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Applied and Environmental Microbiology, October 2001, p. 4512-4519, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4512-4519.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Extracellular Heme Peroxidases in Actinomycetes: a
Case of Mistaken Identity
Maria G.
Mason,*
Andrew S.
Ball,
Brandon J.
Reeder,
Gary
Silkstone,
Peter
Nicholls, and
Michael T.
Wilson
Department of Biological Sciences, University
of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom
Received 26 March 2001/Accepted 18 July 2001
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ABSTRACT |
Actinomycetes secrete into their surroundings a suite of enzymes
involved in the biodegradation of plant lignocellulose; these have been
reported to include both hydrolytic and oxidative enzymes, including
peroxidases. Reports of secreted peroxidases have been based upon
observations of peroxidase-like activity associated with fractions that
exhibit optical spectra reminiscent of heme peroxidases, such as the
lignin peroxidases of wood-rotting fungi. Here we show that the
appearance of the secreted pseudoperoxidase of the thermophilic
actinomycete Thermomonospora fusca BD25 is also associated
with the appearance of a heme-like spectrum. The species responsible
for this spectrum is a metalloporphyrin; however, we show that this
metalloporphyrin is not heme but zinc coproporphyrin. The same
porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that
earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather
than to non-iron-containing porphyrins and that lignin-degrading heme
peroxidases are not secreted by actinomycetes. The porphyrin, an
excretory product, is degraded during peroxidase assays. The low levels
of secreted peroxidase activity are associated with a nonheme protein
fraction previously shown to contain copper. We suggest that the role
of the secreted copper-containing protein may be to bind and detoxify
metals that can cause inhibition of heme biosynthesis and thus
stimulate porphyrin excretion.
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INTRODUCTION |
Biodegradation of lignocellulose by
microorganisms plays an important role in carbon cycling, is of
biotechnological interest to the paper industry, and has potential
application in the field of bioremediation. Some microorganisms secrete
a range of enzymes that completely degrade all the components of
lignocellulose (lignin, hemicelluloses, and cellulose), while others
secrete a narrower range of enzymes that only partially achieve this
degradation (8, 19, 21). White rot fungi secrete both
cellulolytic and ligninolytic enzymes; heme enzymes are major
components of this ligninolytic activity and include the
well-characterized lignin and manganese peroxidases of
Phanerochaete chrysosporium (12).
Some actinomycetes, including thermophilic species and streptomycetes,
secrete cellulose- and hemicellulose-degrading enzymes (2, 4,
11). Since the discovery of extracellular lignin-degrading heme
peroxidases of wood-rotting fungi, considerable effort has been
expended in searching for analogous enzymes in the cellulolytic actinomycetes (9, 22, 24). Indeed, some proteins secreted by the actinomycetes Streptomyces thermoviolaceus
(17), Streptomyces viridosporus T7A (6,
26, 27), and Thermomonospora fusca BD25 (recently
reclassified as Thermobifida fusca [35])
(3, 28) have low peroxidase activity and have been
isolated and partially characterized. The peroxidase-like
proteins from S. thermoviolaceus and S. viridosporus T7A were assigned as heme peroxidases on the
basis of their optical spectra. However, these proteins were not shown
to contain heme directly, nor were they shown to exhibit the
characteristic spectral features associated with changes in the heme
iron redox state, as are seen with P. chrysosporium lignin
peroxidase (32-34) and other heme proteins. Electron
paramagnetic resonance (EPR) spectroscopy has shown that the partially
characterized metalloprotein with peroxidase activity from T. fusca BD25, although displaying heme-like spectra, exhibits no
paramagnetically active heme component in any accessible redox state.
Instead, it contains a nonheme iron center and copper, present in
substantial molar excess over the protein (30).
Here we report that T. fusca exports into the culture medium
a porphyrin, the optical spectrum of which may easily be mistaken for
that of heme. The excretion of this porphyrin parallels the appearance
of peroxidase activity. However, this porphyrin, although associated
with the weak peroxidase activity in the culture medium, is not heme,
and no true peroxidase is present. Furthermore, we confirm that the
weak catalytic peroxidase activity is due to copper associated with the
protein. Our results are compared with those of previous work in this
area, and we conclude that no evidence has been provided to
substantiate previous claims that actinomycetes secrete heme
peroxidases. Thus, we propose that this class of extracellular enzymes
is absent from this family.
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MATERIALS AND METHODS |
Both T. fusca BD25 and S. viridosporus T7A
were maintained as suspensions of spores and hyphal fragments in 20%
(vol/vol) glycerol at 
20°C and routinely cultured on L agar plates
or slants (31). T. fusca liquid cultures were
grown for 7 days as described previously (31) with oat
spelt xylan as the carbon source (8 g liter
1). S. viridosporus T7A was grown under identical conditions except that
the growth temperature was 30°C rather than 50°C. During this
period, the supernatants were monitored for peroxidase activity and for
spectroscopic changes characteristic of metalloporphyrins, i.e., the
appearance of a Soret absorbance band at ~400 nm. Culture supernatants were assayed for heme directly using the pyridine hemochromogen test, i.e., 10-fold dilution of culture supernatants in
0.1 M NaOH-20% pyridine followed by the addition of a few grains of
the reductant sodium dithionite (1). Solutions of the heme proteins horseradish peroxidase, myoglobin, and cytochrome
c, with similar Soret absorbances, were used as positive
controls. Peroxidase activity was measured spectrophotometrically at
510 nm by monitoring the formation of a Schiff base product from the reaction of 4-aminoantipyrene with oxidized 2,4-dichlorophenol. Assays
were performed with defined H2O2 concentrations
and with 5 mM 2,4-dichlorophenol plus 3.2 mM 4-aminoantipyrene as the
colorigenic electron donor system at pH 7 and 30°C.
The kinetic constants Km and maximum turnover
number (TNmax) were determined by fitting the experimental
data to the standard Michaelis-Menten equation using Kaleidagraph
fitting software. Absorbance spectroscopy was carried out using a
Hewlett-Packard diode-array instrument (HP8453), and porphyrin
fluorescence was characterized using a Perkin-Elmer spectrofluorimeter
(LS50B). Reversed-phase high-pressure liquid chromatography (HPLC) was used to isolate the porphyrin from culture supernatants; for this procedure, a Hewlett-Packard HPLC 1100 instrument equipped with a
Zorbax SB-C3 300A column was used. The porphyrin was eluted with an
isocratic gradient system of 35 to 40% acetonitrile in H2O
and 0.1% trifluoroacetic acid (pH 2) (see Fig. 3) at a flow rate of 1 ml/min. Porphyrin concentrations during growth were quantified by HPLC
fractionation of culture supernatants and integration of the major
porphyrin elution peak. Reversed-phase HPLC using a Zorbax 300 SB
C18 column (250 by 4.6 mm) was used to separate porphyrin
standards and thus identify the T. fusca porphyrin by comparing its retention time and UV-visible spectra to those of the
standards under the same conditions. T. fusca and standard porphyrins were eluted with an isocratic gradient system of 5 to 50%,
50 to 95%, and 95 to 5% acetonitrile in H2O and 0.1%
trifluoroacetic acid (pH 2) (see Fig. 4) at a flow rate of 1 ml/min.
Mass spectrometry was carried out by Matrix-assisted laser desorption
ionization-time of flight with a Kratos Analytical Kompact Maldi II instrument.
All standard reagents were from Sigma Chemical Co. Samples of free
porphyrins and copper, zinc, and magnesium metalloporphyrins were
obtained from Porphyrin Products Inc.
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RESULTS |
Characterization of extracellular porphyrin.
Supernatants
obtained from centrifugation of T. fusca cultures possess an
extracellular peroxidase activity and contain a porphyrin species.
Figure 1 compares the spectra of the
culture medium at intervals during growth. The Soret band at 404 nm and the two peaks in the visible region, at 537 and 573 nm, are
characteristic of porphyrins, including heme (iron protoporphyrin IX).
Here they are seen superimposed upon a background spectrum that we
assign to melanin or a melanin-like pigment that renders the
supernatant yellow. The porphyrin concentration reached a maximum on
days 3 and 4, as did the peroxidase activity; thereafter, both declined (Table 1). This result suggested that a
heme peroxidase enzyme could be responsible for the peroxidase
activity, as reported for Streptomyces and for the white rot
fungus P. chrysosporium (12). However, the
species responsible for the porphyrin spectrum shown in Fig. 1 was
insensitive to the powerful reductant sodium dithionite. A direct test
for heme, namely, the formation of a pyridine hemochromogen when heme
is treated with pyridine under alkaline conditions, gave
negative results, indicating that any heme protein present must have
been less than ~1% of that expected for a heme protein displaying a
Soret band of similar absorbance (data not shown). These findings
eliminate the possibility that this species is a heme group.
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FIG. 1.
Time course of porphyrin secretion during the growth of
T. fusca. The UV-visible spectra of the culture medium on
days 1, 2, 3, 4, and 7 of growth are shown (temperature, 25°C; path
length, 1 cm). The Soret band at 404 nm and the two peaks in the
visible region appear on day 2 and reach maxima on day 3. Thereafter,
they decrease; by day 7, they are undetectable.
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In addition, the porphyrin species was found to be fluorescent. Figure
2 shows that photoexcitation at 410 nm
gives rise to
three fluorescence emission bands, at 470, 577, and 630 nm. The
latter two bands are characteristic of a fluorescent nonheme
porphyrin.
Heme groups themselves are not fluorescent. The emission
band
at 470 nm did not maintain a constant intensity ratio with the
two
porphyrin-specific bands, and we tentatively attribute this
result to
emission from a further fluorophore, such as melanin.
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FIG. 2.
Fluorescence emission-excitation spectra. Samples of
culture medium were diluted 200-fold in 100 mM potassium
phosphate at pH 7.0 and 30°C. The emission and excitation band widths
were set at 5 nm. (a) Excitation spectrum of T. fusca
supernatant monitored at 575 nm. (b) Emission spectrum of T. fusca supernatant excited at 410 nm.
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The porphyrin from
T. fusca was isolated from the culture
medium by HPLC. Elution was monitored optically at 400 nm,
close
to the Soret maximum. The total elution profile (Fig.
3A) shows
one major, sharp elution band
at 15.3 min (38% acetonitrile) and
several minor elution bands,
including those at 9.1 and 5.6 min.
The absorbance spectra of the
porphyrin species eluted at 5.6,
9.1, and 15.3 min are shown in Fig.
3B, C, and D, respectively.
The major porphyrin eluted at 15.3 min
(Fig.
3D) is spectroscopically
free of protein or melanin. The
porphyrin eluted at 9.1 min is
complexed with a species absorbing at
300 nm, and the minor component
eluted at 5.6 min contains a complex
absorbing at 278 and 360
nm. The 278-nm absorbance band indicates
aromatic amino acid residues
of proteins, the 300-nm absorbance band
indicates oxidized aromatic
amino acids, and the 360-nm absorbance band
indicates a melanin-like
pigment.
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FIG. 3.
HPLC elution profile and absorbance (Abs) spectra of
T. fusca porphyrin. (a) Elution profile from HPLC
fractionation. Solid line, absorbance at 400 nm (milliabsorbance units
[mAU]); squares, acetonitrile gradient. (b) Absorption
spectrum of 400-nm elution peak at 5.6 min, shown with subtraction of
simulated 360-nm absorbing species. Black line, actual spectrum; broken
line, simulation of 360-nm species; grey line, spectrum minus
simulation, i.e., showing the porphyrin Soret peak. (c) Absorption
spectrum of 400-nm elution peak at 9.1 min. (d) Absorption spectrum of
400-nm elution peak at 15.3 min, showing  ,  , and Soret absorption
bands.
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The
T. fusca porphyrin was fully soluble in aqueous
media at a neutral pH, whereas authentic metal-free and
metallated protoporphyrins
were not. Figure
4 (main panel) shows [see below] the
HPLC elution
profile of the porphyrin standards (Zn
2+
coproporphyrin III and Fe
3+, Mg
2+
Cu
2+, and nonmetallated protoporphyrin IX) together
with the elution
profile of the
T. fusca porphyrin under the
same conditions. Elution
was monitored at 406 nm, the Soret maximum for
the
T. fusca prophyrin.
Both the
T. fusca
porphyrin and the Zn
2+ coproporphyrin III standard eluted
at 9.6 min (64% acetonitrile).
Magnesium protoporphyrin is not seen as
a separate species in
the elution profile because under the HPLC
conditions used (pH
2), the magnesium ion coordination ligands become
protonated and
the magnesium is displaced; the demetallated
protoporphyrin coelutes
with protoporphyrin IX. The broad band eluting
at between 4 and
8 min is due to melanin; porphyrin was not detected in
these fractions.
Figure
4, inset a, compares the spectra in the visible
region
of the
T. fusca porphyrin to those of all the
porphyrin standards.
Figure
4 inset b shows the overlaid UV-visible
spectra of the
T. fusca porphyrin and the Zn
2+
coproporphyrin III standard; these are essentially identical.
All
T. fusca traces have been normalized to the absorbance scale
of the porphyrin standards. The acid-alkali pH dependencies of
the
positions and intensities of the Soret,
and
bands in the
T. fusca porphyrin and the Zn
2+
coproporphyrin III standard were also identical (data not
shown).
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FIG. 4.
Identification of T. fusca porphyrin. HPLC
retention times and UV-visible absorbance (Abs) spectra of T. fusca porphyrin and porphyrin standards are shown. All spectra
were recorded during HPLC separation. T. fusca traces were
normalized to the scale of porphyrin standards. (Main panel) Elution
profile (monitored at 406 minus 470 nm) of T. fusca
porphyrin loaded as culture supernatants (black line) and porphyrin
standards (grey line) and acetonitrile gradient (stippled line). mAU,
milliabsorbance units. (Inset a) Visible spectrum of porphyrin
standards (grey lines) and T. fusca porphyrin (black line).
(Inset b) UV-visible spectra of T. fusca porphyrin (black
line) and Zn coproporphyrin III standard (grey line).
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Kinetics of pseudoperoxidase.
Figure
5 shows a typical time course for the
peroxidase activity of the T. fusca supernatant. This time
course, which monitors product formation, is biphasic. An initial burst
phase is followed by a steady, slow phase. The burst phase is
paralleled by a process in which the porphyrin is degraded, as
demonstrated by a decrease in the absorbance at 403 nm. This result
suggests that porphyrin is acting as a substrate, not as a catalyst.
The amplitude of the burst phase shows that approximately 4 mol of
product is formed per mol of porphyrin degraded.
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FIG. 5.
Correlation between the degradation of T. fusca porphyrin and peroxidase activity during the initial burst
phase. Time courses for peroxidase activity of the T. fusca
supernatant (left ordinate; squares) and porphyrin degradation at an
absorbance (Abs) of 385 to 403 nm (right ordinate; circles).
Measurements were made with 100 mM sodium or potassium phosphate buffer
at pH 7.0 and 30°C.
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The kinetic behavior of the slow, catalytic phase was analyzed by
varying the hydrogen peroxide concentration. Figure
6 compares
the activity of
T. fusca pseudoperoxidase with those of horseradish
peroxidase and
inorganic copper sulfate. The peroxide concentration
is plotted
logarithmically in order to display the full range
utilized. From these
data, the apparent
Km and TN
max were
determined
by fitting the experimental data to the Michaelis-Menten
equation
(see Materials and Methods). Although the peroxidase activity
of the supernatant increases with increasing peroxide concentration
according to the classical hyperbolic relationship, similar behavior
is
seen with inorganic cupric ions as catalysts. Table
2 lists
the values of the kinetic
constants (
Km and TN
max) obtained
from
the data in Fig.
6. The
Km for
H
2O
2 of 18 mM for
T. fusca
pseudoperoxidase
is very similar to the value of 22 mM for free
cupric ions and
quite different from that of 0.74 mM for horseradish
peroxidase,
an authentic heme enzyme.
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FIG. 6.
Kinetics of three peroxidase catalysts. Peroxidase
activities were measured at pH 7.0 and 30°C as described in Materials
and Methods. Points were fitted to a Michaelis-Menten equation with
substrate inhibition. Catalyst concentrations and derived parameters
are listed in Table 2. HRP, horseradish peroxidase.
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Excretion of porphyrins by other microorganisms.
When the
culture medium from S. viridosporus T7A was subjected to the
same analytical procedures as T. fusca, spectral features identical to those shown in Fig. 1 were observed. Again, the
characteristic porphyrin bands were detected and remained unchanged
upon addition of the reductant sodium dithionite. No pyridine
hemochromogen was formed. The spectrum of the S. viridosporus porphyrin was also identical to that of the T. fusca porphyrin after separation by HPLC (Fig. 3).
Furthermore, under certain growth conditions, this same porphyrin was
also produced by
Escherichia coli. The
E. coli
porphyrin
shared the same pH dependencies of spectral band positions
and
intensities as the
T. fusca porphyrin and the
Zn
2+ copropophyrin III standard. Mass spectrometry
of the porphyrin
from
E. coli (available to us in larger
amounts than the
T. fusca product) revealed a molecular mass
for the major fraction of 722
± 2
Da.
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DISCUSSION |
Characterization of T. fusca porphyrin.
Metalloporphyrins containing Fe, Cu, Ni, or Co are nonfluorescent,
whereas metal-free porphyrins and those complexed with Zn, Mg, and Cd
are fluorescent (10). The fluorescence spectrum of the
T. fusca porphyrin and the negative result in the pyridine hemochromogen test confirm that the porphyrin does not contain Fe and
therefore is not a heme species. The UV-visible spectrum, not having
four absorption bands in the visible region (Fig. 4), is not that of a
metal-free porphyrin. There are three categories of metal-porphyrin
bonds in metalloporphyrins, covalent type, ionic type, and intermediate
type. Each type of interaction has characteristic thermodynamic
stability and spectral properties. The spectral properties are related
to the relative intensities of the / band ratios of the visible
region. The covalent-type metal-porphyrin bonds, which include those of
Pt2+, Ni2+, Co2+, and
Cu2+, have / intensity ratios of 
1; ionic types,
including Mg2+, Pb2+, and Sn2+,
have / intensity ratios of 
1; intermediate types, which
include Zn2+ and Cd2+, have / intensity
ratios of approximately unity. Although magnesium is a commonly
available metal ion, the / band ratio in the visible region of
the spectrum excludes magnesium as the metal (25). Furthermore, magnesium porphyrins are not acid stable under the conditions used for HPLC analysis (see Results). Incorporation of metal
ions into porphyrins in aqueous solutions occurs at significant rates
only for Cu2+ and Zn2+ ions. The fact that Cu
produces a covalent-type spectrum (Fig. 4) eliminates copper as the
metal. The strong correlation of the T. fusca and zinc
coproporphyrin spectral properties, i.e., the pH dependencies of peak
positions and intensities, and the / band ratios therefore
support our identification of the remaining possibility, zinc, as the
metal-porphyrin bond species.
The porphyrin nucleus is hydrophobic, and the number of hydrophilic
carboxyl substituents increases its solubility in aqueous
media at a
neutral pH. Coproporphyrin, having four carboxyl substituents,
is
soluble in aqueous media at a neutral pH, whereas protoporphyrin,
with
only two carboxyl groups, is insoluble (
10,
25). The
excretion of coproporphyrin III in microorganisms is well documented
(
7).
The mass spectrometry result obtained with the identical
E. coli product is similar to that expected for zinc coproporphyrin.
Its mass, 722 ± 2 Da, is similar to that of 720 Da
expected for
zinc coproporphyrin. In addition to the spectral
properties, the
chemical properties of the
T. fusca
porphyrin and the Zn
2+ coproporphyrin III standard
were also identical, as shown by
the HPLC elution profile. The other
water-soluble porphyrin, uroporphyrin,
has eight carboxyl residues
(coproporphyrin has four); it is therefore
inconceivable that the
chemical properties and thus the HPLC retention
times would be
identical for zinc coproporphyrins and zinc uroporphyrins.
On the basis
of spectral and chemical properties, we therefore
conclude that the
excreted porphyrin is zinc
coproporphyrin.
The chemical and spectral characteristics of the porphyrin in the
S. viridosporus culture supernatant were identical to those
of the
T. fusca porphyrin; we therefore conclude that this
species
is also likely to be zinc coproporphyrin and not heme, as
previously
claimed (
26).
Porphyrins in other streptomycetes and actinomycetes.
Nonheme
porphyrins have been found in culture supernatants of the soil
streptomycetes Streptomyces misakiensis and
Streptomyces viridochromogenes (23). The
UV-visible spectra of these culture supernatants showed weak 400-nm
porphyrin and 280-nm protein absorption bands, which appeared, as in
the T. fusca spectra, superimposed on a broad melanin-like
absorption band that swept down from the UV spectrum to the visible
spectrum. Although the porphyrin type was not determined, EPR
spectroscopy identified the presence of copper, chelated by ligands
possibly donated by the porphyrin. This result does not exclude the
possibility that zinc porphyrin was also present in the culture
supernatants, as Zn2+ is EPR silent. EPR spectroscopy did
not detect copper porphyrin in the T. fusca culture medium.
The central metal ion in excreted porphyrins may depend upon the ions
available in the growth medium. In any case, the data for S. misakiensis and S. viridochromogenes show that the
presence of a distinct Soret band cannot be assumed to indicate the
presence of heme.
Protein-free fluorescent coproporphyrin III has also been found in the
culture medium of the marine actinomycete
Arthrobacter aurescens RS-2; its excretion was enhanced by aluminium and nickel
(
29). The porphyrin was excreted together with a protein,
possibly
protective in nature and of approximately the same size as the
pseudoperoxidase of
T. fusca (
28).
Porphyrin excretion in other organisms.
The porphyrin that
appears in T. fusca and S. viridosporus cultures
is not unique to actinomycetes; its formation in E. coli indicates that the porphyrin may be a pathological excretory product rather than a functional secretory product.
Aberrant porphyrin excretion has been seen in photosynthetic bacteria
(
5,
14), although in these organisms the porphyrin
was
complexed with proteins, suggesting a role in porphyrin biosynthesis;
again, the major porphyrin excreted was coproporphyrin III. Excretion
or accumulation of coproporphyrin III seems to be typical of blocked
porphyrin synthesis in microorganisms. The conversion of
coproporphyrinogen
III to protoporphyrin IX is a rate-limiting step in
bacterial
heme biosynthesis (
29).
Zinc coproporphyrin excretion is also not peculiar to microorganisms;
it is also recognized clinically as a feature of heme
metabolism in
neonates, whose earliest stools (meconium) have
been shown to contain
porphyrins, the major component of which
is zinc coproporphyrin
(
13,
16). It is recognized hematologically
as one of the
most common by-products of heme biosynthesis (
20),
a
consequence of its chemical stability and aqueous
solubility.
Secreted and intracellular proteins and enzymes.
Streptomycetes are characterized by containing genes for a remarkable
set of families of catalases and peroxidases, only some of which may be
expressed at any time. The Streptomyces coelicolor genome
project (18;
http://www.sanger.ac.uk/Projects/S_coelicolor/) has already revealed
the presence of at least one catalase-peroxidase gene and at least
three paralogues of classical catalase genes (P. Nicholls et al.,
unpublished data). In addition, these organisms contain a unique
family of nonmetal fatty acid-dependent peroxidases apparently derived
evolutionarily from hydrolytic ancestors and whose X-ray crystal
structures have been determined (15). The streptomycete
catalases and peroxidases can exist in more than one form
related by proteolytic degradation. The original holoenzymes contain
not only the heme group characteristic of catalases and peroxidases but also a C-terminal fragment that contains manganese and
that can engage in manganese-dependent peroxidation (36). However, there is no evidence that this enzyme, either in its holoenzyme form or in proteolytically degraded forms, can be secreted by streptomycetes. Moreover, the EPR study of the T. fusca
protein showed no manganese, an element with very characteristic EPR
signals (30).
The emerging genetic analysis of
T. fusca
(
http://www.jgi.doe.gov/JGI_microbial/html/thermobifida/thermob_homepage.html)
also
shows this microorganism to contain several
intracellular catalase
and peroxidase enzymes; these appear to
include the classical
Micrococcus-like catalase,
manganese catalase, glutathione peroxidase,
and a putative
nonheme peroxidase. Preliminary results have shown
the occurrence of
classical catalase activity in broken
T. fusca cells
(Nicholls et al., unpublished). This activity, however,
does not appear
in the growth medium, unless and until severe
damage to the cells and
breakdown of their walls and membranes
have
occurred.
The weak peroxidase activity found in the
T. fusca culture
medium is biphasic; the initial, burst phase correlates with porphyrin
degradation, while the slower, steady phase is comparable to the
peroxidase activity of free cupric ions.
T. fusca
copper-containing
peroxidase has up to 20 copper atoms per protein
molecule (
30),
and the total [Cu
2+] is
therefore 20 times the molar concentration of the protein
(Table
2).
This information implies that approximately 120 nM
bound Cu was present
in the assay system of Fig.
6. Both the apparent
Km and the turnover per Cu atom are therefore
similar for the
"enzyme" and for free CuSO
4.
The heme peroxidase reported for
S. viridosporus
(
26) was not shown directly to contain heme. Our results
show that this,
too, is a nonheme porphyrin associated with melanin and
protein,
like that in
T. fusca. Similarly, the reported heme
peroxidase
of
S. thermoviolaceus, which has a specific
peroxidase activity
comparable to that of
T. fusca, may also
owe its peroxidase activity
to transition metal ions associated, either
specifically or adventitiously,
with protein. An authentic type III
heme peroxidase (members of
the catalase-peroxidase family are
classified as type I) has yet
to be demonstrated in a
prokaryote.
It also improbable that the peroxidase activity of the
T. fusca protein is the function of a true ligninolytic enzyme.
Unlike
the relatively classical peroxidases of the lignin-degrading
fungi,
its turnover is very low and its relative affinity for peroxide
is also rather poor. In a marine
Vibrio sp.
(
14), copper induces
the excretion of extracellular
proteins whose function may be
to bind to and detoxify external copper
ions. The role of the
extracellular Cu protein in
T. fusca
(
30) is thus uncertain.
It may be a peroxidase of
relatively low activity, it may be a
detoxifying Cu-binding protein, or
it may have a function in the
complex process of lignin degradation
that has yet to be
determined.
The appearance of porphyrins into which iron has not been inserted is a
consequence of metal ion inhibition of heme biosynthesis.
The protein
that we have identified as containing copper may have
a role in metal
detoxification. Thus, during rapid growth in the
presence of metals,
such as copper, porphyrins are excreted as
waste products from
incomplete heme synthesis; a protective copper-binding
protein is also
secreted. The peroxidase activity may merely be
the consequence of
binding of the redox-active metal, copper,
to this protein. Our view,
therefore, is that there is no evidence
to support the contention that
actinomycetes secrete heme peroxidases
and that the spectra recorded
from culture media are due to zinc
(or another metal, e.g.,
Cu)-containing porphyrins and not to
heme.
| |
ACKNOWLEDGMENTS |
We thank Neil Barnard for carrying out the mass spectrometry and
Chris Cooper for discussions concerning peroxidase activity and structure.
This work was supported by BBSRC grants to M.T.W. and to A.S.B. and by
a BBSRC scholarship to M.G.M.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom. Phone: 44-1206-873333. Fax: 44-1206-872592. E-mail: mgmaso{at}essex.ac.uk.
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Applied and Environmental Microbiology, October 2001, p. 4512-4519, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4512-4519.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
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