
acid (80 mg l ), Fe(NH
4 2 4 2
) (SO ) (40 mg l ), trace
-
1
element solution (200 µl l ), ampicillin (100 mg
-
1
-1
l ), kanamycin (50 mg l ), and streptomycin (50
-
1
mg l ) until the OD600 reached ~ 0.5. Then, IPTG
was added to a final concentration of 0.1 mM to
induce gene expression, and cells were cultivated
for a further 24 h at 15°C. DEANO was then
added to a final concentration of 0.1 mM, and
cells were further cultivated for an additional 24 h
at 15°C. After centrifugation (20,000 g, 5 min,
room temperature) to remove the cells, HCl (0.15
M) was added to the culture supernatant, followed
by the second centrifugation (20,000 g, 5 min,
(
20,000 g, 5 min, room temperature) and applied
to LC-ESIMS analysis. The compounds were
separated using COSMOSIL 5PYE column (2.0
ID x 150 mm) (Nacalai Tesque) with a linear
gradient of water and acetonitrile containing
-
1
formic acid (0.1%) at a flow rate of 0.4 ml min .
Generation of 3NTyr by a recombinant E. coli
strain expressing rufO and camAB.
1
5

Tyrosine nitration in rufomycin biosynthesis
room temperature) to remove the precipitation.
The supernatant was analyzed with LC-ESIMS as
described above.
1
6

Tyrosine nitration in rufomycin biosynthesis
ACKNOWLEDGEMENTS
This work was partially supported by CREST, JST (grant number JPMJCR13B3), Japan,
Japan Society for the Promotion of Science (JSPS) A3 Foresight Program, Grant-in-Aid for JSPS
Research Fellow (grant number 17J06071), and Amano Enzyme Inc., Japan. The ruf cluster was
deposited to DDBJ under the accession number LC257593. pET28b-camA and pET28b-camB used for
heterologous gene expression were kindly gifted by Dr. Akimasa Miyanaga and Dr. Akira Arisawa.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest with the contents of this article.
AUTHOR CONTRIBUTIONS
H.T. designed the study, performed experiments, analyzed data, and wrote the manuscript.
Y.K. designed the study, analyzed data, and wrote the manuscript. H.M. contributed to the experiment
shown in Fig. S3. Y.O. directed the research, analyzed data, and wrote the manuscript.
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2
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0

Tyrosine nitration in rufomycin biosynthesis
TABLES
Table 1. Predicted functions of ORFs in the ruf cluster.
ORF
rufA
rufB
rufC
rufD
rufE
rufF
rufG
rufH
rufI
Amino acids
339
267
375
48
Proposed function
LacI-like regulatory protein
α/β hydrolase
cytochrome P450
hypothetical protein
type I polyketide synthase
thioesterase
4819
238
33
hypothetical protein
MbtH-like protein
53
380
135
351
277
402
403
394
383
124
296
399
8027
aminotransferase
rufJ
hypothetical protein
ABC-transporter
rufK
rufL
rufM
rufN
rufO
rufP
rufQ
rufR
rufS
rufT
ABC-transporter
cytochrome P450
nitric oxide synthase
cytochrome P450
tryptophan N-dimethylallyltranferase
hypothetical protein
luciferase-like monooxygenase
cytochrome P450
nonribosomal peptide synthetase
2
1

Tyrosine nitration in rufomycin biosynthesis
FIGURES AND FIGURE LEGENDS
A
G H
F
ABCD
E
I JKL M N O PQR S
T
Cytochrome P450
PKS and NRPS
Others
1 kb
B
Loading
module
Module 1
Module 2 Module 3
Module 4
Module 5 Module 6
A
T
C
A
MT
T
C
A
T
C
A
T
C
A
MT
T
C
A
T
C
A
T
TE
Fig. 1. Gene organization of the ruf cluster (A) and domain organization of RufT (B). (A) Genes
encoding cytochrome P450 enzymes and PKS/NRPS are colored in gray and black, respectively.
Proposed functions of each ruf gene product are described in Table 1. (B) A, adenylation domain; T,
thiolation domain (peptidyl-carrier protein); C, condensation domain; MT, methylation domain; TE,
thioesterase domain.
2
2

Tyrosine nitration in rufomycin biosynthesis
A
RufE
RufF
Loading module Module 1
Module 2
ER
DH KR
DH
KR
KSQ AT ACP KS AT
ACP KS AT
ACP TE
S
S
S
O
O
O
OH
OH
OH
hydroxylation
oxidation
O
O
O
NH
2
transamination
O
RufI
RufC
L-trans-2-crotylglycine
trans-2-oxo-
-hexenoic acid
trans-4-
hexenoic acid
4
B
Nid KSQ VVDTGQSSSLV
Tyl KSQ VVDSAQSASLV
Ery KS1 SVDTACSSSLV
Ruf KSQ TVDTGQASSLA
C
Ery KR1B ATLDDGT...SSFASAFGA
Ery KR2A GLPQQVA...SSGAGVWGS
Ruf KR1 GIVQDAT...SSASGTLGA
Ruf KR2 GTTRDAA...SSVAGVTGS
Fig. 2. Putative pathway for trans-2-crotylglycine biosynthesis by the RufEF PKS. (A)
trans-4-Hexenoic acid is synthetized by the type I PKS consisting of RufE and RufF, and then it is
hydroxylated and oxidized by the cytochrome P450 RufC to trans-2-oxo-4-hexenoic acid. Finally,
trans-2-crotylglycine is generated by the transamination catalyzed by RufI. (B) Alignment of partial
Q
Q
amino acid sequences of KS and KS domains from type I PKSs. Nid, KS for niddamycin (AF016585);
Q
Q
Tyl, KS for tylosin (U78289); Ery, KS1 for erythromycin (X62569); Ruf, KS of the loading module
Q
for rufomycin (in this study). The key residue for distinguishing KS and KS is indicated by an arrow.
(
C) Alignment of partial amino acid sequences of KR domains from type I PKSs. Ery KR1B and KR2A,
B-type and A-type KRs for erythromycin (L07626 and X62569), respectively; Ruf KR1 and KR2, KRs
of module 1 and module 2, respectively, for rufomycin (in this study). The key residues for
distinguishing cis- and trans-type KRs are indicated by arrows.
2
3

Tyrosine nitration in rufomycin biosynthesis
rufomycins
Wild-type
4
1
2
3
ꢀ
ꢀ
rufO
rufO
+
3NTyr
25
30
Retention time (min)
Fig. 3. Metabolic analysis of the ΔrufO strain. Compounds were extracted from the whole culture
broth using n-butanol and separated by reverse-phase liquid chromatography. Four rufomycin congeners
(
1–4, see Fig. 7 for their structures) were detected. Top, wild-type; middle, ΔrufO; bottom, ΔrufO
supplemented with 3NTyr.
2
4

Tyrosine nitration in rufomycin biosynthesis
M------RufO CamA CamB
(
kDa)
75
50
37
2
2
5
0
1
5
0
1
Fig. 4. SDS-PAGE analysis of purified RufO, CamA and CamB proteins. After electrophoresis, the
gel was stained with Coomassie Brilliant Blue.
2
5

Tyrosine nitration in rufomycin biosynthesis
0.15
0.10
0.05
0
Fe(II)
0.30
Fe(III)
A
B
Fe(II)-CO
difference
Fe(III)-NO
difference
0.20
3
80
400
420
440
460
480
500
0.10
Wavelength
-0.05
-0.10
-0.15
(nm)
0
380
400
420
440
460
480
500
-0.05
Wavelength (nm)
2
0
0
0
20
0
ꢁM
C
D
0
0
0
0
0
.1 ꢁM
.2 ꢁM
.3 ꢁM
.4 ꢁM
.8 ꢁM
1
10
3
60
400
460
Wavelength
nm)
(
0
-10
0
0.2
0.4
Tyrosine] (ꢁM)
0.6
0.8
[
Fig. 5. UV-Vis spectroscopic study on RufO. (A) CO-binding spectra. Solid line, RufO with sodium
hydrosulfite and CO; dashed line, RufO with sodium hydrosulfite without CO; gray line, difference
spectrum (solid line – dashed line). (B) NO-binding spectra. Solid line, RufO with NO; dashed line,
RufO without NO; gray line, difference spectrum (solid line – dashed line). (C) Tyr-binding analysis. A
type I spectral change was observed following the titration of RufO with Tyr (up to 0.8 µM). (D)
Difference in absorbance between 386 nm and 422 nm versus the Tyr concentration. The data were fit by
a hyperbolic curve, and the dissociation constant was calculated. Assays were conducted in triplicate.
Error bars, standard deviations.
2
6

Tyrosine nitration in rufomycin biosynthesis
A
B
3NTyr
226.88
2
14.87
262.01
84.00
Tyr
authentic
180.90
2
Tyr and 3NTyr
1
50
200
250
300
m/z
C authentic 3NTyr
180.84
+
RufO
209.86
1
00
150
200
250
m/z
reaction product (+RufO)
1
80.85
–
RufO
10
209.86
0
5
Retention time (min)
1
00
150
200
250
m/z
Fig. 6. In vitro analysis of RufO. (A) UV chromatogram of the RufO reaction. Compounds were
separated by reverse-phase liquid chromatography. Top, authentic Tyr and 3NTyr; middle, in vitro
reaction with RufO; bottom, in vitro reaction without RufO. The sample with enzymes was injected and
analyzed prior to that without enzymes and authentic standards. (B) The mass spectrum of the reaction
+
product. 3NTyr (calculated m/z ([M+H] ) = 227.1) was clearly detected in the RufO reaction mixture.
(
C) The tandem mass spectrum of the reaction product was identical with that of authentic 3NTyr.
2
7

Tyrosine nitration in rufomycin biosynthesis
Citrulline
Trp
RufP
Tyr
3 x malonyl-CoA
NO
Arg
RufEF
RufC, RufI
RufN
RufO
OH
OH
O
N
O
2
N
OH
O
NH
2
O
NH
2
HO
NH
2
N-dimethylallyl-
tryptophan
3-nitrotyrosine
trans-2-crotylglycine
Leu
Ala
Leu
Leu
Loading module Module 1
Module 2 Module 3
Module 4
Module 5 Module 6
A
T
C
A
MT
T
C
A
T
C
A
T
C
A
MT
T
C
A
T
C
A
T
TE
RufT
S
S
S
S
S
S
S
O
O
O
O
O
O
O
O
O
O
2
O N
NH
2
N
NH
HN
N
HN
NH
N
O
HO
O
O
O
O
O
O
2
O N
NH
2
N
NH
HN
N
HN
N
O
HO
O
O
2
O N
NH
2
N
NH
HN
N
N
O
HO
O
O
2
O N
NH
2
N
NH
HN
N
O
HO
O
2
O N
NH
2
N
NH
N
O
HO
O
NH
2
N
N
O
NH2
N
O
2
N
HO
epoxidation
hydroxylation
oxidation
O
HN
R
2
cyclization and dehydration of 2
O
NH
N
N
O
O
H
N
O
HN
RufM
RufS
O
O2N
N
H
O
HO
N
O
R
1
O
NH
N
N
HN
O
O
H
N
N
O
rufomycin B (4)
ilamycin B1)
R1
=
, R2 = CH3
O
(
N
H
O
O
N
ilamycin (2)
R1
=
=
, R2 = CHO
O
O
3
1
R
1
, R = COOH
2
Fig. 7. Proposed pathway for the biosynthesis of rufomycin. Initially, three unusual amino acids,
N-dimethylallyltryptophan, 3NTyr, and trans-2-crotylglycine are synthesized. Subsequently, an NRPS
(
RufT) assembles seven amino acids using N-dimethylallyltryptophan as a starter amino acid, and then
the cyclic heptapeptide rufomycin B (4) is released. Two cytochrome P450 enzymes modify the peptide
occasionally to synthetize other derivatives.
2
8

Identification and characterization of a bacterial cytochrome P450 monooxygenase
catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis
Hiroya Tomita, Yohei Katsuyama, Hiromichi Minami and Yasuo Ohnishi
J. Biol. Chem. published online August 3, 2017
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