Streptomyces clavuligerus HlmI is an intramolecular disulfide-forming dithiol oxidase in holomycin biosynthesis

Article abstract of DOI:10.1021/bi200321c

Holomycin and related dithiolopyrrolone antibiotics display broad-spectrum antimicrobial activities and contain a unique 5,5-bicyclic ring structure with an N-acylated aminopyrrolone fused to a cyclic ene-disulfide. Here we show that the intramolecular disulfide bridge is constructed from the acyclic ene-dithiol at a late stage in the pathway by a thioredoxin oxidoreductase-like enzyme HlmI from the holomycin producer Streptomyces clavuligerus. Recombinant HlmI was purified from E. coli with bound flavin adenine dinucleotide (FAD) and converts reduced holomycin to holomycin utilizing O₂ as cosubstrate. As a dithiol oxidase, HlmI is functionally homologous to GliT and DepH, which perform a similar dithiol to disulfide oxidation in the biosynthesis of fungal natural product gliotoxin and epigenetic regulator compound FK228, respectively. Deletion of the hlmI gene in the wild type S. clavuligerus and in a holomycin-overproducing mutant resulted in decreased level of holomycin production and increased sensitivity toward holomycin, suggesting a self-protection role of HlmI in the holomycin biosynthetic pathway. HlmI belongs to a new clade of uncharacterized thioredoxin oxidoreductase-like enzymes, distinctive from the GliT-like enzymes and the DepH-like enzymes, and represents a third example of oxidoreductases that catalyzes disulfide formation in the biosynthesis of small molecules.

Full text of DOI:10.1021/bi200321c

ARTICLE
pubs.acs.org/biochemistry
Streptomyces clavuligerus HlmI Is an Intramolecular Disulfide-Forming
Dithiol Oxidase in Holomycin Biosynthesis
Bo Li and Christopher T. Walsh*
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston,
Massachusetts 02115, United States
S Supporting Information
b
ABSTRACT: Holomycin and related dithiolopyrrolone anti-
biotics display broad-spectrum antimicrobial activities and
contain a unique 5,5-bicyclic ring structure with an N-acylated
aminopyrrolone fused to a cyclic eneꢀdisulfide. Here we show
that the intramolecular disulfide bridge is constructed from the
acyclic eneꢀdithiol at a late stage in the pathway by a thior-
edoxin oxidoreductase-like enzyme HlmI from the holomycin
producer Streptomyces clavuligerus. Recombinant HlmI was
purified from E. coli with bound flavin adenine dinucleotide
(FAD) and converts reduced holomycin to holomycin utilizing O2 as cosubstrate. As a dithiol oxidase, HlmI is functionally
homologous to GliT and DepH, which perform a similar dithiol to disulfide oxidation in the biosynthesis of fungal natural product
gliotoxin and epigenetic regulator compound FK228, respectively. Deletion of the hlmI gene in the wild type S. clavuligerus and in a
holomycin-overproducing mutant resulted in decreased level of holomycin production and increased sensitivity toward holomycin,
suggesting a self-protection role of HlmI in the holomycin biosynthetic pathway. HlmI belongs to a new clade of uncharacterized
thioredoxin oxidoreductase-like enzymes, distinctive from the GliT-like enzymes and the DepH-like enzymes, and represents a third
example of oxidoreductases that catalyzes disulfide formation in the biosynthesis of small molecules.
atural product antibiotics holomycin and its variants, such as
thiolutin, aureothricin, xenorhabdin, and thiomarinol, share
respective biosynthetic gene clusters (Figure 1B).11,12 Prior to
the work reported herein, it was unclear which enzyme may be
responsible for generation of the unusual disulfide present in
holomycin.
N
a common N-acylated bicyclic dithiolopyrrolone scaffold (also
known as pyrrothine) (Figure 1A).1ꢀ4 These dithiolopyrrolone
antibiotics have been reported to interfere with bacterial RNA
synthesis;5ꢀ7 however, the mode of action and the relevance of
the disulfide to activity remain unclear as direct inhibition of
RNA polymerases has yet to be reconstituted in vitro.7
We recently identified the holomycin biosynthetic gene
cluster through genome mining of the producing bacterium
S. clavuligerus.13 In addition to a non-ribosomal peptide synthe-
tase (NRPS) module which activates and loads the essential
building block L-Cys and an acetyl transferase for introduction of
the N-acetyl moiety, four predicted flavin-dependent oxidore-
ductases are encoded in the gene cluster (Figure 2A). The
presence of four such oxidoreductases is consistent with a
pathway where L-Cys-L-Cys undergoes oxidations removing a
net total of eight electrons in four two-electron oxidation steps,
each catalyzed by one of the flavoenzymes (Figure 2B). One of
the flavoenzymes, HlmI, displays homologies to the large family
of thioredoxin reductases, involved in dithiolꢀdisulfide redox
interconversions in protein substrates. We demonstrate that
HlmI is a dithiol oxidase, converting the reduced dithiol form
of holomycin (red-holomycin), 2, to the disulfide form, 1
(Figure 1B). Heterologous expression of the holomycin gene
cluster in S. albus was recently reported by Deng and co-workers,
Disulfides are present in a wide variety of natural products, in
larger ribosomally encoded constructs, e.g., conotoxins and cyclo-
tides, as well as smaller, non-ribosomal peptides such as gliotoxin
and the antitumorigen FK228. In all of these natural products,
the disulfide is essential for the activity of the molecule. In the
cyclotides, cystine disulfides impart increased thermal stability,
conformational rigidity, and cell permeability.8 Similarly, in
FK228, the disulfide has been shown to promote cell permeability
while masking the active form containing free thiol of the
epigenetically active prodrug.9 The mycotoxin gliotoxin employs
the disulfide in two complementary and damaging activities: (1)
redox cycling to produce destructive superoxide free radicals and
(2) cross-linking and inhibition of cellular proteins via formation
of mixed disulfides with the reduced form.10 Given the importance
of the disulfide linkage to stability and mobility of these molecules,
it is of considerable interest as to when and how the disulfides are
installed in the mature natural products. Formation of disulfides in
FK228 and gliotoxin is catalyzed by thioredoxin reductase-like
enzymes DepH and GliT, both of which are encoded in their
Received: March 2, 2011
Revised:
April 17, 2011
Published: April 19, 2011
r
2011 American Chemical Society
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cell disruptor. The lysis mixture was clarified by ultracentrifuga-
tion, and the supernatant was incubated with 2 mL of nickel-
NTA agarose resin (Qiagen) at 4 °C with gentle mixing for 1 h.
The resin with bound protein was loaded on to a column to drain
the flowthrough, washed with 50 mL of column buffer and 20 mL
of column buffer containing 20 mM imidazole, and eluted with
10 mL of elution buffer (300 mM imidazole in column buffer).
The elution fractions were analyzed by SDS-PAGE, and the
fractions containing HlmI were pooled and concentrated to
5 mL with a 15 mL Amicon (Millipore) (10 kDa MW cutoff).
Concentrated protein was desalted with a PD-10 desalting column
(GE Healthcare) twice to remove excess imidazole. The resulting
protein was flash frozen with liquid N2 and stored at ꢀ80 °C.
Molecular Cloning of gliT, Overexpression, and Purifica-
tion of the GliT Protein. Aspergillus fumigatus Af293 cDNA was a
gift from Robert A. Cramer Jr. (Durham, NC).15 Using this
cDNA as template, gliT was amplified by PCR and cloned into
pET24b between NdeI and XhoI sites. The sequence was verified
by DNA sequencing. BL21 (DE3) cells containing the con-
structed vector were grown overnight at 37 °C. 6 L of LB
containing 40 μg/mL kanamycin and 5 mM MgCl2 was inocu-
lated with 1% of the overnight culture. When OD600 nm of the
culture reached 0.7, protein expression was induced with 0.1 mM
IPTG and the culture was grown for an additional 12 h at 15 °C.
Cells were harvested and resuspended in 28 mL of lysis buffer
(25 mM Tris, pH 8.0, 500 mM NaCl), lysed with two passes on
an Emulsiflex-C5 cell disruptor (Avestin). Cell lysate was cleared
at 35 000 rpm (95 000g) for 35 min and incubated with 1.1 mL of
Ni-NTA resin (2.2 mL 50% slurry) that was prewashed with lysis
buffer for 1.5 h at 4 °C. Resin was then transferred to column, and
a gradient imidazole elution was performed: 25 mL for 0 and
5 mM imidazole, 15 mL for 25 mM, 10 mL for 200 mM, and 5 mL
for 500 mM imidazole. GliT was eluted in the 25, 200, and
500 mM imidazole elution fractions, which were combined and
concentrated to less than 2 mL and injected for gel filtration in
20 mM Tris, pH 8, 50 mM NaCl. Fractions containing GliT
were pooled and concentrated, and 10% glycerol was added. A
total of 4.5 mL of GliT was obtained, and its concentration was
determined by Bradford as 294 μM.
Determination of HlmI Oligomeric State and FAD Content
in HlmI and GliT. Purified HlmI was analyzed by size exclusion
chromatography using a 10/300 Superdex 200 gel-filtration
column and running buffer containing 50 mM HEPES, 150 mM
NaCl, and 5% glycerol at pH 7.5. The calculated molecular weight
for HlmI, including the His tag, is 39 163 Da, and the apparent
molecular weight for HlmI is ∼80 kDa based on the standard
curve on the gel filtration column, suggesting that HlmI exists as a
dimer. Both HlmI and GliT were denatured by boiling for 5 min to
release the flavin cofactor. The supernatant was injected on
Accurate-Mass Q-Tof LC/MS instrument (Agilent Technologies
6520), and the flavin cofactor was identified as FAD. To measure
the FAD content, UV scans between 200 and 600 nm (Cary
UVꢀvis) were taken for HlmI and GliT and also the FAD-
containing supernatant from the denatured samples. Absorbance
at 450 nm for the latter samples was used to determine the con-
Figure 1. (A) Structures of holomycin and related dithiolopyrrolone
antibiotics. (B) Conversion of the reduced form of holomycin, gliotoxin,
and FK228 into the disulfide form by thioredoxin oxidoreductase-like
enzymes.
and deletion of the hlmI gene abolished holomycin production,
suggesting a role for HlmI in holomycin biosynthesis.14
’ MATERIALS AND METHODS
Molecular Cloning of hlmI and Overexpression and Pur-
ification of the HlmI Protein. The hlmI gene was cloned
into pET30-Ek/LIC vector (Novagen) using primers listed in
Table S1, and the gene sequence was verified by DNA sequen-
cing. This vector was introduced into BL21-CodonPlus (DE3)-
RIPL chemical competent cells (Stratagene). A 2 L LB broth was
inoculated with 20 mL of overnight culture that was started from
a single colony, and protein overexpression was induced at an
OD600 nm of 0.6 with 0.2 mM isopropyl-β-D-thiogalactopyrano-
side (IPTG) at 18 °C for 18 h. The cells were harvested by
centrifugation, resuspended in 20 mL of column buffer (50 mM
HEPES, 300 mM NaCl, 10% glycerol, pH 7.5), and lysed with a
centration of FAD using extinction coefficient 11300 Mꢀ1 cmꢀ1
,
and the background absorbance at 280 nm from FAD was
subtracted from the A280 nm values of the nondenatured HlmI
and GliT samples. The adjusted A280 nm values were used for
calculation of protein concentrations using theoretical extinction
coefficients 26595 Mꢀ1 cmꢀ1 for HlmI and 30160 Mꢀ1 cmꢀ1 for
GliT. Duplicate experiments were performed, and the average
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Figure 2. (A) Holomycin (hlm) gene cluster with genes colored by function. hlmA, acyltranferase; hlmB, acyl-CoA dehydrogenase; hlmC, thioesterase;
hlmD, glucoseꢀmethanolꢀcholine oxidoreductase; hlmE, NRPS (cyclizationꢀadenylationꢀthiolation, Cy-A-T); hlmF, lantibiotic decarboxylase;
hlmG, globin; hlmH, major facilitator family transporter; hlmI, thioredoxinꢀdisulfide oxidoreductase; hlmJ, hlmM, transcriptional regulators; hlmK,
inactive thioesterase domain; hlmL, condensation domain. (B) Proposed holomycin biosynthetic pathway. HlmI, the focus of this study, is shown in a
highlighted box and is proposed to be involved in the late stages of holomycin biosynthesis.
FAD content was 20% for HlmI and 35% for GliT. To reconstitute
the binding of FAD in HlmI and GliT, both proteins were
incubated with excess FAD on ice for 30 min. Excess FAD was
removed by passing the proteins through Micro Bio-Spin 6
chromatography columns pre-equilibrated in buffer containing
20mMTris, 10 mMNaCl, 5%glycerol (pH 8.5). HlmIwas further
purified using a MonoQ 10/100 GL anion exchange column (GE
Healthcare). A linear gradient from 10 mM NaCl to 1 M NaCl was
runoversevencolumnvolumes at1 mL/min. Fractions containing
HlmI were combined and concentrated using Amicon Ultra cen-
trigual filters (0.5 mL, 3 kDa molecular weight cutoff). The resulting
protein was shown to be over 95% pure by SDS-PAGE. The FAD
content was determined using the same method as described above
and increased to 80% and 76% in GliT and HlmI, respectively.
Enzymatic Assays To Measure Steady-State Kinetic Para-
meters. Holomycin was isolated from overproducing strain
S. clavuligerus ΔORF15 and purified by HPLC as previously
described.13 The concentration of holomycin was calculated
based on UV absorbance at 388 nm using a reported extinction
velocity was calculated from the decrease in absorbance of
reduced holomycin at 340 nm with a extinction coefficient at
15 900 Mꢀ1 cmꢀ1. This extinction coefficient was obtained by
fully converting a solution of holomycin of known concentration
to reduced holomycin with excess TCEP and measuring the
absorbance at 340 nm. Background oxidation by O2 was taken
for each substrate concentration without enzyme. The enzyme
catalyzed rate was obtained by subtracting the nonenzymatic
rate from the overall rate. These experiments were carried out
in duplicates for calculation of error. Data were analyzed and
plotted using GraphPad Prism. Kinetic measurement was also
performed with the FAD-reconstituted HlmI and GliT, which
contained a much higher percentage of FAD, at a concentration
of 25 nM (76% FAD, 19 nM effective concentration) and
12.5 nM (80% FAD, 10 nM effective concentration), respectively.
Determination of Half-Life of Reduced Holomycin and
Reduced Holothin. Holothin was generated by microwave-
assisted deacetylation of holomycin dissolved in 1,3-dioxane and
heated at 100 °C for 10 min in the presence of 2 N HCl. The water
layer of product mixture was vacuum-dried and resuspended
in water and further purified by HPLC. A sample of 50 μM
holomycin or holothin was mixed with 1 equiv of TCEP in the
presence of 100 mM phosphate buffer (pH 6.5) to generate
reduced holomycin or reduced holothin. The conversion from the
reduced form to the disulfide form was monitored continuously
by UV. Absorbance at 340 and 335 nm was used respectively to
calculate the half-life of reduced holomycin and reduced holothin.
Data were analyzed and plotted using GraphPad Prism.
coefficient of 11 220 Mꢀ1 cmꢀ1 16
Reduced holomycin was
.
generated by reduction of holomycin with 1 mol equiv of TCEP
in the presence of 100 mM phosphate buffer (pH 6.5), and a
sample of 3 μL of 2 μM HlmI (50 nM final total concentration,
10 nM effective concentration) or 3 μL of 1 μM GliT (25 nM
final total concentration, 8.75 nM effective concentration) was
added to initiate the reaction (total reaction volume 120 μL).
Because of the detectable nonenzymatic oxidation by O2, multi-
ple concentrations for HlmI and GliT were tested to find
oxidation rates that are well above background yet still in the
linear range, and the optimal concentrations for HlmI and GliT
are 50 and 25 nM, respectively. Initial velocity was measured
in a continuous assay by taking a UV scan (Cary UVꢀvis) from
200 to 600 nm every 0.2 min for a total of 10 min at reduced
holomycin concentrations of 5, 10, 25, 50, and 100 μM. Initial
Genetic Deletion of hlmI Gene in S. clavuligerus. Deletion of
hlmI gene was achieved using ReDirect PCR-targeting strategy.17
The hlmI gene with a 2 kb extension of the chromosomal seque-
nce on each end was cloned into a PCR-Blunt vector (KanR,
Invitrogen). This vector was introduced into E. coli BW25113/
pIJ790 strain allowing for λ red-mediated recombination. The
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of 10 μL was injected on the Accurate-Mass Q-Tof LC/MS
instrument (Agilent Technologies 6520) for detection of holo-
mycin production via electrospray ionization (ESI).
Agar Diffusion Assays To Test Holomycin Sensitivity of
S. clavuligerus Strains. Spore stocks were prepared for wild
type, ΔhlmI, ΔORF15, and ΔORF15/ΔhlmI strains. The spore
concentrations were determined by serial dilutions of the stock
and counting of colony forming units. Approximately 5 ꢁ 105
spores were plated for each strain, and a sample of 5 μL of
7 mg/mL holomycin was added to the center of the MYM plate
(4.0 g of yeast extract, 10.0 g of malt extract, 2.0 g of dextrose,
and 20.0 g of agar per liter of media). The plates were incubated
at 30 °C for 4ꢀ7 days, and the diameter of the inhibition zone
was determined as a measurement for the sensitivity of these
strains toward holomycin.
Phylogenetic Analysis of HlmI. HlmI was aligned with a
number of bacterial and fungal thioredoxinꢀoxidoreductase like
enzymes from the protein database using ClustalX. Alignment
was edited using MacClade and imported into RAxML for
generation of dendrogram by maximum likelihood.20 The result-
ing dendrogram was further colored by Adobe Illustrator.
’ RESULTS AND DISCUSSION
Figure 3. (A) LC-MS analysis of holomycin treated with 2 equiv of
TCEP. UV trace at 390 nm is shown with a clear separation of red-
holomycin (5.0 min) and holomycin (7.2 min). The presence of
holomycin in the UV trace is due to the reoxidation of red-holomycin
by O2 during the sample injection and column separation. (B) UV
spectra of red-holomycin and holomycin.
Holomycin Biosynthetic Gene Cluster Nomenclature. Pre-
viously, we demonstrated that ORFs 3483-3496 from the
S. clavuligerus genome (locus tag SSCG_03483 through
SSCG_03496) constitute a likely holomycin biosynthetic gene
cluster by both genetic manipulation and biochemical character-
ization of ORFs 3483 and 3488.13 In this work we renamed ORFs
3483-3496 as hlmA-hlmM (Figure 2A), where hlm stands for
holomycin and is a three-letter acronym not used elsewhere in
bacterial gene assignments. ORF3492 as the focus of this work is
renamed as hlmI.
resulting strain was transformed with PCR fragments containing
the desired antibiotic resistance cassette including a 36-nt exten-
sion homologous to the target region directly upstream and
downstream of the hlmI gene. Correct recombined vectors con-
taining the desired disruption cassettes were selected based on
antibiotic resistance. The apramycin resistance cassette was con-
structed using pIJ773 (ApraR, acc(3)IV-oriT) vector as template
for disruption in S. clavuligerus wild type. The vector pIJ10700
(HygR, hyg-oriT) was used as template for generating the hygro-
mycin B resistance cassette to target the ORF15::apr holomycin-
overproducing mutant, which already contains an apramycin
resistance marker. Both pIJ vectors were obtained from John
Innes Centre, Norwich, UK. TheE. coliWM6062straincontaining
the disruption vectors was conjugated with the wild type or
ORF15::apr S. clavuligerus strains following stardard protocols.18
Correct exoconjugants resulting from homologous recombination
were selected based on antibiotic resistance and verified by PCR
using an internal primer for the disruption cassette (pIJ773-
RP603: 50-GAG TTG TCT CTG ACA CAT TCT GGC G-30;
or pIJ10700-FP1000: 50-GGG AAC ACC GTG CTC ACC-30)
and an external primer that anneals to hlmF gene (Scl-HlmF-
LICFP: 50-GGT ATT GAG GGT CGC ATG GAA AAT CCG
ATC CCC TCT CCT TC-30). The PCR products were also
purified and verified by DNA sequencing.
Preparation and Characterization of Reduced Holomycin.
The disulfide formation is proposed to occur at late stages of
holomycin assembly via oxidation of the dithiol groups in reduced
holothin (red-holothin) or red-holomycin, 2 (Figure 2B). Because
a large quantity of holomycin was readily available from a
previously described overproducing strain of S. clavuligerus,21 we
first reduced holomycin with tris(2-carboxyethyl)phosphine
(TCEP) to generate red-holomycin, 2, as a substrate for enzymatic
analysis. LC-MS analysis of the TCEP reduction mixture showed
chromatographic separation of 1 ([M þ H]þ expected 214.9943,
observed 214.9954) from 2 ([M þ H]þ expected 217.0100,
observed 217.0111), which was eluted earlier than holomycin on
the C18 column and showed two mass unit increase in molecular
weight (Figure 3A) as anticipated. The conversion of the disulfide
form to the dithiol form of holomycin by TCEP resulted in a
significant blue shift of the λmax of UV absorbance from 385 nm in
2 to 340 nm in 1 with an ∼50% increase in extinction coefficient to
15900 Mꢀ1 cmꢀ1 (Figure 3B). The reduction of holomycin to
red-holomycin is consistent with prior findings from Hertweck and
colleagues that TCEP generates the reduced form of gliotoxin.11
The change in UV absorbance affords an analytical method for
monitoring the reductionꢀoxidation reaction described below.
S. clavuligerus HlmI Is an FAD-Dependent Oxidase for
Reduced Holomycin. Among the four predicted flavoenzymes in
the S. clavuligerus cluster, HlmI shares sequence homology with
thioredoxin oxidoreductase superfamily (Figure S1), in particular
the recently characterized gliotoxin dithiol oxidase GliT.11 To
evaluate a comparable function of HlmI as a red-holomycin oxidase,
Analysis of Holomycin Production by hlmI Deletion Mu-
tants. The correct ΔhlmI deletion mutants were grown in TSB
media for 24ꢀ48 h. A sample of 10 mL of liquid GSPG
(glycerolꢀsucroseꢀprolineꢀglutamate) medium19 was inocu-
lated with 10 μL of the starter culture and grown with shaking
at 30 °C for 3ꢀ5 days. The resulting culture supernatant was
filtered with Spin-X centrifuge tube filters (Corning), and a sample
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Figure 4. (A) UVꢀvis absorbance spectrum of HlmI containing 20% of FAD. (B) Time course of oxidation of 100 μM red-holomycin by 50 nM HlmI in
10 min (UV spectra scan was taken every 0.4 min). Arrow indicates the direction of UV spectra progression. (C) MichaelisꢀMenten kinetics curve of
HlmI (20% FAD) and GliT (35% FAD) with varying concentrations of red-holomycin substrate. Standard deviation is shown as error bars. (D)
Anaerobic assay of 50 μM of red-holomycin and 50 nM HlmI in the presence of 1 mM NADþ (NADþ, no O2). HlmI was unable to convert 2 to 1
without oxygen. When the assay was opened to the atmosphere (NADþ, O2), facile oxidation of 2 to 1 by HlmI was observed. Substitution of NADþ
with NADPþ yielded the very similar results and therefore is not shown in the graph.
addition of excess TCEP did not further increase the UV absor-
bance at 340 nm, indicating that nearly all holomycin in solution
had been reduced. Subsequent addition of HlmI led to rapid loss of
the A340 nm peak as the dithiol substrate was oxidized to intramo-
lecular disulfide in holomycin. A family of progression curves could
be recorded to monitor reaction progress and enabled determina-
tion of the catalytic parameters of HlmI as a dithiol oxidoreductase
for red-holomycin (Figure 4B).
Concentration of 2 (from quantitative TCEP-mediated re-
duction of 1 in situ) was varied at a fixed concentration of HlmI
(50 nM, effective concentration 10 nM based on 20% active
enzyme by FAD content) to determine the steady-state kinetic
parameters. A MichaelisꢀMenten curve was generated yielding
a kcat of 333 ( 28 minꢀ1 and a KM of 4.6 ( 1.9 μM for 2
(Figure 4C). Kinetic measurements using FAD recontituted
HlmI with 0.76 equiv of FAD generated similar results (Figure S3).
In control experiments under the same incubation conditions
without addition of enzyme, the nonenzymatic rate of reoxida-
tion of 2 to 1 was monitored and a half-life of 7 min was obtained
for 2, corresponding to a kuncat of about 0.1 minꢀ1 (Figure S4).
The kcat/kuncat ratio for HlmI is 3330/1, suggesting a 3 orders of
magnitude rate enhancement in accelerating the disulfide for-
mation. The half-life of reduced holomycin was not altered in
the presence of EDTA, suggesting metal-mediated thiol oxida-
tion was not responsible for the nonenzymatic formation of the
intramolecular disulfide.
Figure 5. Proposed mechanism of catalysis by HlmI to convert red-
holomycin to holomycin.
HlmI was expressed heterologously in E.coli and purified as a yellow
protein at a yield of 20 mg/L (Figure 4A and Figure S2a). The
bound cofactor was determined as FAD by LC/MS after heat
denaturation of HlmI (Figure S2b,c) and was present in 0.2 mol
equiv to the 40 kDa protein. In vitro reconstitution by incubation
with excess FAD resulted in an increase in FAD content to 0.76
equiv. The apparent oligomeric state of HlmI was determined by
gel filtration elution profile as dimeric.
To assay the activity of purified HlmI, holomycin, 1, was reduced
in situ in buffer solutions with 1 equiv of TCEP to generate red-
holomycin, 2. The reduction process appeared to be complete as
No electron receptors were added in the HlmI oxidation reaction,
suggesting that molecular oxygen is the terminal electron acceptor.
Addition of nicotinamide cofactors (NADþ or NADPþ) did not
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KM and kcat values were obtained for GliT reconstituted with 0.8
equiv of FAD (Figure S3). The kcat/KM catalytic efficiency ratios
are 72 minꢀ1 μMꢀ1 for HlmI and 16 minꢀ1 μMꢀ1 for GliT for the
oxidation of red-holomycin, respectively. Therefore, HlmI is over-
all more efficient than GliT to make the cyclic disulfide in
holomycin, but GliT is substantially promiscuous.
red-Holothin vs red-Holomycin as HlmI Substrates. During
the initial characterization of the holomycin biosynthetic path-
way, we demonstrated that an acetyltransferase HlmA encoded
in the gene cluster catalyzes N-acetylation of holothin
(deacetylated holomycin, see Figure 2B for structure) as a late
step in the pathway.13 Acetylation of the exocyclic amino group
of the dithiolopyrrolone was proposed to be involved in stabiliz-
ing the heterocyclic scaffold and/or be a self-protection strategy.
We attempted to evaluate whether the reduced dithiol form of
holothin could also be a substrate for HlmI. Holothin was
generated by microwave-mediated deacetylation of holomycin
and isolated in its oxidized form.13 Subsequent reduction of
holothin by TCEP resulted in a blue shift in the UV spectrum
consistent with the maximum absorbance around 340 nm ob-
served in red-holomycin (Figure S5a). The dithiol group in red-
holothin was much more prone to nonenzymatic oxidation to
disulfide than red-holomycin. The half-life of red-holothin under
aerobic conditions was very short, about 0.3 min (Figure S5b).
HlmI qualitatively accelerated the reoxidation of red-holothin to
holothin, but the high nonenzymatic background rate precluded
kinetic measurements. The significantly faster nonenzymatic
oxidation of red-holothin compared to red-holomycin indicates
that acetylation of the exocyclic amine of the aminopyrrolone
ring confers a substantial electronic effect on the dithiol/disulfide
redox properties. In both the N-acetylated red-holomycin and the
free amine-containing red-holothin, the intramolecular dithiol
groups are set up geometrically for facile closure to cyclic
disulfide.
Phenotype of an HlmI deletion in S. clavuligerus. To assess
the importance of HlmI in holomycin biosynthesis, we con-
ducted targeted deletion of the hlmI gene in both the wild
type S. clavuligerus and in the holomycin-overproducing strain
(ΔORF15) in which ORF15 in the clavulanate biosynthetic
pathway has been disrupted resulting in 10ꢀ100-fold over-
production of holomycin.21 The deletion of hlmI in both strains
was verified by PCR analysis (Figure S6), and holomycin
production was decreased by 102ꢀ103-fold in both strains
(Figure 6A). Agar diffusion assays with pure holomycin demon-
strated that hlmI deletions in wild type and ΔORF15 S. clavuli-
gerus strains rendered both strains more susceptible toward
holomycin (Figure 6B). This result suggests that HlmI protects
the producer against the deleterious effects of holomycin at a
high concentration. Similarly, it was reported that deletion of gliT
in gliotoxin pathway abolished gliotoxin production and led to
increased sensitivity of the producing strain toward gliotoxin.11,22
Similar to gliotoxin, holomycin may also exist in the inactive
disulfide form and become reduced in the cellular environment
yielding the active form of the antibiotic. The dithiol oxidase
HlmI may act as a protective catalyst against holomycin in the
producing S. clavuligerus strain.
Figure 6. Phenotypes of hlmI deletion mutants in wild type S. clavuli-
gerus and holomycin-overproducing strain (ΔORF15). (A) Holomycin
production by four S. clavuligerus strains as determined by LC-MS. Mass
extraction results are shown for holomycin ([M þ H]þ, 214.9943),
allowing for 20 ppm error. The level of holomycin produced by ΔhlmI
and ΔhlmI/ΔORF15 strains was decreased by 102ꢀ103-fold in compar-
ison to that of wild type, and the ΔORF15 mutant and therefore did not
show in the trace when the mass intensity is normalized to the level of
holomycin production in ΔORF15. (B) Sensitivity of four S. clavuligerus
strains toward holomycin determined in an agar diffusion inhibition
assay. A sample of 5 μL of 7 mg/mL holomycin was added onto a lawn of
freshly plated bacteria and incubated at 30 °C for 5 days.
increase the oxidation rate. Furthermore, HlmI failed to convert 2 to
1 under anaerobic conditions in the presence of NADþ or NADPþ
but readily catalyzed the disulfide formation once the reaction is
opened to the atmosphere (Figure 4D). These results indicate that
HlmI acts as a dithiol oxidase, similar to GliT. GliT has been
proposed to use its active site disulfide as an initial redox port of
entry for electrons from the dithiol substrate into FAD.11 The
resulted FADH2ꢀenzyme intermediate would be rapidly reoxidized
by O2, presumably through the canonical FAD-C4a-OOH inter-
mediate. Such a pathway for electron flow is also likely for HlmI,
which contains a conserved CXXC motif (Figure S1). We propose
that the FAD reductive half-reaction is initiated by attack of the thiol
group or the eneꢀthiol group of 2 on the active site disulfide in
HlmI to yield a covalent disulfide adduct with the enzyme.
Intramolecular attack of the second thiol upon proton abstraction
by an active site base would lead to formation of the cyclic disulfide
in 1 with generation of the reduced form of the enzyme (it is not
known which thiol of 2 would initiate attack on the disulfide in the
active site of HlmI; the enethiol, as a tautomer of a thioaldehyde, is
likely to be less nucleophilic, but nothing is known about the
orientation of 2 in the enzyme active site) (Figure 5). FADox then
regenerates the active site disulfide in the enzyme to form FADH2.
Oxidation of FADH2 by O2 yields a transient FAD-C4a-OOH on
the way to release of hydrogen peroxide and regeneration of FADox,
presumably constituting the oxidative half-reaction. The utilization
of O2 differentiates HlmI and GliT from DepH, which makes use of
NADþ or NADPþ as electron receptors.12
GliT Has Reduced Holomycin Oxidase Activity. Recombi-
nant GliT was purified from E. coli (Dr. Carl Balibar thesis)a and
contains 0.35 equiv of bound FAD. We assayed GliT at 25 nM
(effective concentration 8.75 nM with bound FAD) to determine
whether it can oxidize 2 in addition to its normal substrate reduced
gliotoxin (red-gliotoxin). This fungal dithiol oxidase can indeed
oxidize 2 back to 1 (Figure 4C) with a KM for 2 at 198 ( 64 μM,
20ꢀ130-fold higher than HlmI. Interestingly, the measured kcat
was about 10-fold higher than HlmI, at 3166 ( 742 minꢀ1. Similar
DithiolꢀDisulfide Reactivity in Holomycin. It is not clear
whether holomycin plays physiological roles other than self-
defense for its producing strain. The exact mechanism of action
against target microbes has yet to be determined. Although
holomycin was shown to block RNA synthesis in whole bacteria
and RNA polymerases have been suggested as holomycin targets,
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Biochemistry
ARTICLE
may ensure accumulation of the natural product in the oxidized
and acetylated state with low adventitious redox reactivity. Future
studies will involve the examination of the formation of any mixed
disulfides between holomycin and intracellular proteins as part of
the antibacterial effect of holomycin and related dithiolopyrrolone
scaffolds.
An alternative mode of reactivity is also possible involving
conjugated addition of cellular nucleophiles to the dienone
chromophore for holomycin. Amine nucleophiles such as
N-acetylcysteamine to a certain degree perturbed the UV spec-
trum of holomycin by changing its λmax at 385 nm in buffered
solutions at pH 9 (Figure S8), although no adduct formation has
been observed by LC-MS, suggesting that the conjugated
Michael addition of amines with holomycin may be transient.
Phylogeny Analysis of HlmI. Alignment of HlmI with a
number of bacterial and fungal proteins that are likely to catalyze
dithiolꢀdisulfide redox chemistry revealed four distinctive phy-
logeny groups: the thioredoxin oxidoreductase (TrxB) group
and three small molecule oxidoreductase groups (Figure 7 and
Figure S8). The TrxB group catalyzes the reduction of disulfides
in thioredoxins, which are small proteins involved in a wide range
of cellular processes such as redox-sensitive signal transduction,
stress response, and detoxification.23 This group includes both
bacterial and fungal thioredoxin oxidoreductases. Interestingly,
among the small molecule oxidoreductases, HlmI, GliT, and
DepH belong to three different groups. The GliT group consists
of fungal proteins from gene clusters for biosyntheses of mol-
ecules closely related to gliotoxin and is likely differentiated from
the HlmI and DepH groups because of its fungal origin. The
DepH group contains a number of bacterial putative oxidore-
ductases including TdpH encoded in a FK228 homologous gene
cluster from Burkholderia thailandensis E264. The products of
this gene cluster have recently been identified as burkholdac A
and B, which are new HDAC inhibitors containing an intramo-
lecular disulfide.24 Many hypothetical proteins in the HlmI group
are predicted dithiolꢀdisulfide oxidoreductases, and they may
be involved in similar cellular processes such as small molecule
biosynthesis or detoxification. Further interrogation of the
genome context of these hypothetical proteins may lead to the
discovery of novel natural product biosynthetic gene clusters.
Figure 7. Phylogenetic analysis of HlmI and HlmI homologues (HlmI
group), DepH and homologues (DepH group), GliT and homologues
from other ETP fungal clusters (GliT group), and thioredoxin reduc-
tase-like proteins from bacteria and fungi (TrxB group). This dendro-
gram is generated by RAxML and bootstrap support values and the
distance scale are displayed.
E. coli RNA polymerases are not susceptible toward holomycin in
in vitro assays.7 The disulfide embedded in a conjugated bicyclic
framework is likely to engage in dithiolꢀdisulfide redox chem-
istry with holomycin targets. We have conducted an initial
examination of holomycin reactivity with the most abundant
cellular thiol, glutathione, as a prototypic marker of redox
behavior. We first incubated the disulfide-containing holomycin
with the reduced form of glutathione (GSH) and monitored for
reduction of holomycin by gain of UV absorbance at 340 nm. No
changes were detected over a wide range of GSH concentrations
and molar excess over 1. When we assayed in the opposite
direction, for the ability of 2 to reduce oxidized glutathione
(GS-SG), a rapid decrease in UV absorbance at 340 nm was
observed, corresponding to the oxidation of 2 by GS-SG. The
rate of oxidation of 2 as a function of oxidized GS-SG is shown in
Figure S7.
Subsequent studies will be necessary to determine redox
equilibria and ultimately reduction potentials of holomycin under
conditions where competing oxidation by molecular O2 is sup-
pressed. It is clear that red-holomycin is kinetically and thermo-
dynamically competent to reduce the intermolecular disulfide in
oxidized glutathione. Thus, the reduced form of holomycin and
presumably holothin (perhaps even more so) may be strong
thiol reductants in both microbial producers and target cells.
The combination of oxidation by HlmI and acetylation by HlmA
’ SUMMARY
HlmI joins GliT as a member of FAD-dependent dithiol
oxidases, using O2 as cosubstrate to accelerate the formation of
intramolecular disulfide bridges in the late steps of small mole-
cule biosynthesis. These two enzymes differ from the FK228
dithiol oxidase DepH, which utilizes NADPþ as the electron
acceptor. Both HlmI and GliT were shown to play a role in
protecting the producer organisms from the action of their own
natural products. Similar to gliotoxin and FK228, the reduced
dithiol form of holomycin may be the active form of the
antibiotic, and the redox properties of the thiol/enethiol pair in
reduced holomycin remain to be examined. HlmI and related
proteins constitute a new phylogenetic group of oxidoreductases
that catalyze disulfide formation in small molecules and are
different from the GliT and DepH dithiol oxidase groups. These
small molecule dithiol oxidases are phylogenetically separated
from the peptide oxidoreductases, suggesting they have evolved
for specific and efficient catalysis of small molecule substrates.
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ARTICLE
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: christopher_walsh@hms.harvard.edu. Phone: 617.432.
1715. Fax: (þ1) 617.432.0483.
Funding Sources
This research was financially supported by NIH Grant GM49338
(C.T.W.).
’ ACKNOWLEDGMENT
We thank Dr. Carl Balibar for the generous gift of purified
GliT, Dr. Kapil Tahlan for providing S. clavuligerus ORF15::apr
mutant, Dr. Emily Balskus and Dr. Rebecca Case for assistance in
constructing the phylogeny tree, and Dr. Albert Bowers for
suggestions regarding the manuscript.
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’ ABBREVIATIONS
red-holomycin, reduced holomycin; red-holothin, reduced holothin;
red-gliotoxin, reduced gliotoxin; red-FK228, reduced FK228; HDAC,
histone deacetylase; FAD, flavin adenine dinucleotide; NRPS, non-
ribosomal peptide synthetase; IPTG, isopropyl-β-D-thiogalactopyr-
anoside; TCEP, tris(2-carboxyethyl)phosphine; GSH, glutathione;
GS-SG, oxidized glutathione; NADþ, nicotinamide adenine dinu-
cleotide; NADPþ, nicotinamide adenine dinucleotide phosphate;
SDS-PAGE, sodium dodecyl sulfateꢀpolyacrylamide gel electro-
phoresis; HPLC, high-performance liquid chromatography; LC-MS,
liquid chromatographyꢀmass spectrometry.
’ ADDITIONAL NOTE
a Purified GliT was generously provided by Dr. Carl Balibar,
a previous graduate student in the Walsh group.
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