A stereoselective vanadium-dependent chloroperoxidase in bacterial antibiotic biosynthesis

Article abstract of DOI:10.1021/ja201088k

Halogenases catalyze reactions that introduce halogen atoms into electron-rich organic molecules. Vanadium-dependent haloperoxidases are generally considered to be promiscuous halogenating enzymes that have thus far been derived exclusively from eukaryotes, where their cellular function is often disputed. We now report the first biochemical characterization of a bacterial vanadium-dependent chloroperoxidase, NapH1 from Streptomyces sp. CNQ-525, which catalyzes a highly stereoselective chlorination-cyclization reaction in napyradiomycin antibiotic biosynthesis. This finding biochemically links a vanadium chloroperoxidase to microbial natural product biosynthesis.

Full text of DOI:10.1021/ja201088k

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pubs.acs.org/JACS
A Stereoselective Vanadium-Dependent Chloroperoxidase
in Bacterial Antibiotic Biosynthesis
Peter Bernhardt,† Tatsufumi Okino,‡,† Jaclyn M. Winter,† Akimasa Miyanaga,† and Bradley S. Moore*,†,§
†Scripps Institution of Oceanography and the §Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California,
San Diego, California 92093, United States
S Supporting Information
b
Scheme 1. Napyradiomycin Biosynthesis Starting from
ABSTRACT: Halogenases catalyze reactions that intro-
duce halogen atoms into electron-rich organic molecules.
Vanadium-dependent haloperoxidases are generally consid-
ered to be promiscuous halogenating enzymes that have
thus far been derived exclusively from eukaryotes, where
their cellular function is often disputed. We now report the
first biochemical characterization of a bacterial vanadium-
dependent chloroperoxidase, NapH1 from Streptomyces sp.
CNQ-525, which catalyzes a highly stereoselective chlorina-
tion-cyclization reaction in napyradiomycin antibiotic bio-
synthesis. This finding biochemically links a vanadium
chloroperoxidase to microbial natural product biosynthesis.
Malonyl-CoA and Leading to A80915C 2 Proceeds via a
V-CPO-Catalyzed Chlorination-Cyclization of SF2415B1 3
To Form SF2415B3 4
any halogenated natural products have therapeutic poten-
M
tial for human health, and there is great interest in
discovering and exploiting enzymes that trigger halogenation
reactions. Nature has developed an extensive repertoire of
halogenation strategies to incorporate fluorine, chlorine, bro-
mine, and iodine into organic molecules,1,2 Although many
studies in recent years have increased our mechanistic under-
standing of halogenase biochemistry, vanadium-dependent ha-
loperoxidases (V-HPOs) remain poorly understood in the
context of natural product biosynthesis.3 Herein, we describe
the first functional characterization of a bacterial V-HPO and
show that the enzyme stereoselectively converts SF2415B1 into
SF2415B3, two napyradiomycin antibiotics isolated from acti-
nomycetes (Scheme 1).4-6
into single stereoisomers of known halogenated metabolites R-,
β-, and γ-snyderol and (þ)-3β-bromo-8-epicaparrapi oxide.13-18
We recently identified three genes (napH1, H3, and H4),
homologous to eukaryotic V-HPOs, in a bacterial gene cluster
encoding the formation of chlorinated polyketide-terpenoid anti-
biotics, called napyradiomycins.19 The presence of these genes in a
bacterial gene cluster presented us with a unique opportunity to
characterize a V-HPO in its biosynthetic context.
Napyradiomycins constitute a class of over 30 natural products
produced by terrestrial and marine streptomycetes.4,5,20,21 Com-
paring metabolites and gene clusters isolated from Streptomyces sp.
CNQ-525 and S. aculeolatus led us to propose a pathway starting
from the aromatic polyketide tetrahydroxynaphthalene 1 leading to
the trichlorinated meroterpenoid A80915C 2 via two intermediate
meroterpenoids, SF2415B1 3 and SF2415B3 4 (Scheme 1).19
We hypothesized that at least one of the three V-HPO-encoding
genes catalyzes the conversion of 3 into 4, through a C-C and
C-Cl bond-formation reaction. This type of reaction occurs in
the V-BPO-catalyzed bromonium-ion-mediated cyclization of
(E)-(þ)-nerolidol by red algal V-BPOs.15
V-HPO utilizes hydrogen peroxide and a covalently bound
vanadate cofactor to oxidize halides (X-) into a mixture of
activated halogenating agents, often represented by hypohalous
acid (HOX), which can then react with electron-rich organic
substrates. All currently characterized V-HPOs are eukaryotic in
origin and isolated as vanadium bromoperoxidase (V-BPO) or
chloroperoxidase (V-CPO) in red and brown algae and dema-
tiaceous hyphomycete fungi.7-12
The cellular function of (eukaryotic) V-HPOs is often spec-
ulative as it is challenging to establish authentic substrates for the
reactions they catalyze. In some cases, the producing organism
does not produce any known halogenated metabolites.3 There is
indirect evidence, however, that electron-rich organic substrates
bind to V-BPOs isolated from the marine red algae Corallina,
Laurencia, and Plocamium. For example, V-BPOs from Corallina
convert the commercially available terpenoid (E)-(þ)-nerolidol
We cloned the genes coding for NapH1, H3, and H4 into the
Escherichia coli expression vector pHis8.22 After heterologous
expression and purification using Ni-NTA affinity chromatogra-
phy, we analyzed the purification steps and found that while all
Received: February 3, 2011
Published: March 08, 2011
r
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reconstitute insoluble NapH4 failed, we focused our attention
on NapH1 and H3.
Next, we isolated SF2415B1 3, the putative substrate
(Scheme 1), from S. aculeolatus NRRL 18422, and verified its
structure by comparing its spectral properties with literature
values.6 Gratifyingly, when this molecule was incubated with
NapH1 in the presence of hydrogen peroxide and chloride in
aqueous buffered (pH 6) solution, a single new peak appeared in
the chromatogram (Figure 1A). Isolation and spectral analysis
(HRMS and NMR) of the enzymatic product showed that it was
indeed a single stereoisomer, SF2415B3 4 (SI). NapH1 catalyzed
this reaction with an optimum pH of 6 (Figure S2, SI) and
following Michaelis-Menten kinetics with chloride and hydrogen
peroxide (Figure 1B). The maximum rate of the reaction (Vmax) at
pH 6 was 3.9 ( 0.2 and 4.3 ( 0.1 μmol/min/mg for hydrogen
peroxide and chloride, respectively. The Michaelis constant (Km)
was 1.2 ( 0.4 μM and 4.0 ( 0.1 mM for hydrogen peroxide and
chloride, respectively. We were unable to determine the Km for
SF2415B1 3, but estimate the value to below the detection limit of
our assay system (Km < 1 μM).
NapH3 and C. officinalis V-BPO (CoBPO) failed to catalyze
the formation of SF2415B3 4, in the presence of chloride, or
bromo-SF2415B3 5, in the presence of bromide. Furthermore,
NapH1 did not accept (()-nerolidol or lapachol (4-hydroxy-3-
(3-methylbut-2-enyl)naphthalene-1,2-dione) as alternate sub-
strates (Figure S3, SI). Since lapachol contains the hydroxy and
isoprene units in the analogous positions to substrate SF2415B1
3, and since NapH1 did not convert lapachol, we conclude that 3
likely interacts intimately with NapH1 during catalysis. In con-
trast, CoBPO catalyzed the complete conversion of lapachol into
numerous products (Figure S3, SI). The high in vitro selectivity
of NapH1 distinguishes this enzyme from other characterized
vanadium haloperoxidases, and strongly supports a link between
the napH1-containing gene cluster and production of napyra-
diomycins in S. aculeolatus and Streptomyces sp. CNQ-525. It also
provides the first evidence that V-CPOs can catalyze stereose-
lective and substrate-specific chlorination-cyclization reactions.
When chloride and bromide were added in an equimolar ratio,
NapH1 catalyzed the preferential bromination of SF2415B1 3 to
form bromo-SF2415B3 5 (Figure S4, SI). However, while the
chlorination reaction rapidly proceeded to completion, the
reaction with bromide, in comparison, resulted in only a partial
conversion into bromo-SF2415B3 5 (Figure 1A). This partial
conversion is likely due to a modification to the enzyme as further
conversion occurred once additional NapH1 supplemented the
reaction (Figure S5, SI). In addition to bromo-SF2415B3 5,
NapH1 triggered the formation of two new compounds with
identical molecular formulas C26H34O6BrCl based on HRMS
analysis. This observation is consistent with the formation of two
bromohydrin stereoisomer derivatives of SF2415B1, presently of
unknown structure, that form upon hydrolysis of a common
intermediate bromonium ion (Figure 1A, and SI).
Figure 1. HPLC and spectrophotometric assay of heterologously
expressed NapH1. (A) HPLC assay that monitors the chlorination
and bromination of SF2415B1 3 catalyzed by wild-type (WT) or
Ser427His NapH1. The negative control is heat-inactivated WT NapH1
incubated in a complete assay mixture containing chloride. Bromohy-
drins a and b are of unknown structure. (B) Pseudo-first-order approx-
imation steady-state kinetic analysis of NapH1 catalysis in the
chlorination or SF2415B1 3. Three independent measurements at each
substrate concentration gave the displayed nonlinear regression fit (line)
to the averaged data (points). (C) Spectrophotometric assay that
monitors the bromination and chlorination of monochlorodimedone
(MCD), which results in a decrease in absorbance at 290 nm. The assay
mixture is equilibrated at 30 °C (initial minutes) and then started by
addition of either NapH1 (above) or Corallina officinalis V-BPO
(CoBPO) (below). After no activity was observed in the presence of
chloride (0.2 M), bromide (0.2 M) was added, and the reaction started
and continued until all MCD was depleted.
Since NapH1 appeared to perform a nonspecific bromination of
3, we tested the activity of NapH1 in the general haloperoxidase
assay that relies on detecting the chlorination or bromination of
an electron-rich indicator substrate, monochlorodimedone
(MCD), using spectrophotometry (Scheme 1C).23 Our assay
results clearly demonstrated that NapH1 is inactive when chloride
is the only halide source and only active after addition of bromide.
This brominating activity is dramatically lower compared to the
nonspecific brominating activity of C. officinalis V-BPO (CoBPO)
(Figure 1C, Figure S6, SI). The nonspecificity in bromination
three enzymes expressed well, only NapH1 (Figure S1, Support-
ing Information [SI]) and NapH3 were expressed in the soluble
fraction as octahistidyl-tagged proteins. Since all attempts to
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may be a result of production of diffusible hypobromous acid.
Our results suggest that although the MCD assay is a benchmark
assay used to quantify and isolate new general-activity halopero-
xidases,23 it may not be suitable for isolating new V-CPOs in
bacterial natural product biosynthesis.
UCSD Chemistry and Biochemistry Mass Spectrometry Facility
and NMR Facility, respectively, for instrument expertise. This
work was generously supported by research grants from the NIH
(AI047818) and the California Sea Grant Program (R/NMP-
100) to B.S.M., postdoctoral fellowships from the NIH
(GM096711) to P.B. and JSPS to A.M., a Ruth L. Kirschstein
National Research Service Award to J.M.W. from the NIH
Training Program in Marine Biotechnology (GM067550), and
the Naito Foundation for sabbatical support to T.O.
To obtain insight into the substrate binding of NapH1, we
compared amino acid sequence alignments with available crystal
structures of the more promiscuous V-HPOs, C. inaequalis
V-CPO (CiCPO) and CoBPO (SI).24,25 From this, we selected
two residues in NapH1, His420 and Ser427, that had previously
been proposed to be involved in determining the specificity of
V-HPOs.3,19 His420 is a histidine and phenylalanine in CoBPO
and CiCPO, respectively, while Ser427 is a histidine in CoBPO
and CiCPO. After constructing the His420Phe and Ser427His
mutants of NapH1 using site-directed mutagenesis, we expressed
and purified the resulting enzyme variants. Purified His420Phe
NapH1 was inactive in the MCD assay and in the chlorination and
bromination of SF2415B1 3 (Figure S7, SI). Interestingly, how-
ever, while Ser427His NapH1 showed near wild type activity at
approximately 70% in the MCD assay, this mutant enzyme was
unable to catalyze the conversion of substrate 3 into SF2415B3 4 or
bromo-SF2415B3 5. Instead, Ser427His NapH1 exclusively cata-
lyzed the formation of SF2415B1-based bromohydrins (Figure 1A).
Hence the activity with SF2415B1 3 can be completely abolished by
a single point mutation to NapH1, Ser427His, without compromis-
ing the ability to produce activated brominating species.
V-BPO is known to endogenously contain bromine atoms on
side-chains,26 presumably acquired in post-translation through
self-halogenation. It is possible that the activated brominating
species produced by NapH1 reacts with an amino acid side-chain,
and thus creates an alternative means for the activated brominat-
ing species to brominate MCD, or initiate the formation of
bromohydrins. Future experiments will seek to further understand
the interplay of specific and nonspecific halogenation events.
In summary, we have described the first highly substrate-
selective vanadium haloperoxidase in natural product biosynth-
esis, where the chloroperoxidase NapH1 carries out a stereoselective
chlorination-cyclization reaction with a known polyketide-terpe-
noid natural product.
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’ ASSOCIATED CONTENT
S
Supporting Information. Figures S1-S7, experimental
b
methods, sequence alignment, HRMS data, NMR spectra of
SF2415B3. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
bsmoore@ucsd.edu
Present Addresses
‡Faculty of Environmental Earth Science, Hokkaido University,
Sapporo, 060-0810, Japan.
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’ ACKNOWLEDGMENT
S. aculeolatus NRRL 18422 was provided by Drs. McAlpine and
Zhao (Thallion Pharmaceuticals, Canada), while the SF2415B3
standard was a kind gift from Dr. Ohyama (Meiji Seika Kaisha,
Ltd., Japan). We gratefully acknowledge Drs. Su and Mrse at the
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