From the Department of Biochemistry, University of
Illinois at Urbana-Champaign, Urbana, Illinois 61801 and the
§ Department of Applied Microbiology and Gene Technology,
TNO Nutrition and Food Research Institute, Utrehtseweg 48, 3704 HE
Zeist, The Netherlands
Received for publication, October 24, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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Site-directed mutagenesis has been used to
investigate the role of glutamic acid 183 in chloroperoxidase
catalysis. Based on the x-ray crystallographic structure of
chloroperoxidase, Glu-183 is postulated to function on distal side of
the heme prosthetic group as an acid-base catalyst in facilitating the
reaction between the peroxidase and hydrogen peroxide with the
formation of Compound I. In contrast, the other members of the heme
peroxidase family use a histidine residue in this role. Plasmids have
now been constructed in which the codon for Glu-183 is replaced with a
histidine codon. The mutant recombinant gene has been expressed in
Aspergillus niger. An analysis of the produced mutant gene
shows that the substitution of Glu-183 with a His residue is
detrimental to the chlorination and dismutation activity of
chloroperoxidase. The activity is reduced by 85 and 50% of wild type
activity, respectively. However, quite unexpectedly, the epoxidation
activity of the mutant enzyme is significantly enhanced ~2.5-fold.
These results show that Glu-183 is important but not essential for the
chlorination activity of chloroperoxidase. It is possible that the
increased epoxidation of the mutant enzyme is based on an increase in
the hydrophobicity of the active site.
Chloroperoxidase (CPO1;
EC 1.11.1.10) is a heavily glycocylated heme peroxidase secreted from
the filamentous fungus Caldariomyces fumago (1-3). Natural
product research in the 1940s discovered the biosynthesis of
chlorinated products by C. fumago (4). Caldariomycin
(2,2-dichloro-1,3-cyclopentanediol) is the major chlorinated compound
found in C. fumago cultures. Much later, enzyme and tracer
experiments delineated the role of CPO in the biosynthesis of
caldariomycin (5, 6). CPO is undoubtedly the most versatile member of
the heme peroxidase family. In addition to catalyzing chlorination,
bromination, and iodination reactions (7-9), CPO catalyzes
typical one electron oxidations (10) and possesses catalase (11)
and monooxygenase activities (12). CPO is especially adept in
catalyzing a number of chiral oxidations. The chiral epoxidation of
olefins (13-16), sulfides (17-19), and indole (20-22) are
carried out in high yield and enantioselectivity. CPO also catalyzes
chiral benzylic (23) and propargylic hydroxylations (24-25). The
stereoselective abilities of CPO in chiral catalysis suggest that
native CPO and/or engineered CPO mutants have the potential to become
important industrial catalysts.
The initial reaction of CPO with hydrogen peroxide generates an
oxidized intermediate, Compound I. This oxidized intermediate contains
one oxygen atom and both of the oxidizing equivalents originally
present in hydrogen peroxide. One of the oxidizing equivalents is
associated with the conversion of heme ferric iron to an oxyferryl iron
species, and the other is generated by the removal of one of the
electrons from the heme porphyrin, generating a porphyrin It is assumed that the versatile functions of CPO must be based on a
CPO unique active site structure that includes a proximal thiolate
ligand to the heme iron and a glutamic acid residue distal to the heme
(28). It has been proposed that Glu-183 functions as a general
acid-base catalyst in facilitating the formation of Compound I. In
contrast, most if not all other peroxidases have histidines functioning
in this role. In previous experiments, the proximal thiolate ligand
(Cys-29) has been replaced with a histidine residue and the mutant
enzyme was expressed in C. fumago (29). In this mutant, the
kcat for chlorination and peroxidation was
reduced ~95%. The catalase and epoxidation activity in the mutant
were reduced ~70%. It is obvious that cysteine 29 plays an important
role in CPO catalysis. However, unlike the comparable cytochrome P450
Cys to His mutant, the replacement of CPO cysteine 29 with histidine
does not totally abolish CPO activities. The distal Glu-183 also plays
an important role in the catalytic reactions of CPO. Attempts to
express a Glu-183 to His mutant in C. fumago were
unsuccessful probably because the mutation is a lethal event. Previous
attempts to produce CPO knock-outs in C. fumago also systematically failed. Because it is highly unlikely that CPO, a
secreted enzyme, is an essential gene product in C. fumago, there must be an alternate explanation for these failures. The opposite
strand DNA to the DNA coding for CPO contains an open reading frame;
thus, it is quite possible that CPO mutations could prove to be lethal
by producing a mutation in the opposite strand DNA. Recently, Conesa
et al. (30) have shown that the wild type CPO gene can be expressed in Aspergillus niger
with the recombinant protein retaining the catalytic properties of the
native C. fumago enzyme. Therefore, this expression system
was chosen for the production of the Glu-183 to His mutant. This paper
describes the preparation, expression, properties, and catalytic
activities of this CPO mutant.
Reagents
Restriction enzymes were purchased from New England
Biolabs (Beverly, MA) and Invitrogen. T4 DNA ligase was purchased from Promega (Madison, WI). Purified oligonucleotides were purchased from
Keck Biotechnique Center at University of Illinois. Unless otherwise
noted, chemicals were obtained from commercial sources and used without
further purification.
Site-directed Mutagenesis
Mutations were carried out using the QuikChange site-directed
mutagenesis kit of Stratagene (La Jolla, CA). The mutations were
introduced via PCR amplification of pTZC using synthetic oligonucleotide primers. The primers contained a silent mutation site
that could be used in a convenient mutant-screening assay. Primers for
site-directed mutagenesis were designed with the aid of the Wisconsin
GCG Package, version 9.1 (Madison, WI). PCR reactions were carried out
in a PerkinElmer thermal cycler (PerkinElmer Life Sciences). Standard
"hot-start" PCR was performed using high fidelity pfu
polymerase. The PCR products were extracted and purified from agarose
gels using kits purchased from Qiagen (Valencia, CA). The desired
mutations were identified by restriction analysis and confirmed by DNA sequencing.
Construction of pgpd/CPOE183H
Expression vector pgpd/CPOE183H was constructed via a three-way
ligation. The three DNA sequences used in the construction were: 1) a
230-bp AflIII/XmnI fragment derived from pCPO3
(30) that contained the N-terminal CPO coding sequence, 2) a ~900-bp XmnI/BamHI fragment derived from pTZC (31) that
contained the remaining CPO coding sequence, and 3) an A. niger expression vector, pAN52-5Not (GenBankTM
accession number Z32750), that contained an A. nidulans gpd promoter and the A. niger trpC terminator sequences (32).
The XmnI/BamHI fragment contained the E183H
mutation. The A. niger vector was digested with
NcoI and BamHI to create an insertion site for
the CPO sequences (Fig. 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cation radical (26, 27).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 1.
The E183H expression vector for
co-transformation of A. niger.
Expression and Selection of Mutant Clones
Plasmid gpd/CPOE183H was co-transformed with pAB4-1 in A. niger MGG029 according to previously described procedures (33). Transformants were screened in situ by immunodetection as described previously (34).
Culture of Mutant Clones
The mutant clones were grown in roller bottles at 22 °C for
5-7 days. Each bottle contained 300 ml of 5% maltose, 5% yeast extract, 0.5% casein amino acids, 50 µM hemin (Sigma),
and 100 µM -aminolevulinic acid (Sigma) and the Hutner
metal ion supplement (35).
Partial Purification of the Mutant CPO
To stabilize the mutant chlorination and epoxidations activities, all of the purification buffer solutions contained 50 µm hemin. Crude culture medium containing the secreted enzyme (1.5 liters) was concentrated 50-fold and applied to a DEAE-Sepharose CL-6B ion-exchange chromatography column (4 × 30 cm) that had been equilibrated with 50 mm Bis/Tris at pH 6.5. The flow rate was adjusted 1 ml/min. Chromatography was carried out at 4 °C. The protein was eluted from the column with a step gradient of 50 mM Bis/Tris buffer at pH 6.5 containing 10-70 mM ammonium sulfate. Each fraction was assayed for chlorination activity to identify the active fractions. The active fractions appeared in the eluent fractions containing 28 mM ammonium sulfate. The enzyme was concentrated by ultrafiltration (Amicon, Newburyport, MA).
SDS-PAGE and Isoelectric Focusing (IEF)
The purity of the mutant enzyme was examined by 12.5% SDS-PAGE gel (Invitrogen) under denaturing condition (36). The enzymes were visualized by staining with Coomassie Brilliant Blue R-250. IEF electrophoresis was performed using the Pharmacia Phastsystem with IEF PhastGels (pH 3-9) and the Pharmacia IEF Mix 2.8-6.5 kit. IEF gels were visualized by silver staining.
Protein Determination
Heme proteins were quantitatively determined using the hemochromogen analysis developed by Deeb and Hager (37). This information provided a basis for comparing the relative enzymatic activities of the mutant and native CPO.
Immuno-dot Blot Assay
Because both apomutant and holomutant enzymes were secreted in the A. niger cultures, the total concentration of mutant enzyme was measured in a dot blot assay. Aliquots of the partially purified mutant enzyme or aliquots of the crude culture medium were loaded onto nitrocellulose membranes. The membrane was incubated with anti-CPO rabbit polyclonal IgG antibody for 1 h at room temperature. After incubation, the membrane was washed and then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G for 1 h. After this second incubation step, the membrane was washed and then reacted with diaminobenzidine and hydrogen peroxide for 10 min for color development. The total concentration of apomutant plus holomutant enzyme was estimated by comparing the dot blot results with the results obtained from serial dilutions of known concentrations of native CPO.
Circular Dichroism
Far-UV-CD spectra were recorded on a JASCO J-720 spectropolarimeter at room temperature. Two spectra were recorded from 250 to 200 nm at 0.1-nm intervals with a spectral bandwidth of 1 nm (the spectra were averaged). The concentration of partially purified samples for both the mutant and native CPO control was identical (50 µg/ml).
Optimum pH for the Chlorination Activity
The relation between activity and pH was examined from pH 2.5 to 7.0 using the potassium phosphate buffers.
Enzyme Activity Assays
All UV-visible absorption measurements were determined in a Shimadzu UV-1201 spectrophotometer using 1-cm path length quartz cuvettes.
1). Chlorination Assay-- The chlorination of monochlorodimedone to dichlorodimedone was used for measuring the chlorination activity of the mutant CPO (7). The reaction mixture contained 20 mM potassium phosphate buffer, pH 2.75, 20 mM KCl, 0.5 mM monochlorodimedone, 2 mM H2O2, and a suitable aliquot of the enzyme in a total volume of 3 ml. The reaction was initiated by the addition of enzyme, and the decrease in absorbance at 278 nm was monitored at room temperature. One unit of chlorination activity was defined as the formation of 1 µmol of dichlorodimedone per second.
2). Epoxidase Assay-- The epoxidation of p-nitrostyrene was used to measure epoxidation activity according to the procedure developed by Rai et al. (38). The reaction mixture contained 100 mM sodium acetate buffer, pH 4.5, 0.2 mM p-nitrostyrene, and 2 mM H2O2 plus a suitable aliquot of the enzyme in a total volume of 3 ml. The activity was determined by monitoring the decrease in absorbance at 312 nm as a function of time. One unit of epoxidation activity was defined as the formation of 1 µmol of p-nitrostyrene oxide per second.
3). Catalase Assay--
The oxidation of reduced methylene blue
was used to measure catalase activity (38). The reaction mixture
contained 100 mM sodium acetate buffer, pH 4.5, 0.2 mM dithionite reduced methylene blue, 2 mM
H2O2, and a suitable aliquot of enzyme in a
total volume of 3 ml. One unit of catalase activity was defined as the
formation of 1 µmol of the oxidized form of methylene blue per second.
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RESULTS |
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Isolation of the E183H Mutant--
A. niger produces
the E183H mutant enzyme at levels comparable to the expression of wild
type CPO in A. niger. However, a majority of the mutant CPO
expressed in A. niger is in the form of apoenzyme having no
heme incorporated. By comparing the relative levels of apomutant and
holomutant enzymes estimated from a hemochromogen assay and a dot blot
immunoassay, the ratio of apoenzyme to holoprotein was ~1.5 to 1. A
similar observation has been made in the expression of recombinant wild
type CPO in A. niger (30). Most disturbingly, the E183H
presented serious problems in early attempts to purify and characterize
the mutant enzyme. In early studies, the Glu-183 mutant lost most of
its chlorination and epoxidation activity in simple dialysis and
ultrafiltration steps. Acetone precipitation, a regular purification
step used with native CPO, could not be used because the mutant lost
almost all activity when exposed to acetone at 20 °C.
Subsequently, it was discovered that the loss of the chlorination and
epoxidation activity during dialysis and ultrafiltration could be
prevented by the addition of iron protoporyphyrin IX to the mutant
enzyme preparations. The presence of heme greatly stabilized the E183H
extracts, and in addition, 90% of the mutant protein now appeared in
the form of holoenzyme. In contrast, holoenzyme represented only
one-third of the mutant enzyme population in untreated extracts.
Conesa et al. (30) found that secreted wild type CPO in A. niger was subject to partial degradation by secreted protease activity. In our initial problems concerning the instability of the E183H mutant enzyme, we considered protease degradation as a potential cause of the instability. This turned out not to be true. Ten different protease inhibitors either singly or in kit form were tested and were found to have no effect. The inhibitors tested were antipain-dihydrochloride, bestatin, chymotatin, E-64, leupeptin, pepstatin, phosphoramidon, pefablioc SC, aprotinin, and phenylmethylsulfonyl fluoride. Other reagents such as bovine serum albumin, glycerol, and sorbitol had no effect on the stability of the E183H enzyme.
Increasing the Yield of the E183H by the Addition of
-Aminolevulinic Acid (ALA)--
In regular culture broth, the
secretion level of the mutant protein (apoenzyme plus holoenzyme) was
quite low (~2 mg/liter). The addition of ALA to the culture medium
increased the secretion level ~4-fold. ALA synthase is thought to be
a crucial enzyme to regulate heme synthesis. There are a few reports
regarding an increase in heme protein production by ALA inducement. For example, Herbaud et al. (39) found that the highest
cytochrome c3 production was obtained when
Escherichia coli was grown in LB medium supplemented with
-aminolevulinic acid under aerobic condition (39). Further studies
showed that the regulative effects of ALA and hemin on ALA synthase
were done through transcriptional regulation (40). In these studies on
the production of mutant CPO by A. niger, the addition of
-aminolevulinic acid significantly enhanced the production of the
E183H mutant enzyme.
Partial Purification of E183H Mutant--
The instability of the
mutant enzyme precluded the normal initial acetone precipitation
routinely used to purify native CPO. Even the presence of heme did not
protect Glu-183 from denaturation by acetone. DEAE ion exchange
chromatography was used exclusively for purification of the mutant
enzyme. As described under "Experimental Procedures," all
purification buffers contained 50 µm of hemin to stabilize the mutant
enzyme. The specific activity of the partially purified E183H increased
by 145-fold, and the enzyme showed a major band in SDS-PAGE (Fig. 2) and had a RZ value of
1.2 (Fig. 3).
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Chlorination and Dismutation Activities of the E183H
Mutant--
To evaluate the effect of the distal substitution on the
various activities of CPO, the activities of the E183H mutant were measured in three different assays. The chlorination activity of the
mutant enzyme decreased to a level of ~15% of the wild type
activity, and the catalase activity decreased to 50% of the native CPO
catalytic activity (Fig. 4).
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Epoxidation Activity in the E183H Mutant-- As outlined under "Experimental Procedures," the epoxidation of p-nitrostyrene was used to assay the epoxidation activity of the E183H mutant according to the procedure developed by Rai et al. (38). In contrast to the results obtained for the chlorination and dismutation mutant activities, epoxidation activity unexpectedly increased 2.5-fold (Fig. 4).
Optimum pH and IEF--
To further elucidate the role of Glu-183
as a general acid-base catalysis, the pH optimum of the E183H mutant
was examined. The enzyme activity was measured at various pH values
ranging from pH 2.5 to 7.0. The concentration of
H2O2 used in these studies was optimized for
each pH value. The mutant was most active at pH 3.0, whereas its native
counterpart was at pH 2.75 (Fig. 5). The
E183H mutant showed a slightly higher pI value based on their isoelectric focusing gel properties (Fig.
6), but this was expected for the
replacement of a negatively charged by a positively charged one.
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Circular Dichroism--
CD spectrum was used to exclude the
possibility that activities of mutant enzyme were decreased by large
structure changes. The CD data showed that there were some small
differences in intensity in the peptide bond region between the mutant
and native CPO; however, the overall shapes of the two spectra were the
same (Fig. 7). The CD spectra of the
mutant proteins (80.5% -helix, 6.2%
-turn, and 15.7%
unordered) were very similar to that of wild type (79.6%
-helix,
0.6%
-sheet, 6.4%
-turn, and 16.2% unordered). The
result indicated that the amino acid substitutions had no significant
effect on the overall folding of the mutant enzyme.
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DISCUSSION |
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The discovery that hemin protects the mutant enzyme during purification and the observation that the apoform of the mutant can be converted to the holoenzyme in the presence of hemin were extremely important observations because hemin allowed the purification and characterization of this mutant enzyme. This discovery may also offer a new approach to the production of mutant CPOs. Wild type apoCPO can be produced in E. coli in large quantities; however, under the very best conditions, only small amounts of holoenzyme could be obtained by reconstitution procedures (41). Because the E183H apomutant readily reconstitutes to holoenzyme and because the E183H mutant is more active than wild type CPO in chiral epoxidation catalysis, it appears logical to assume that further mutation of E183H in E. coli is an obvious choice for examining the active site requirements for chiral catalysis.
Histidine plays a key role as a general acid-base catalyst in the
formation of Compound I in the plant peroxidases. Poulos and Kraut (42)
proposes that the distal histidine assists the peroxide anion to bind
the heme iron by acting as a general base catalyst while accepting a
proton from the neutral hydrogen peroxide when it binds to the iron and
by acting as a general acid catalyst by donating a proton to the
departing hydroxide anion (42). Several mutant peroxidases have
revealed histidine as an important active site residue. In wild type
CPO, a glutamic acid residue replaces the plant peroxidase histidine
distal residue and is postulated to function as the acid-base catalyst,
facilitating the formation of Compound I. By analogy to the experiments
with horseradish peroxidase where replacement of the distal histidine leads to large losses in activity (43), it would be expected that
replacement of the distal glutamic acid in CPO also should be
deleterious. This is certainly true in terms of the halogenation activity of CPO. The rate of enzymatic chlorination in the Glu-183 to
His mutant falls by ~85%. This indicates that the distal Glu residue
in native CPO is important but not essential for the halogenation activity of the enzyme. The van der Waals volume of histidine (98.8 cm3 mol1) is larger than that of histidine
(85.9 cm3 mol
1), so some distortion of the
mutant active site should be expected. The fact that the mutant heme
prosthetic group is more loosely held supports the conclusion that
there has been a change in the geometry of the active site. The x-ray
structure of native CPO identifies a proton shuttle among Glu-183,
His-105, and Asp-106 (44). When Glu-183 is replaced with a histidine
residue, the normal orientation of this triad could be partially
destroyed. Such a change could obviously impair the chlorination
activity of the mutant. In studies with horseradish peroxidase mutants, it has been shown that the loss of a hydrogen bond between the distal
His-42 and Asp-70 induces severe functional defects in peroxidase
activity (45). In addition, previous experiments have shown that the
modification of a single histidine residue with diethyl pyrocarbonate
totally inactivates CPO with respect to chlorination activity (46).
A quite different picture emerges from a comparison of the catalytic
activity of the mutant and native CPOs in the epoxidation reaction. In
this case, the mutant is ~2.5-fold more active than the native
enzyme. In focusing on the origin of this increase in activity, it is
highly unlikely that the increase in activity could be associated with
the formation of Compound I, the first step in all CPO oxidations. The
rate constant for the formation of Compound I in the reaction of native
enzyme with hydrogen peroxide is 2.4 × 106
M1sec
1 (47), whereas the rate
constants for both the chlorination and epoxidation activity of native
CPO are magnitudes lower. Thus, the overall rate for both chlorination
and epoxidation reactions must reside in the second step involving the
reaction between Compound I and the oxidizable substrate. It seems
probable that the distortion of the active site that inhibits the
chlorination activity must actually facilitate the reaction between the
olefinic substrate and the oxygen atom in the oxyferryl Compound I
species, perhaps by allowing a closer approach of the olefinic
substrate to the oxygen atom of the oxyferryl group in Compound I. Clearly, the olefinic substrate must approach the Compound I
intermediate from the distal side of the heme. In the mechanism-based
suicide reaction between CPO Compound I and
cis-
-methyl styrene, an oxygen-carbon bond is formed in
the inactive intermediate (48). In a similar vein, the proton shuttle
among Glu-183, His-105, and Asp-106 that is important for chlorination
activity appears unimportant for epoxidation activity. The diethyl
pyrocarbonate treatment that kills chlorination activity does not
impair epoxidation activity.
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ACKNOWLEDGEMENT |
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We thank Dr. Shaomin Tian for CD data processing.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grant GM 07768.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, University of Illinois, 600 S. Mathews Ave., Urbana, IL 61801. Tel.: 217-333-9686; Fax: 217-244-5858; E-mail: l-hager@uiuc.edu.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M210906200
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ABBREVIATIONS |
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The abbreviations used are:
CPO, chloroperoxidase;
IEF, isoelectric focusing;
ALA, -aminolevulinic
acid.
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