Characterization of SfmD as a heme peroxidase that catalyzes the regioselective hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine in saframycin A biosynthesis

Article abstract of DOI:10.1074/jbc.M111.306316

Saframycin A (SFM-A) is a potent antitumor antibiotic that belongs to the tetrahydroisoquinoline family. Biosynthetic studies have revealed that its unique pentacyclic core structure is derived from alanine, glycine, and non-proteinogenic amino acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me- OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic pathway of SFM-A, was hypothesized to be responsible for the generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr based on previously heterologous expression results. We now report the in vitro characterization of SfmD as a novel heme-containing peroxidase that catalyzes the hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydrogen peroxide as the oxidant. In addition, we elucidated the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of SfmD in combination with biochemical assays of SfmM2, a methyltransferase within the same pathway. Furthermore, SacD, a counterpart of SfmD involved in safracin B biosynthesis, was also characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of SFM-A and its analogs. Finally, we found that the conserved motif HXXXC is crucial for heme binding using comparative UV-Vis and Magnetic Circular Dichroism (MCD) spectra studies of SfmD wild-type and mutants. Together, these findings expand the category of heme-containing peroxidases and set the stage for further mechanistic studies. In addition, this study has critical implications for delineating the biosynthetic pathway of other related tetrahydroisoquinoline family members.

Full text of DOI:10.1074/jbc.M111.306316

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 7, pp. 5112–5121, February 10, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Characterization of SfmD as a Heme Peroxidase That
Catalyzes the Regioselective Hydroxylation of
3-Methyltyrosine to 3-Hydroxy-5-methyltyrosine in
□
S
Saframycin A Biosynthesis*
Received for publication, September 22, 2011, and in revised form, December 19, 2011 Published, JBC Papers in Press, December 20, 2011, DOI 10.1074/jbc.M111.306316
Man-Cheng Tang, Cheng-Yu Fu1, and Gong-Li Tang2
From the State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, China
Background: SfmD is a hypothetical protein thought to be a hydroxylase in saframycin A biosynthesis.
Results: SfmD binds heme and hydroxylates tyrosine analogs using H2O2 as oxidant.
Conclusion: SfmD is a heme peroxidase that catalyzes hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using
H2O2 as oxidant.
Significance: This study reveals a novel type of heme peroxidase and has significance for the biosynthesis of analogues of
saframycin A.
Saframycin A (SFM-A) is a potent antitumor antibiotic that tions for delineating the biosynthetic pathway of other related
tetrahydroisoquinoline family members.
belongs to the tetrahydroisoquinoline family. Biosynthetic
studies have revealed that its unique pentacyclic core structure
is derived from alanine, glycine, and non-proteinogenic amino
acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me-
OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic
pathway of SFM-A, was hypothesized to be responsible for the
generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr
based on previously heterologous expression results. We now
Tetrahydroisoquinoline alkaloids are a growing class of anti-
biotics possessing a characteristic tetrahydroisoquinoline
structure (1). Members of this family display a range of antitu-
mor, antimicrobial, and other biological activities depending on
their structures. Saframycin A (SFM-A,3 Fig. 1A), isolated from
report the in vitro characterization of SfmD as a novel heme- Streptomyces lavendulae NRRL 11002, is a representative
member of this family with potent antitumor activity that is
derived from iminium ion via a carbinolamine moiety (2). The
most well-known member of this family of compounds, ectei-
nascidin 743 (ET-743, Fig. 1A), which is a highly potent analog
of SFM-A, has been approved as an anticancer drug for the
treatment of advanced soft tissue sarcoma (1, 3). It shares a
central pentacyclic core with SFM-A, with the exception of the
oxidation state of their terminal rings and an additional macro-
lactone bridge found in ET-743. Moreover, most of the tetra-
hydroisoquinoline family members, such as saframycins,
safracins, naphthyridinomycins, and ecteinascidins, all possess
the same core quinone structure (Fig. 1B), which implies that
there may be some common precursors involved in their bio-
synthesis (1).
containing peroxidase that catalyzes the hydroxylation of
3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydro-
gen peroxide as the oxidant. In addition, we elucidated the bio-
synthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of
SfmD in combination with biochemical assays of SfmM2, a
methyltransferase within the same pathway. Furthermore,
SacD, a counterpart of SfmD involved in safracin B biosynthesis,
was also characterized as a heme-containing peroxidase, sug-
gesting that SfmD-like heme-containing peroxidases may be
commonly involved in the biosynthesis of SFM-A and its ana-
logs. Finally, we found that the conserved motif HXXXC is cru-
cial for heme binding using comparative UV-Vis and Magnetic
Circular Dichroism (MCD) spectra studies of SfmD wild-type
and mutants. Together, these findings expand the category of
heme-containing peroxidases and set the stage for further
mechanistic studies. In addition, this study has critical implica-
A series of biosynthetic studies have indicated that the qui-
none moiety is derived from the non-proteinogenic amino acid
3 The abbreviations used are: SFM-A, Saframycin A; ALA, 5-aminolevulinic
acid; Dopa, 3,4-dihydroxyphenylalanine; EPR, electron paramagnetic reso-
nance; ESI-MS, electrospray ionization-mass spectrometry; ET-743, Ectei-
nascidin 743; HPLC, high performance liquid chromatography; IDO,
indoleamine 2,3-dioxygenase; IPTG, isopropyl ␤-D-thiogalactopyranoside;
MCD, magneticcirculardichroism;3-Me-Tyr, 3-methyltyrosine;3-Me-OMe-
Tyr, 3-methyl-O-methyltyrosine; 3-OH-5-Me-Tyr, 3-hydroxy-5-methylty-
rosine; 3-OH-5-Me-OMe-Tyr, 3-hydroxy-5-methyl-O-methyltyrosine; OMe-
Tyr, O-methyltyrosine; SAC-B, Safracin B; SacD, a putative hydroxylase
involved in the biosynthesis of safracin B; SAM, S-adenosylmethionine;
SfmM2, a putative C-methyltransferase involved in the biosynthesis of
saframycin A; TCA, trichloroacetic acid; TDO, tryptophan 2,3-dioxygenase.
* This work was supported in part by grants from the National Basic Research
Program of China (973 Program) (2010CB833200 and 2012CB721100), the
National Natural Science Foundation of China (20832009 and 20921091),
and the Chinese Academy of Science (KJCX2-YW-H08).
□S
This article contains supplemental Figs. S1–S9 and Tables S1–S3.
1 Present address: Shaanxi Entry-Exit Inspection and Quarantine Bureau, 10
North Hanguang Road, Xi’an, Shaanxi 710068, China.
2 To whom correspondence should be addressed: Shanghai Institute of
Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Rd., Shang-
hai, 200032, China. Tel.: 86-21-54925113; Fax: 86-21-64166128; E-mail:
gltang@sioc.ac.cn.
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Characterization of SfmD as a Heme-containing Peroxidase
FIGURE 1. Structures of representative tetrahydroisoquinoline family members with their common features and the proposed biosynthetic pathway
of 3-OH-5Me-OMe-Tyr. A, structures of the representative members of tetrahydroisoquinoline family natural products. B, core quinone structure of these
members and its deduced biosynthetic precursors. C, proposed biosynthetic pathway of 3-OH-5-Me-OMe-Tyr involved in SFM-A or SAC-B biosynthesis.
precursor 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5- of Tyr and its derivatives using hydrogen peroxide (H2O2) as
Me-OMe-Tyr), which originates from tyrosine (Tyr) (4–9). the oxidant. From kinetic studies of SfmD and enzyme assays of
Recently, a three-gene cassette, sfmD/sfmM2/sfmM3 from the SfmM2, the true substrate of SfmD was identified as 3-methyl-
biosynthetic gene cluster of SFM-A or sacD/sacF/sacG from tyrosine (3-Me-Tyr), and the biosynthetic pathway of 3-OH-5-
the biosynthetic gene cluster of SAC-B, was reported to be Me-OMe-Tyr was also elucidated. This type of heme-contain-
responsible for the biosynthesis of 3-OH-5-Me-OMe-Tyr ing peroxidase was further demonstrated to be a potentially
(6–8). The hypothetical proteins, SfmD and its homolog SacD, common factor in the biosynthesis of SFM-A and its analogues,
were deduced to be hydroxylases responsible for the 3-hydroxy which possess the same core quinone structure as SFM-A, as
group construction based on heterologous expression results determined by characterization of SacD in vitro. Furthermore,
(7, 8). However, these proteins have no sequence similarity to comparative analyses of UV-Vis and Magnetic Circular Dichr-
the known characterized proteins, and little is known about oism (MCD) spectra of SfmD wild-type and SfmD mutants
these enzymes or the reactions that they catalyze.
revealed that the conserved motif HXXXC plays a crucial role in
Generally, several types of hydroxylases that catalyze heme binding.
hydroxylation on the aromatic ring of Tyr or its derivatives have
EXPERIMENTAL PROCEDURES
General Methods, Biochemicals, and Chemicals—DNA isola-
been reported (supplemental Fig. S1). One major type is pteri-
dine dependent, which is represented by Tyr hydroxylase that is
involved in the biosynthesis of catecholamine neurotransmit- tion and manipulation in Escherichia coli were performed
ters (10). Another type is tyrosinase, which is copper dependent according to standard methods (13). PCR amplifications were
and involved in the biosynthesis of melanin pigments (11). The carried out on an authorized thermal cycler (Eppendorf AG
third type is two-component, FAD-dependent monooxyge- 22331, Hamburg, Germany) using PrimeSTAR HS DNA
nase, such as SgcC, which was reported to catalyze the hydroxy- polymerase (TaKaRa, Japan). Primer synthesis and DNA
lation of ␤-Tyr tethered to a carrier protein during the biosyn- sequencing were performed at Shanghai Invitrogen Biotech
thesis of the enediyne antitumor antibiotic C-1027 (12). A Co., Ltd. (China). The E. coli DH5␣ cells were purchased from
common feature of these types of Tyr hydroxylases is that all of Invitrogen (Carlsbad, CA), and E. coli BL21 (DE3) cells were
them are non-heme-dependent monooxygenases and catalyze purchased from Novagen (Madison). Restriction enzymes were
the hydroxylation using oxygen as the oxidant.
purchased from TaKaRa Biotechnology Co., Ltd. (Dalian,
Distinct from these types of Tyr hydroxylases, we herein China). All chemicals and reagents were purchased from Sig-
report the in vitro biochemical characterization of SfmD as a ma-Aldrich or GL Biochem (Shanghai) Ltd. unless noted oth-
heme-containing peroxidase that catalyzes the hydroxylation erwise. Analytical HPLC was carried out on an Agilent 1200
FEBRUARY 10, 2012•VOLUME 287•NUMBER 7
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Characterization of SfmD as a Heme-containing Peroxidase
HPLC system with PDA detector. LC-MS analysis was carried
out on an Agilent 1200 HPLC instrument connected to LCQ
Fleet electrospray ionization (ESI) mass spectrometer (Thermo
Fisher Scientific Inc.). The electrospray ionization-mass spec-
troscopies (ESI-MS) were performed with a Shimadzu LCMS-
2010 EV mass spectrometer. NMR data were collected using a
Bruker 400 MHz spectrometer. Tyr, 3,4-dihydroxyphenylala-
nine (Dopa), O-methyltyrosine (OMe-Tyr), and DL-m-Tyr were
purchased from Sigma-Aldrich.
A modificatory procedure to overproduce SfmD in the pres-
ence of 5-aminolevulinic acid (ALA) was similar to the afore-
mentioned procedure, with the exception that ALA (1 mM, final
concentration) and (NH4)2Fe(SO4)2 (1 mM, final concentra-
tion) were added to the culture at the same time as IPTG. The
heme content of purified SfmD was determined by the pyridine
hemochrome assay as previously described (17).
In Vitro Enzymatic Assays of SfmD—The assay conditions
used to test whether SfmD is a heme-containing oxygenase are
similar to the assay conditions that reported for heme-contain-
ing oxygenases in the presence of oxygen, such as conditions for
cytochrome P450 (18), secondary amine oxygenase (19), heme
oxygenase (20), prostaglandin H Synthase (21), and tryptophan
2, 3-dioxygenase TioF (22).
Chemical Synthesis of Tyr Derivatives—The 3-methyl-O-
methyltyrosine (3-Me-OMe-Tyr) and 3-OH-5-Me-OMe-Tyr
were each prepared as described (6, 14, 15). Synthesis of 3-Me-
Tyr and 3-hydroxy-5-methyltyrosine (3-OH-5-Me-Tyr) were
achieved by following a previously reported method (6, 14, 15).
The experimental details and characterization of the interme-
diates are supplied under supplemental materials. The 1H NMR
The assay conditions used to test SfmD as a heme-containing
peroxidase are as follows. Standard assay (at 25 °C) mixtures (50
␮l) were composed of 100 mM Tris-HCl, pH 9.0, 1 mM ascorbic
acid, 2 mM H2O2, 1 mM 3-Me-Tyr (Tyr, OMe-Tyr, or 3-Me-
OMe-Tyr), and 50 ␮M SfmD. In the H218O2 assays, only H2O2
was replaced by H218O2. The reactions were started by adding
H2O2 and terminated by adding 0.5 ␮l of trichloroacetic acid
(TCA). Identical assays with boiled SfmD were carried out as
negative controls. Following centrifugation to remove protein,
the reactions were analyzed by HPLC or LC-MS using an ana-
lytic Inertsil ODS-EP column (5 ␮m, 4.6 ϫ 250 mm, GL Sci-
ences). The LC conditions were as follows. Solvent A was H2O,
and Solvent B was CH3CN; both solvent contained 0.1% TFA
(v/v) in the HPLC analysis or 0.1% HCO2H (v/v) in the LC-MS
analysis. The column was equilibrated with 100% solvent A and
followed by the following linear gradient program: 100% A/0%
B, 0–3 min; ramp to 28% B, 3–15 min; ramp to 95% B, 15–17
min; and return to 100% A/0% B, 17–20 min. The flow rate was
1 ml minϪ1, and elution was monitored at 276 nm.
1
assignments of 3-Me-Tyr are as follows: H NMR (400 MHz,
D2O): ␦ 2.18 (s, 3H), 3.06ϳ3.25 (m, 2H), 4.21ϳ 4.24 (m, 1H),
6.85 (d, 1H, J ϭ 8.0Hz), 7.01 (d, 1H, J ϭ 8.0Hz), 7.08 (s, 1H); MS
ϩ
1
(ESI): m/z 196.18 ([MϩH] ). The H NMR assignments of
1
3-OH-5-Me-Tyr are as follows: H NMR (400 MHz, D2O): ␦
2.21 (s, 3H), 3.04ϳ3.23 (m, 2H), 4.24ϳ4.34 (m, 1H), 6.67 (s, 1H),
ϩ
6.70 (s, 1H); MS (ESI): m/z 212.0 ([MϩH] ).
Cloning, Expression, and Purification of SfmD—The sfmD
gene was PCR-amplified from plasmid pTL2027 (8) as a tem-
plate with the primers listed in supplemental Table S1. The
purified PCR product was cloned into the EcoRI/HindIII site of
pSP72 to generate pTL2039. When the sequence was verified, a
1.1 kb NdeI/HindIII fragment recovered from pTL2039 was
ligated into the same site of pET37b to afford the expression
plasmid pTL2040. C-terminal His8-tagged SfmD was overex-
pressed in E. coli BL21 (DE3). A 3 ml starter culture was grown
Ϫ1
overnight from a single colony in LB media with 50 ␮g ml
The optimized reaction conditions for SfmD were deter-
mined by varying the assay pH (5.0–9.5). Kinetic studies of the
protein were performed by changing the substrates (Tyr or
3-Me-Tyr) concentrations from 25 to 2000 ␮M under opti-
mized conditions with 4 mM H2O2. Product formation was
determined using HPLC. Each data point represents a mini-
mum of three replicate end point assays; kinetic constants were
obtained by nonlinear regression analysis using OriginLab
OriginPro (OriginLab software, Northampton, MA).
kanamycin at 37 °C with shaking and then used to inoculate 1
liter of LB medium at 37 °C with 50 ␮g mlϪ1 kanamycin for 2 h.
The cultures were then cooled to 16 °C for 1 h, induced with 0.1
mM IPTG when the A600 reached 0.5, and incubated at 16 °C for
an additional 24 h.
All protein purification steps took place at 4 °C. Protein puri-
fication buffers contained 500 mM NaCl, 50 mM sodium phos-
phate adjusted to pH 8.0, 10% glycerol and increasing concen-
trations of imidazole. Buffers A (lysis), B (wash), and C (elution)
contained 10, 50, and 250 mM imidazole, respectively. The cells
were harvested by centrifugation (6000 rpm for 5 min at 4 °C)
and resuspended in buffer A. The cells were lysed by sonication
(10 ϫ 60 s pulsed cycle), and the debris was removed by centrif-
ugation (12,000 rpm for 60 min at 4 °C). Soluble protein was
collected and purified on a Ni-NTA column by washing with
several volumes of buffer B and eluting with 10 ml of buffer C.
The eluant was concentrated and desalted into 50 mM Tris-HCl
buffer (pH 8.0), 50 mM NaCl, and 10% glycerol using a PD-10
column (GE Healthcare). The purified proteins were concen-
trated using an Amicon Ultra-4 (10K, GE Healthcare), stored as
10% glycerol stocks at Ϫ80 °C, and utilized without further
modifications. Protein purity was assessed by 12% acrylamide
SDS-PAGE. Protein concentration was determined by the
Bradford method (16) using a BSA calibration curve.
Cloning, Expression, Purification, and Biochemical Charac-
terizations of SacD—The construction procedure for the
expression plasmid of sacD was similar to sfmD. Plasmid
pTL2031 (8) was used as the PCR template. The resulting
expression plasmid pTL2044 is a derivative of pET37b, in which
SacD will be overproduced as a C-terminal His8-tagged fusion
protein. The expression and purification of SacD were carried
out in a manner similar to the modificatory procedure of SfmD.
The purified proteins were utilized without further modifica-
tions. The biochemical characterizations of SacD were carried
out similar to the procedures of SfmD.
Cloning, Expression, and Purification of SfmM2—The con-
struction procedure for the expression plasmid of sfmM2 was
similar to sfmD. Plasmid pTL2027 (8) was used as the PCR
template. The resulting expression plasmid pTL2042 is a deriv-
ative of pET37b. C-terminal His8-tagged SfmM2 was overex-
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Characterization of SfmD as a Heme-containing Peroxidase
pressed in E. coli BL21 (DE3). The expression and purification
procedures of recombinant SfmM2 were carried out in a man-
ner similar to that described for SfmD. The purified proteins
were utilized without further modifications.
Characterization of the C-methylation Function of SfmM2 in
Vitro—Standard reactions (50 ␮l) consisted of 100 mM Tris-
HCl (pH 8.0), 2 mM SAM, 1 mg mlϪ1 BSA, 1 mM Tyr (OMe-Tyr,
3-Me-OMe-Tyr, or DL-m-Tyr), and 50 ␮M SfmM2 at 30 °C. The
reactions were started by adding SfmM2 and terminated by
adding 0.5 ␮l of TCA. Identical assays with boiled SfmM2 were
carried out as negative controls. Following centrifugation to
remove protein, the reactions were analyzed by HPLC or
LC-MS using an analytic Inertsil ODS-EP column (5 ␮m, 4.6 ϫ
250 mm, GL Sciences). The LC conditions were as follows. Sol-
vent A was H2O, and Solvent B was CH3CN; both solvent con-
tained 0.1% formic acid (v/v) in the HPLC or LC-MS analysis.
The column was equilibrated with 100% solvent A and followed
by a linear gradient program: 100% A/0% B, 0–3 min; ramp to
16% B, 3–20 min; ramp to 95% B, 20–23 min; and return to
100% A/0% B, 23–26 min. The flow rate was 1 ml minϪ1 and
elution was monitored at 276 nm.
Construction of SfmD Mutants—The specified mutants of
SfmD were performed by site-specific mutagenesis using the
QuickChange Muti-Site Directed Mutagenesis Kit (Stratagene)
according to the manufacture’s introductions. The pTL2039
vector was used as the template, and the primers listed in sup-
plemental Table S2 were used for the specified mutant. Conse-
quently, the mutated versions of sfmD were confirmed by
sequencing and then cloned into pET37b using the same strat-
egy to make pTL2040 for native SfmD expression respectively.
Protein expression and purification of these mutants were car-
ried out in a manner similar to the modification procedure
described for SfmD wild type (WT). The enzyme assays of
mutants were also similar to the assays of SfmD WT.
FIGURE2. ProteinpurificationandcofactoridentificationofSfmD. A, puri-
ficationofrecombinantC-His8-taggedSfmDasmonitoredbySDS-PAGE. Lane
M, molecular weight marker; lane 1, total proteins after IPTG induction; lane 2,
total soluble proteins; lane 3, purified protein. B, UV-Vis spectra of SfmD. SfmD
without (i) and with (ii) addition of ALA when the protein was induced to
express. C, characterization of SfmD by LC-MS. SfmD without (i) and with (ii)
addition of ALA during the protein expression. D, pyridine hemichrome and
hemochrome assays of SfmD, the amount of heme bound to SfmD was cal-
culated by following the absorbance change at 553 nm using a difference
extinction coefficient of 23.76 mMϪ1 cmϪ1 (17). (i) the hemochrome of SfmD;
(ii) the hemichrome of SfmD.
Spectroscopic Techniques—UV-Visible absorption spectra
were recorded with a Jasco V530 spectrophotometer at room
temperature. MCD spectra were measured in 0.1-cm cuvettes
at a magnetic field of 0.75T with the Jasco J810 spectropolarim-
eter at 4 °C as previously described (23). MCD manipulations
were carried out as previously reported (23) using JASCO soft-
ware. The electron paramagnetic resonance (EPR) spectra of
SfmD WT were carried out on a Bruker EMX plus 10/12 spec-
trometer system (Bruker Co., Ltd., Germany) at the High Mag-
netic Field Laboratory, Chinese Academy of Sciences, Hefei, was obtained, a structural homology search was performed
China. Samples were prepared in Tris-HCl (100 mM, pH 7.5) using the online program HHpred, which revealed that the C
buffer to appropriate concentrations for SfmD WT and its terminus of SfmD was structurally homologous to tryptophan
mutants.
2, 3-dioxygenase (TDO) from Ralstonia metallidurans and
indoleamine 2, 3-dioxygenase (IDO) from human. Both of these
proteins are heme-containing oxygenases that catalyze the oxi-
RESULTS
Bioinformatics Analysis of SfmD—BLAST analysis of the dative cleavage of tryptophan to N-formyl kynurenine. How-
365-amino acid sequence of SfmD revealed that SfmD had no ever, further investigation was needed to determine whether
sequence similarity to any functionally characterized proteins SfmD is a heme-containing protein.
in the NCBI database, and no putative conserved domain was
Enzyme Purification and Cofactor Identification of SfmD—
identified. There were only three hypothetical proteins that Recombinant SfmD was expressed in E. coli BL21 (DE3) for in
showed homology to SfmD: SacD, which is involved in safracin vitro characterization. The C-terminal His8-tagged enzyme was
biosynthesis (51% similarity), AZL_f00720 from Azospirillum purified by Ni-NTA affinity chromatography and migrated as a
sp. B510 (45% similarity), and KSE_08540 from Kitasatospora single band at the predicted size of 39.8 kDa based on SDS-
setae KM-6054 (43% similarity). Because no useful information PAGE analysis (Fig. 2A). The purified SfmD was green and
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Characterization of SfmD as a Heme-containing Peroxidase
exhibited absorbance maxima at 404 nm in UV-Vis spectra (Fig. 3-Me-Tyr and resulted in an [Mϩ2] product as expected (Fig.
2B), which were similar to other heme-containing proteins 3D). Therefore, these data provided in vitro validation that
reported in literature (24, 25). When subjected to LC-MS anal- SfmD is a heme-containing peroxidase that catalyzes the regi-
ysis, the major peak was found to be 39,854.6 Da (Fig. 2C), oselective hydroxylation of aromatic amino acid using H2O2 as
which is in accordance with the calculated molecular weight the oxidant.
(MW) of SfmD (39,854.12 Da) without any cofactor. Mean-
Substrate Specificity and Kinetics of SfmD—To identify
while, another minor peak, 40,469.9 Da, which represents an enzyme substrate specificity, the four plausible substrates were
additional 615.3 Da, was also detected, indicating that parts of assayed. As shown in supplemental Fig. S2, both Tyr and 3-Me-
the proteins may be co-purified with cofactors. Among the Tyr could be hydroxylated, indicating that SfmD had some sub-
known cofactors, it was close to the calculated MW of heme strate flexibility. For kinetic analysis, assay conditions were
(616.43 Da). We then overexpressed and purified SfmD again studied at varying pH values (ranging from 5.0 to 9.5 in 0.5
using a modified procedure where 5-aminolevulinic acid steps). The optimized condition was found to be at pH 9.0 (sup-
(ALA), a biosynthetic precursor of heme, was added to the cul- plemental Fig. S3). Under this condition, the kinetic studies
ture broth at the time of protein expression induction. As toward Tyr and 3-Me-Tyr were carried out in the presence of
expected, not only was the absorption intensity at 404 nm in excess H2O2 (Fig. 4). SfmD showed a conversion with a Km of
UV-Vis spectra remarkably increased (Fig. 2B), but the abun- 0.64 Ϯ 0.09 mM and kcat of 17.8 Ϯ 1.2 minϪ1 toward 3-Me-Tyr
dance of the peak at 40,470 Da in the mass spectrum was also and Km of 1.0 Ϯ 0.2 mM and kcat of 12.3 Ϯ 1.2 minϪ1 toward Tyr.
increased (Fig. 2C). Together, these data indicated that SfmD is The higher specificity (kcat/Km, shown in Fig. 4) of SfmD toward
a heme-containing protein. To quantify the amount of heme 3-Me-Tyr over Tyr indicated that 3-Me-Tyr should be the true
bound to the protein, pyridine hemochrome assays (17) were substrate of SfmD in vivo.
carried out. The results showed that 5 ␮M SfmD bound 4.6 Ϯ
0.2 ␮M heme (mean Ϯ S.D., n ϭ 3) (Fig. 2D), indicating that one Me-OMe-Tyr—To further investigate the biosynthetic pathway
SfmD binds one molecular of heme. of 3-OH-5-Me-OMe-Tyr, SfmM2, the putative C-methyltrans-
Confirming the Biosynthetic Pathway of 3-OH-5-
Characterization of SfmD as a Heme-containing Peroxi- ferase, was overexpressed and purified as an N-His8 tagged
dase—Heme-containing proteins generally fall into two large recombinant protein. The enzyme migrated as a protein with a
superfamilies: heme-containing oxygenases, which usually use MW of ϳ42 kDa by SDS-PAGE analysis, which was close to the
oxygen as the oxidant, and heme-containing peroxidases, predicted MW of SfmM2 (41.8 kDa) (supplemental Fig. S4A).
which usually use peroxides as the oxidant (25, 26). Because In vitro biochemical assays using this recombinant protein indi-
SfmD showed structural homology to TDO and IDO, we first cated that only Tyr can be converted to the corresponding
tested whether SfmD was an oxygen-dependent protein. Vari- methylated product 3-Me-Tyr among the tested substrates
ous assays of SfmD with the four plausible substrates (Tyr, (supplemental Fig. S5). Based on the assay results of SfmD and
3-Me-Tyr, OMe-Tyr, and 3-Me-OMe-Tyr) were carried out SfmM2, we confirmed the biosynthetic pathway of 3-OH-5-
using the assay procedures reported in the literature for heme- Me-OMe-Tyr as follows (Fig. 1C): Tyr is first methylated to
containing oxygenases in the presence of oxygen (see “Experi- 3-Me-Tyr by SfmM2, and then SfmD catalyzes the hydroxyla-
mental Procedures”). Unfortunately, no corresponding tion of 3-Me-Tyr to 3-OH-5-Me-Tyr. Finally, 3-OH-5-Me-Tyr
hydroxylation products were detected under these conditions is transformed to 3-OH-5-Me-OMe-Tyr by the putative
(data not shown). We then examined whether SfmD can use O-methyltransferase SfmM3.
H2O2 as the oxidant. To our surprise, when SfmD was added
Enzymatic Characterizations of SacD—From the biosyn-
into the solution of H2O2, a slow dismutation of H2O2 occurred thetic pathway of SAC-B, there is a homolog of SfmD that was
(Fig. 3A), demonstrating that SfmD has very weak catalase named as SacD. To test whether the properties or functions of
Ϫ1
activity (the turnover number was only ϳ0.6 s calculated SacD were similar to SfmD, we overexpressed the protein in
from the oxygen electrode experiment, see supplemental infor- E. coli and purified it as a C-His8 tagged recombinant protein
mation). Additionally, low temperature (10 K) EPR was used to (supplemental Fig. S4B). The purified SacD showed a maxima
characterize the properties of SfmD. Spectra were recorded absorbance at 404 nm in UV-Vis spectra (Fig. 5A) and a weak
before and after the addition of H2O2 to the enzyme in the ability to dismutate H2O2 (data not shown). In the in vitro assays,
absence of reducing substrates. The spectrum of SfmD was SacD also used H2O2 as the oxidant, displayed an optimal activity
dominated by the high-spin ferric signals (g ϭ 5.9 and gʈ ϭ 2.0, at pH 9.0 (supplemental Fig. S6), and converted 3-Me-Tyr to the
Fig. 3B), which was similar to the EPR spЌectra of the resting corresponding hydroxylation product, 3-OH-5-Me-Tyr (Fig. 5B).
peroxidases (27). After the addition of H2O2, the spectra of These results indicated that, like SfmD, SacD is also a heme-con-
peroxide-activated SfmD showed a free radical with g ϳ1.99, taining peroxidase that catalyzes the hydroxylation of aromatic
which was in agreement with what was previously assigned to amino acids using H2O2 as the oxidant.
the porphyrin radical in heme peroxidase Compound I (28).
Determination of the Heme-binding Site of SfmD—In heme-
Moreover, when we incubated 3-Me-Tyr with SfmD in the containing proteins, there are usually one or more conserved
presence of H2O2, it was completely converted to a new com- motifs that are crucial for heme binding, such as FXXGXXX-
pound that had an HPLC retention time and MS that were CXG in P450s (29), CXXCH in c-type cytochromes (30), HPGG
identical to the authentic product 3-OH-5-Me-Tyr (Fig. 3C). in cytochrome b5 (31), CPs in proteins regulated by heme (32),
To confirm that the oxygen atom that was inserted in the prod- GXXDG in DyP-type peroxidases (24), and others. Sequence
uct originated from H2O2, H218O2 assays were conducted with alignment of SfmD and its analogous (SacD, AZL_f00720, and
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Characterization of SfmD as a Heme-containing Peroxidase
FIGURE 3. Characterization of SfmD as a heme-containing peroxidase. A, H2O2 dismutation experiments recorded in UV-Vis spectra at 240 nm. The solution
of H2O2 in the absence (i) or presence (ii) of SfmD (25 ␮M). B, low temperature EPR spectra of SfmD (50 ␮M). Spectra of SfmD resting state (top) indicating the
high-spin ferric signals (g ϭ 2.0 and g ϭ 5.9) and its peroxide-activated form (Compound I) (bottom). C, HPLC analysis of the hydroxylation of 3-Me-Tyr to
3-OH-5-Me-Tyr catalyzed by SfmD: authentic standard of 3-OH-5-Me-Tyr (i) and 3-Me-Tyr (ii), reaction with O2 (iii) and H2O2 (iv) as the oxidant, negative control
with boiled enzyme (v); (●), 3-OH-5-Me-Tyr, (␱), 3-Me-Tyr. D, LC-MS analysis of the production of 3-OH-5-Me-Tyr using H2O2 (i) and H218O2 (ii) as the oxidant.
KSE_08540) did not identify any of these conserved motifs, but and these mutants demonstrated that the signal intensity of
three other conserved motifs were found, including WXXXH, SfmD WT, SfmD H191A, and SfmD H274A were comparable
SGXXXXDH, and HXXXC (Fig. 6). To test whether these motifs and the signal intensity of SfmD H313A and SfmD C317A were
are important for heme binding, we constructed several very weak (Fig. 7B), indicating that the mutants H313A and
mutants of SfmD, such as H191A, H274A, H313A, and C317A, C317A had very little heme-Fe binding ability. Together, these
by site-specific mutagenesis, since the residues His and Cys are data confirmed that the conserved motif HXXXC found in
usually essential for heme binding. These mutants were over- SfmD is crucial for heme binding. In addition, we found that all
expressed and purified in E. coli using the same conditions as the four mutants of SfmD lost the ability to hydroxylate 3-Me-
SfmD WT (supplemental Fig. S7). When the UV-Vis spectra Tyr (supplemental Fig. S8), suggesting that the other two con-
were measured, the absorbance intensity was dramatically served motifs also play important roles in the catalytic cycles.
decreased in SfmD H313A and SfmD C317A compared with
DISCUSSION
SfmD WT, SfmD H191A and SfmD H274A (Fig. 7A), suggest-
ing that the heme content of SfmD H313A and SfmD H317A
Tyr and its derivatives not only play important physiolog-
may be very low. Furthermore, the MCD spectra of SfmD WT ical roles in living cells but also are precursors to many sec-
FEBRUARY 10, 2012•VOLUME 287•NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 5117

Characterization of SfmD as a Heme-containing Peroxidase
FIGURE 5. UV-Vis spectra of SacD (A) and HPLC analysis of the hydroxyla-
tion of 3-Me-Tyr to 3-OH-5-Me-Tyr catalyzed by SacD (B). (i) Authentic
standard of 3-Me-Tyr; (ii) authentic standard of 3-OH-5-Me-Tyr; (iii) negative
control with boiled enzyme; (iv) reaction mixture; (●), 3-OH-5-Me-Tyr, (E),
3-Me-Tyr.
FIGURE 4. Kinetic analysis of the hydroxylation reaction catalyzed by
SfmD with substrate 3-Me-Tyr (A) or Tyr (B). The reaction mixtures consist
of 50 ␮M protein, excess H2O2 (4 mM) and variable substrate (25 ␮M-2 mM)
under optimized conditions.
tives) hydroxylases. Based on the UV-Vis spectra and LC-MS
ondary metabolites with bioactivities. For example, L-Dopa, analysis of purified SfmD expressed in the absence or presence
the aromatic ring hydroxylation product of Tyr, is marketed of ALA, we found that SfmD could bind heme as a cofactor.
as a psychoactive drug for use in the clinical treatment of Moreover, the results of the pyridine hemochrome assays indi-
Parkinson’s disease and dopamine-responsive dystonia. In cated that SfmD binds heme as a 1:1 complex. These findings
addition, it is also an important precursor to the cate- confirmed that SfmD is a heme-containing protein that is dif-
cholamine neurotransmitters exemplified by dopamine and ferent from the known Tyr (or its derivatives) hydroxylase.
adrenaline (10) as well as the bioactive secondary metabo-
Heme-containing proteins generally fall into two superfami-
lites exemplified by lincomycin (33) and CC-1065 (34). Sev- lies: heme-containing oxygenases, which usually use oxygen as
eral types of enzymes have been reported to catalyze the the oxidant, and heme-containing peroxidases, which usually
hydroxylation of the aromatic ring of Tyr or its derivatives, use peroxide as the oxidant. No hydroxylation activity of SfmD
such as pteridine-dependent Tyr hydroxylase, copper-de- was detected using the conditions reported for oxygenases with
pendent tyrosinase, FAD-dependent SgcC, and others. A the plausible substrates in the presence of oxygen (see “Exper-
common feature of these enzymes is that all of them are imental Procedures”). To our surprise, SfmD showed some cat-
oxygen-dependent non-heme-containing monooxygenases. alase activity that could dismutate H2O2. Moreover, in the pres-
Recent reports have found that the non-proteinogenic amino ence of H2O2, SfmD could catalyze the hydroxylation of 3-Me-
acid precursor 3-OH-5-Me-OMe-Tyr originates from Tyr in Tyr to 3-OH-5-Me-Tyr. Further analysis using an H218O2 assay
the biosynthesis of SFM-A (6, 8). SfmD, a hypothetical protein confirmed that the oxygen inserted into the product originated
thought to be responsible for the generation of the 3-hydroxy from H2O2. These data indicate that SfmD is a H2O2-depen-
group (8), shows no homology to the known Tyr (or its deriva- dent heme-containing protein. As reported in the literature,
5118 JOURNAL OF BIOLOGICAL CHEMISTRY
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Characterization of SfmD as a Heme-containing Peroxidase
FIGURE6. MultiplesequencealignmentofSfmDanditsanalogs. AminoacidsequenceswereobtainedfromGenBankTM, including:SfmDfromStreptomyces
lavendulae NRRL 11002; SacD from Pseudomonas fluorescens A2–2; AZL_f00720 from Azospirillum sp. B510; KSE_08540 from Kitasatospora setae KM-6054.
there are two types of heme-containing proteins that are H2O2- (peroxidase-cyclooxygenase and peroxidase-catalase super-
dependent: heme-containing peroxidases and H2O2-depend- family) and three families (dyp-type peroxidases, heme-halo-
ent P450s. To determine the type that SfmD belongs to, the peroxidases and di-heme peroxidases) (36). Among these,
classical carbon monoxide (CO) binding experiments (35) were heme-thiolate peroxidases, such as heme-thiolate haloperoxi-
carried out. As shown in supplemental Fig. S9 and Table S3, the dases, and heme-thiolate aromatic peroxidases, were reported
Soret band of SfmD changed to 418 nm, not ϳ450 nm, after to possess the ability to transfer oxygen from peroxides to var-
treatment with Na2S2O4 and CO, which ruled out the H2O2- ious organic substrates including aromatic compounds (37, 38).
dependent P450 family of proteins. Moreover, SfmD showed And also, horseradish peroxidase was reported to be effective in
properties similar to the heme-containing peroxidase in low oxidizing Tyr to Dopa in the presence of dihydroxyfumaric acid
temperature EPR studies. Therefore, we concluded that SfmD and oxygen (39). But until now, none of the heme-containing
is a heme-containing peroxidase that catalyzes the hydroxyla- peroxidases listed in PeroxiBase has been shown to catalyze the
tion of Tyr or its derivatives using H2O2 as the oxidant.
aromatic ring hydroxylation of Tyr (or its derivatives) in the
Heme-containing peroxidases are widely distributed among presence of H2O2. SfmD shows no homology to these heme-
bacteria, archaea, and eukarya, and catalyze one- and two-elec- containing peroxidases listed in the PeroxiBase, and is a novel
tron oxidation reactions of a great diversity of inorganic and heme-containing peroxidase reported to catalyze the aromatic
organic compounds with H2O2. All currently available gene ring hydroxylation of Tyr or its derivatives using H2O2 as the
sequences of these metalloenzymes (listed in the PeroxiBase oxidant. Very recently, Orf13 involved in the biosynthesis of
website) can be phylogenetically divided into two superfamilies anthramycin was also reported to be a heme-containing perox-
FEBRUARY 10, 2012•VOLUME 287•NUMBER 7
JOURNAL OF BIOLOGICAL CHEMISTRY 5119

Characterization of SfmD as a Heme-containing Peroxidase
SfmD-like heme-containing peroxidase. Together with the
assay results from C-methyltransferase SacF, where only Tyr
can be methylated,4 the biosynthetic pathway of 3-OH-5-Me-
OMe-Tyr involved in the biosynthesis of SAC-B was found to
be identical to the aforementioned pathway (Fig. 1C). In addi-
tion, it has been shown that naphthyridinomycin, which pos-
sesses the same core quinone structure as SFM-A, can incorpo-
rate Tyr, 3-Me-Tyr, and 3-OH-5-Me-Tyr, but not Dopa (5).
These data are consistent with our results that were character-
ized in vitro. Together, these findings indicate that the SfmD-
like heme-containing peroxidase may be commonly involved in
the biosynthesis of tetrahydroisoquinoline family members
that feature the same core quinone structure with SFM-A, and
imply that the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr
might be similar in the biosynthesis of these members.
In addition to SacD, there are two other homologs of SfmD in
the NCBI data base, AZL_f00720 and KSE_08540, both of
which are hypothetic proteins from genome sequences. Both
are all located in putative secondary metabolite gene clusters,
although the functions are yet to be established. A sequence
alignment of SfmD with its analogs identified three conserved
motifs within the C-terminal domain. Mutation studies con-
firmed that the conserved motif HXXXC was crucial for heme
binding. This motif is different from the known heme binding
motifs reported in other heme-containing proteins, such as
FXXGXXXCXG, CXXCH, HPGG, CP, and GXXDG, which sug-
gests that SfmD and its analogs may belong to a novel type of
heme-containing protein family. In addition, these SfmD
mutants lost the ability to hydroxylate 3-Me-Tyr, suggesting
that the other two conserved motifs also play important roles in
the catalytic cycle. However, the actual functions of these
motifs in the catalytic cycle are still unknown and will require
additional structural investigations.
FIGURE 7. UV-Vis spectra (A) and MCD spectra (B) of SfmD wild type and
mutants. SfmD WT (black solid), SfmD H191A (purple dot), SfmD H274A (green
dot), SfmD H313A (red dot), SfmD C317A (blue dash).
In conclusion, we found that SfmD, along with its analog
SacD, are clearly heme-containing peroxidases that catalyze the
regioselective hydroxylation of 3-Me-Tyr to 3-OH-5-Me-Tyr
idase catalyzing the hydroxylation of Tyr to Dopa in the pres- using H2O2 as the oxidant. These findings not only have signif-
ence of H2O2 around the time we submitted this article (40). icance for the biosynthesis of other tetrahydroisoquinoline
And Orf13 also had the ability to catalyze the hydroxylation of family members that possess the same core quinone structure
Tyr to Dopa by a molecular oxygen-dependent pathway in the with SFM-A, but also set the stage for elucidation of the mech-
presence of dihydroxyfumaric acid or ascorbic acid (40). But anism for these newfound heme-containing peroxidases.
SfmD showed no hydroxylation activity under the same condi-
Acknowledgments—We thank Prof. Ben Shen, Departments of Chem-
istry and Molecular Therapeutics and Natural Products Library Ini-
tiative at The Scripps Research Institute, and Dr. Xu-Dong Qu of
Shanghai Institute of Organic Chemistry for valuable advice and sug-
gestions. We also thank Prof. Jian-Hua Ju and Dr. Bo Wang of South
China Sea Institute of Oceanology, Chinese Academy of Sciences to
help us order H218O2; Prof. Zi-Xin Deng’ Laboratory of Shanghai Jiao-
Tong University for support in obtaining MS data of proteins; Prof.
Yu-Heng Zhang and Wei Tong of High Magnetic Field Laboratory,
Chinese Academy of Science for assistance with EPR analysis.
tions as reported for Orf13 using oxygen as the oxidant (data
not shown). And meanwhile, SfmD could dismutate H2O2,
while Orf13 would be inactivated when only reacted with H2O2.
In addition, these two proteins show no homology to each
other. All these indicate that distinct from Orf13 SfmD belongs
to another type of heme-containing peroxidase.
Among the plausible substrates, both Tyr and 3-Me-Tyr can
be hydroxylated by SfmD. Kinetic study results indicated that
the preferred substrate of SfmD is 3-Me-Tyr. In combination
with the assay results from SfmM2, we ascertained the biosyn-
thetic pathway of 3-OH-5-Me-OMe-Tyr. As shown in Fig. 1C,
Tyr is methylated to 3-Me-Tyr, followed by SfmD-mediated
hydroxylation to 3-OH-5-Me-Tyr, and finally transformed to
3-OH-5-Me-OMe-Tyr. Moreover, three similar proteins,
SacD/SacF/SacG, were also found to be involved in the biosyn-
REFERENCES
1. Scott, J. D., and Williams, R. M. (2002) Chemistry and biology of the
tetrahydroisoquinoline antitumor antibiotics. Chem. Rev. 102,
thesis of SAC-B. In this study, we also characterized SacD as a 4 G. Tang, unpublished data.
5120 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 7•FEBRUARY 10, 2012

Characterization of SfmD as a Heme-containing Peroxidase
1669–1730
version of heme to bilirubin by microsomal heme oxygenase. Proc. Natl.
2. Arai, T., Takahashi, K., and Kubo, A. (1977) New antibiotics saframycins
A, B, C, D, and E. J. Antibiot. 30, 1015–1018
Acad. Sci. U.S.A. 61, 748–755
21. Smith, W. L., and Marnett, L. J. (1991) Prostaglandin endoperoxide syn-
thase: structure and catalysis. Biochim. Biophys. Acta 1083, 1–17
3. Cuevas, C., and Francesch, A. (2009) Development of Yondelis (trabect-
edin, ET-743). A semisynthetic process solves the supply problem. Nat. 22. Sheoran, A., King, A., Velasco, A., Pero, J. M., and Garneau-Tsodikova, S.
Prod. Rep. 26, 322–337
(2008) Characterization of TioF, a tryptophan 2,3-dioxygenase involved in
3-hydroxyquinaldic acid formation during thiocoraline biosynthesis. Mol.
Biosyst. 4, 622–628
4. Mikami, Y., Takahashi, K., Yazawa, K., Arai, T., Namikoshi, M., Iwasaki, S.,
and Okuda, S. (1985) Biosynthetic studies on saframycin A, a quinone
antitumor antibiotic produced by Streptomyces lavendulae. J. Biol. Chem. 23. Huff, A. M., Chang, C. K., Cooper, D. K., Smith, K. M., and Dawson, J. H.
260, 344–348
(1993) Imidazole- and alkylamine-ligated iron (II, III) chlorin complexes
as models for histidine and lysine coordination to iron in dihydroporphy-
rin-containing proteins: characterization with magnetic circular dichro-
ism spectroscopy. Inorg. Chem. 32, 1460–1466
5. Palaniswamy, V. A., and Gould, S. J. (1986) The incorporation of 3Ј-meth-
yltyrosine and 5Ј-methyl DOPA into naphthyridinomycin. J. Am. Chem.
Soc. 108, 5651–5652
6. Li, L., Deng, W., Song, J., Ding, W., Zhao, Q.-F., Peng, C., Song, W.-W., 24. Sugano, Y. (2009) DyP-type peroxidases comprise a novel heme peroxi-
Tang, G.-L., and Liu, W. (2008) Characterization of the saframycin A gene
dase family. Cell. Mol. Life Sci. 66, 1387–1403
cluster from Streptomyces lavendulae NRRL 11002 revealing a nonribo- 25. Sono, M., Roach, M. P., Coulter, E. D., and Dawson, J. (1996) Heme-
somal peptide synthetase system for assembling the unusual tetrapeptidyl
skeleton in an iterative manner. J. Bacteriol. 190, 251–263
containing Oxygenases. Chem. Rev. 96, 2841–2888
26. Poulos, T. L. (2010) Thirty years of heme peroxidase structural biology.
Arch. Biochem. Biophys. 500, 3–12
7. Velasco, A., Acebo, P., Gomez, A., Schleissner, C., Rodríguez, P., Aparicio,
T., Conde, S., Muñoz, R., de la Calle, F., Garcia, J. L., and Sánchez-Puelles, 27. Palmer, G. (1983) in Iron Porphyrins: Part II (Lever, A. B. P., and Gray,
J. M. (2005) Molecular characterization of the safracin biosynthetic path-
H. B., eds) pp. 43–88, Addison-Wesley, Reading, MA
way from Pseudomonas fluorescens A2–2: designing new cytotoxic com- 28. Khindaria, A., and Aust, S. D. (1996) EPR detection and characterization of
pounds. Mol. Microbiol. 56, 144–154
lignin peroxidase porphyrin pi-cation radical. Biochemistry 35,
8. Fu, C. Y., Tang, M. C., Peng, C., Li, L., He, Y. L., Liu, W., and Tang, G. L.
13107–13111
(2009) Biosynthesis of 3-hydroxy-5-methyl-o-methyltyrosine in the safra- 29. Khatri, Y., Hannemann, F., Ewen, K. M., Pistorius, D., Perlova, O., Kagawa,
mycin/ safracin biosynthetic pathway. J. Microbiol. Biotechnol. 19,
439–446
N., Brachmann, A. O., Müller, R., and Bernhardt, R. (2010) The CYPome
of Sorangium cellulosum So ce56 and identification of CYP109D1 as a new
fatty acid hydroxylase. Chem. Biol. 17, 1295–1305
9. Koketsu, K., Watanabe, K., Suda, H., Oguri, H., and Oikawa, H. (2010)
Reconstruction of the saframycin core scaffold defines dual Pictet-Spen- 30. Allen, J. W., Leach, N., and Ferguson, S. J. (2005) The histidine of the
gler mechanisms. Nat. Chem. Biol. 6, 408–410
c-type cytochrome CXXCH haem-binding motif is essential for haem
attachment by the Escherichia coli cytochrome c maturation (Ccm) appa-
ratus. Biochem. J. 389, 587–592
10. Fitzpatrick, P. F. (1999) Tetrahydropterin-dependent amino acid hy-
droxylases. Ann. Rev. Biochem. 68, 355–381
11. Sánchez-Ferrer, A., Rodríguez-López, J. N., Garcia-Cánovas, F., and 31. Guillou, H., D’Andrea, S., Rioux, V., Barnouin, R., Dalaine, S., Pedrono, F.,
García-Carmona, F. (1995) Tyrosinase: a comprehensive review of its
mechanism. Biochim. Biophys. Acta 1247, 1–11
Jan, S., and Legrand, P. (2004) Distinct roles of endoplasmic reticulum
cytochrome b5 and fused cytochrome b5-like domain for rat ␦6-desatu-
rase activity. J. Lipid Res. 45, 32–40
12. Lin, S., Van Lanen, S. G., and Shen, B. (2008) Characterization of the
two-component, FAD-dependent monooxygenase SgcC that requires 32. Mense, S. M., and Zhang, L. (2006) Heme: a versatile signaling molecule
carrier protein-tethered substrates for the biosynthesis of the enediyne
controlling the activities of diverse regulators ranging from transcription
factors to MAP kinases. Cell Res. 16, 681–692
antitumor antibiotic C-1027. J. Am. Chem. Soc. 130, 6616–6623
13. Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory 33. Neusser, D., Schmidt, H., Spizèk, J., Novotnà, J., Peschke, U., Kaschabeck,
Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York
14. Trost, B. M., and Rudd, M. T. (2003) Chemoselectivity of the ruthenium-
catalyzed hydrative diyne cyclization: total synthesis of (ϩ)-cylindricine C,
D, and E. Org. Lett. 5, 4599–4602
S., Tichy, P., and Piepersberg, W. (1998) The genes lmbB1 and lmbB2 of
Streptomyces lincolnensis encode enzymes involved in the conversion of
L-tyrosine to propylproline during the biosynthesis of the antibiotic linco-
mycin A. Arch. Microbiol. 169, 322–332
15. Deboves, H. J., Montalbetti, C. A., and Jackson, R. F. (2001) Direct synthe-
sis of Fmoc-protected amino acids using organozinc chemistry: applica-
34. Hurley, L. H., and Rokem, J. S. (1983) Biosynthesis of the antitumor anti-
biotic CC-1065 by Streptomyces zelensis. J. Antibiot. 36, 383–390
tion to polymethoxylated phenylalanines and 4-oxoamino acids. J. Chem. 35. Hannemann, F., Bichet, A., Ewen, K. M., and Bernhardt, R. (2007) Cyto-
Soc. Perkin Trans. 1, 1876–1884
chrome P450 systems-biological variations of electron transport chains.
Biochim. Biophys. Acta 1770, 330–344
16. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of protein-dye 36. Zamocky, M., and Obinger, C. (2010) in Biocatalysis Based on Heme Per-
binding. Anal. Biochem. 72, 248–254
oxidases, (Torres, E., and Ayala, M., eds) Springer-Verlag, Berlin,
Heidelberg
17. Berry, E. A., and Trumpower, B. L. (1987) Simultaneous determination of
hemes a, b, and c from pyridine hemochrome spectra. Anal. Biochem. 161, 37. Hofrichter, M., and Ullrich, R. (2006) Heme-thiolate haloperoxidases: ver-
1–15
satile biocatalysts with biotechnological and environmental significance.
Appl. Microbiol. Biotechnol. 71, 276–288
18. Chowdhury, G., Murayama, N., Okada, Y., Uno, Y., Shimizu, M., Shibata,
N., Guengerich, F. P., and Yamazaki H. (2010) Human liver microsomal
cytochrome P450 3A enzymes involved in thalidomide 5-hydroxylation
and formation of a glutathione conjugate. Chem. Res. Toxicol. 23,
1018–1024
38. Hofrichter, M., Ullrich, R., Pecyna, M. J., Liers, C., and Lundell, T. (2010)
New and classic families of secreted fungal heme peroxidases. Appl. Mi-
crobiol. Biotechnol. 87, 871–897
39. Smith, P. I., and Swan, G. A. (1976) A study of the supposed hydroxylation
of tyrosine catalysed by peroxidase. Biochem. J. 153, 403–408
19. Alberta, J. A., and Dawson, J. H. (1987) Purification to homogeneity and
initial physical characterization of secondary amine monooxygenase. 40. Connor, K. L., Colabroy, K. L., and Gerratana, B. (2011) A heme peroxi-
J. Biol. Chem. 262, 11857–11863
dase with a functional role as an L-tyrosine hydroxylase in the biosynthesis
of anthramycin. Biochemistry. 50, 8926–8936
20. Tenhunen, R., Marver, H. S., and Schmid, R. (1968) The enzymatic con-
FEBRUARY 10, 2012•VOLUME 287•NUMBER 7
JOURNAL OF BIOLOGICAL CHEMISTRY 5121

Enzymology:
Characterization of SfmD as a Heme
Peroxidase That Catalyzes the
Regioselective Hydroxylation of
3-Methyltyrosine to
3-Hydroxy-5-methyltyrosine in Saframycin
A Biosynthesis
Man-Cheng Tang, Cheng-Yu Fu and Gong-Li
Tang
J. Biol. Chem. 2012, 287:5112-5121.
doi: 10.1074/jbc.M111.306316 originally published online December 20, 2011
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