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(Received for publication, September 28,
1995; and in revised form, January 26, 1996) From the
Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six
electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The
enzyme from the bacterium Myxococcus xanthus has been cloned,
expressed, purified, and characterized. The protein has been expressed
in Escherichia coli using a Tac promoter-driven expression
plasmid and purified to apparent homogeneity in a rapid procedure that
yields approximately 10 mg of purified protein per liter of culture.
Based upon the deduced amino acid sequence the molecular weight of a
single subunit is 49,387. Gel permeation chromatography in the presence
of 0.2% n-octyl-
The penultimate step in the heme biosynthetic pathway, the six
electron oxidation of protoporphyrinogen IX to protoporphyrin IX, is
catalyzed by the enzyme protoporphyrinogen oxidase (EC
1.3.3.4)(1, 2, 3) . In eukaryotes this enzyme
is located on the cytosolic side of the inner mitochondrial membrane
and utilizes molecular oxygen as its terminal electron acceptor. The
enzyme from two prokaryotes have been cloned, sequenced, expressed, and
partially characterized. The enzyme from Bacillus subtilis(4, 5) is similar to the eukaryotic enzyme in
that it contains a flavin and utilizes molecular oxygen as terminal
electron acceptor. However, its substrate specificity is much broader
than the eukaryotic enzyme since it will oxidize not only
protoporphyrinogen IX, but also the pathway intermediate
coproporphyrinogen III. In addition it is resistant to inhibition by
the herbicide acifluorfen which strongly inhibits the eukaryotic
enzyme. Protoporphyrinogen oxidase has also been cloned and expressed
from Escherichia coli(6) . The cloned protein is
smaller in size than the B. subtilis and eukaryotic enzymes,
does not contain an FAD binding motif, and is obligatorily coupled to
the cell's respiratory chain. Based upon previous data from Desulfovibrio gigas(7) it would appear that the
cloned E. coli enzyme is a subunit of a multi-protein complex. Data base searches using the B. subtilis protoporphyrinogen
oxidase derived amino acid sequence yielded two similar bacterial
sequences(8) . In an effort to expand our knowledge about this
enzyme and to discover if the herbicide resistance and broad substrate
specificity found with the B. subtilis enzyme are
characteristic of the prokaryotic, oxygen dependent enzymes, we
expressed, purified, and characterized protoporphyrinogen oxidase of
the bacterium Myxococcus xanthus.
To purify the enzyme, a 3-ml bed
volume Qiagen Ni-NTA agarose column was prepared and equilibrated with
50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 0.2% n-octyl-
SDS gel electrophoresis was carried out with
Mini-Protean II Ready Gels (Bio-Rad, Hercules, CA). Visible/UV spectra
were recorded with a Varian 219 spectrophotometer. Metal analysis by
plasma emission was carried out by the Chemical Analyses Laboratory at
the University of Georgia. Femtomole sequencing (Promega) with Quantitation of H For flavin
determination samples of purified enzyme were treated with 5% (w/v)
trichloroacetic acid or 50% acetonitrile. Either of these procedures
precipitated the protein and released the flavin into solution.
Identification of FAD was made based upon the pH-dependent change in
fluorescence(15) . For quantitation a molar extinction
coefficient of 13,000 M/cm at 450 nm was employed. To
determine flavin to protein stoichiometry, protein concentration was
determined spectrophotometrically using a molar extinction coefficient
of 21,700 at 275 nm which is based upon the amino acid composition. All
enzyme preparations that were used for flavin determination were
subjected to SDS gel electrophoresis to ensure that the particular
enzyme preparation was homogeneous by this standard.
Figure 1:
Amino acid sequence of
protoporphyrinogen oxidase of M. xanthus. The protein sequence
was derived from the previously published nucleotide sequence of Li et al.(9) (GenBank
The success of the Ni-chelate column in the purification was
found to be quite dependent upon buffer pH. Although the enzyme bound
to the matrix at higher pH (i.e. 8.0) its elution required
higher imidazole concentrations and never resulted in recovery of only
a single protein. The expression and purification described above
yields a single protein band on SDS gel electrophoresis (Fig. 2)
of estimated molecular weight of 50,000. This corresponds well to the
molecular weight of 49,387 based upon predicted amino acid sequence.
Figure 2:
SDS gel electrophoresis of purified
recombinant M. xanthus protoporphyrinogen oxidase. Consecutive
fractions from the elution of enzyme from the Ni
Figure 3:
Visible/ultraviolet spectrum of purified M. xanthus protoporphyrinogen oxidase. Enzyme (30
µM) for the scan is in 20 mM sodium phosphate, pH
7.4, 0.2% n-octyl-
Figure 4:
Molecular weight determination for
native M. xanthus protoporphyrinogen oxidase. Details are in
the text. The molecular weight markers shown are cytochrome c,
The M.
xanthus enzyme is strongly inhibited by the herbicide acifluorfen (Fig. 5). This level of inhibition is similar to what is seen
with the mammalian enzyme and unlike what was reported for the B.
subtilis protein. Preliminary kinetic experiments suggest that
acifluorfen may be a slow binding competitive inhibitor (data not
shown). The M. xanthus enzyme uses protoporphyrinogen IX as
substrate with an apparent K
Figure 5:
Inhibition of M. xanthus protoporphyrinogen oxidase by the herbicide acifluorfen. The
enzyme was incubated in the assay reaction mixture, without
porphyrinogen substrate, for 5 min prior to addition of
substrate.
The enzymatic conversion of protoporphyrinogen IX to
protoporphyrin IX was first unequivocally demonstrated by Poulson and
Polglase in 1975(2) . While a number of papers have appeared on
the characterization of the purified eukaryotic
enzyme(12, 16, 17, 18) , little
biophysical or accurate kinetic data were available since the enzyme is
present in low amounts in cells and is difficult to purify. In the
current study we have presented data on the expression and
characterization of protoporphyrinogen oxidase from the Gram-negative
bacterium M. xanthus. Interestingly the DNA sequence for this
enzyme was reported in 1992 as part of a study from Shimkets'
group on genes involved in the developmental cycle of this
myxobacterium(9) . The sequence of an unidentified open reading
frame that was not involved in development, which is upstream and in an
opposite orientation from the csgA gene was reported. A later computer
data base search by others (8) suggested that the encoded
protein may be similar to protoporphyrinogen oxidase from B.
subtilis(4, 5) . Previous work has shown that
bacteria apparently utilize one of two different enzymes to catalyze
this
step(3, 4, 5, 6, 7, 8) .
Protoporphyrinogen oxidase activity in E. coli has been found
to involve at least two distinct gene products (hem G (6) and
hem K(19) ). Neither of these encoded proteins resemble the FAD
containing protoporphyrinogen oxidase of mouse, human and B.
subtilis although similar derived amino acid sequences have been
found in the Haemophilus influenzae( Data presented above
demonstrate that the previously published open reading frame from the
myxobacterium M. xanthus codes for the enzyme
protoporphyrinogen oxidase. The expressed protein is an oxygen
dependent, flavin containing enzyme that is similar to the mammalian
enzyme and the enzyme from B. subtilis. The protein which has
two amino-terminal his residues was expressed in E. coli using
a vector in which four additional his residues were added to create a
6-his tag for purification via Ni-chelate chromatography. The expressed
enzyme is found in both membrane and cytoplasmic fractions, but the
purified enzyme rapidly precipitates out of solution in the absence of
detergent and purification requires at least 0.2% octyl glucoside. Once
purified the protein is stable for weeks at 4 °C. The enzyme has
a molecular weight of 49,387 as determined from the derived amino acid
sequence which is in good agreement with what is found by SDS gel
electrophoresis. The pure enzyme in solution containing 0.2% detergent
exists as a homodimer with no detectable monomer form. Visible spectra
of purified protein shows that it possesses a flavin cofactor as
suggested from the sequence which contains a dinucleotide (FAD) binding
consensus motif(22) . The stoichiometry of the FAD to protein
in all preparations obtained to date is only about 0.5 and this may
reflect that the flavin readily dissociates, or that the dimer form of
the enzyme possesses only one FAD per dimer. While the possibility
exists that the cells are unable to synthesize sufficient FAD to
provide two FAD per dimer, this seems less likely since addition of 1
µM riboflavin to the bacterial culture during the last 2 h
of induction did not have a discernable affect upon the FAD content of
the purified enzyme. Similar findings of less than stoichiometric
amounts of cofactor have been reported for monoamine oxidase which is
also a dimeric FAD containing oxidase although its FAD is covalently
bound(23) . Kinetic analysis of M. xanthus protoporphyrinogen oxidase demonstrates that it is more similar to
the previously characterized eukaryotic enzymes (16, 17, 18) than to the only other
characterized prokaryotic enzyme from B.
subtilis(5, 8) . Unlike the bacillus enzyme, the M. xanthus enzyme does not oxidize coproporphyrinogen III and
is strongly inhibited by acifluorfen. These data show that the bacillus
enzyme's properties are not representative of all oxygen
dependent prokaryotic protoporphyrinogen oxidases. While it will be
necessary to characterize the enzyme from additional bacteria before
determining which of these enzymes is most widely distributed among
prokaryotes, the available data on B. subtilis ferrochelatase,
the terminal heme biosynthetic pathway enzyme, demonstrate that this
bacillus enzyme also possesses some properties such as protein
solubility and metal specificities that are unique among the currently
characterized ferrochelatases(24, 25) . These
observations suggest that a class of bacteria represented by B.
subtilis may have evolved a slightly altered way of dealing with
the arrangement and intracellular compartmentation of the terminal
segment of the heme biosynthetic pathway. The findings that the M.
xanthus protoporphyrinogen oxidase is as sensitive to acifluorfen
as the eukaryotic enzymes (26, 27, 28) demonstrates that the basis for
the B. subtilis enzyme's resistance to this herbicide
must be due to a property unique to bacillus and not a more general
structural difference between the prokaryotic and eukaryotic enzymes.
Since it has been suggested that acifluorfen is a competitive inhibitor
of protoporphyrinogen oxidase because it bears a structural resemblance
to one-half of the porphyrinogen macrocycle, it will be of interest to
see if acifluorfen resistance and broadened substrate specificity are
necessarily coupled. Comparison of the derived amino acid sequence
for protoporphyrinogen oxidase from M. xanthus with B.
subtilis(4) , mouse(29) , and human (20, 30) show that there is only 15% identity among
all sequences. Between the two bacterial sequences there is 23%
identity. If one considers conservative amino acid substitutions then
there is about 35-40% homology among the four sequences. While
the regions of identity appear to be relatively randomly distributed
throughout the proteins, there are several discrete regions of
homology. The most obvious of these regions is the dinucleotide binding
motif (22) found at the amino-terminal end of all four
sequences. The structural/functional purposes of the remaining regions
are currently unknown, but the lack of an identifiable membrane
spanning region in any of the sequences rules out that possibility. The reaction catalyzed by protoporphyrinogen oxidase is a six
electron oxidation. Previously we have shown that three O
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8714-8718
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE CLONED
ENZYME (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-D-glucopyranoside yields a
molecular weight of approximately 100,000 while SDS gel electrophoresis
shows a single band at 50,000. The native enzyme is, thus, a homodimer.
The purified protein contains a non-covalently bound FAD but no
detectable redox active metal. The M. xanthus enzyme utilizes
protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and
produces 3 mol of H
O/mol of protoporphyrin. The
apparent K
and k
for protoporphyrinogen in assays under atmospheric concentrations
of oxygen are 1.6 µM and 5.2 min
,
respectively. The diphenyl ether herbicide acifluorfen at 1 µM strongly inhibits the enzyme's activity.
Plasmid Construction
The plasmid pLJS43 (9) was kindly supplied by L. Shimkets. The DNA for the
putative protoporphyrinogen oxidase coding region was obtained by using
polymerase chain reaction with the oligonucleotide primers: sense,
5`-CC
ATGGACCATCACCATCACCACCACATGCCGAGGACA-3`;
antisense,
5`-CAAGCTTCACTACGGGGCGTGGGAGGT-3`.
The 5` end primer was designed to contain a NcoI restriction
site as well as codons for an additional four His residues so that the
expressed protein would contain a His-6 tag for Ni-chelate
chromatography. The 3` end was extended to create a HindIII
site. This fragment was cloned into the Tac-driven expression vector
pTF20E which is a derivative of pBTac-1 (Boehringer Mannheim) in which
an optimally spaced ribosomal binding site, T7 enhancer and ATG start
site (10) were placed immediately downstream from the Tac
promoter. The expression vector pMx-PPO was transformed into E.
coli JM109 for protein expression.Expression and Purification
JM109 cells containing
pMx-PPO were inoculated into 1 liter of Circlegrow medium (BIO101) with
100 µg/ml ampicillin and incubated at 37 °C with 250 rpm
shaking overnight. Cells were harvested by centrifugation (10,000
g, 10 min, 4 °C), and the pellet was collected and
suspended in 60 ml of 50 mM sodium phosphate, pH 7.4. The
cells were lysed by sonication and the cell membrane fraction separated
from the cytoplasmic fraction by centrifugation at 100,000 g, 60 min, 4 °C. Enzyme was solubilized from the isolated
membrane fraction by resuspension of the pellet in 60 ml of 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 0.5% n-octyl--D-glucopyranoside followed by
sonication for 15 s. This was then centrifuged (100,000 g, 60 min, 4 °C) to separate the solubilized enzyme from
the remaining membrane fraction.-D-glucopyranoside. The solubilized
fraction was passed through this column before the column was washed
with 50 ml of the equilibration buffer with 20 mM imidazole.
Protoporphyrinogen oxidase was eluted with equilibration buffer
containing 150 mM imidazole.Procedures
Protoporphyrinogen oxidase was assayed
as described previously(11, 12) . The assay buffer
contained 100 mM sodium phosphate, pH 7.4, 0.1 mM EDTA, and 0.1% Tween 20. Porphyrinogen substrate was prepared via
reduction with sodium amalgam and was used immediately. In assays where
acifluorfen (Chem Service, Inc.) was present the acifluorfen was added
from a stock solution in dimethyl sulfoxide. Addition of an equivalent
amount of dimethyl sulfoxide alone to the assays had no effect on
observed enzyme activity. All assay procedures were carried out in a
darkened room.
S-dATP was employed for DNA sequencing. Additional DNA
sequencing was also carried out by the Molecular Genetics Facility at
the University of Georgia.O produced was achieved using scopoletin(13) . Briefly,
assays were set up where complete conversion of protoporphyrinogen to
protoporphyrin was achieved within 5 min. Duplicate samples were
prepared for porphyrinogen concentrations of 0, 10, 25, and 50
µM. Complete conversion of substrate to product was
verified spectrofluorometrically before HO concentration was determined. A standard curve for scopoletin
quantitated HO was constructed using a
HO solution whose concentration had been
verified with 4-aminoantipyrine(14) .
cDNA Sequence of M. xanthus Protoporphyrinogen
Oxidase
The direct translation of the previously published M. xanthus DNA sequence for an unidentified open reading frame
(which we have now shown codes for protoporphyrinogen oxidase) yields a
protein whose derived amino acid sequence is smaller than the protein
we have expressed and characterized herein. It was suggested by Hansson
and Hederstedt (8) that a T insertion at base pair 388 of the
published DNA sequence would yield a sequence that had homology to the
protoporphyrinogen oxidase of B. subtilis. To confirm and
identify any insertion or deletion we sequenced the region of M.
xanthus DNA from plasmid pLJS45 that encodes protoporphyrinogen
oxidase. It was found that there was not a T insertion at bp 388 as
suggested but rather a single C insertion immediately after bp 376
(data not shown). This correction has been entered with GenBank
(accession no. M73709). The sequence of the correct translation
product is shown in Fig. 1along with the three other known
oxygen dependent protoporphyrinogen oxidases.
M73709). Also shown
in this figure are sequences for human (GenBank U26446),
mouse (GenBank U25114), and B. subtilis (GenBank M97208) protoporphyrinogen oxidases (Ppo). This alignment was generated by the GCG program Pileup.
The underlined region represents the putative dinucleotide
binding motif (C. M. Frazier, GenBank accession nos.
U39704 and L43967).
Expression and Purification
The addition of four
His residues after amino acid number 2 to yield six consecutive His
residues allowed the use of a Ni-chelate column for purification. For
high yields it was found important to keep protease inhibitors present
in all buffers and to carry out the chromatography step immediately
following cell fractionation. While the expressed enzyme was found
membrane associated in E. coli, at high levels of expression
it was also found in the cytoplasmic fraction. Thus, the addition of
the four his residues at the amino terminus did not appear to affect
the cellular location of the protein. The possibility that this
extension has an adverse affect on this particular enzyme seems
unlikely since the amino terminus of known protoporphyrinogen oxidases
(along with monoamine oxidases which are similar to this enzyme) are
highly variable in length and amino acid composition in the region
upstream from the putative dinucleotide binding region. The B.
subtilis protoporphyrinogen oxidase has been expressed by this
laboratory both with (
)and without (5) the His tag,
and both forms of the enzyme have identical kinetic parameters.
NTA
column are shown in lanes 1-3 (
3 µg), and 5-7 (1 µg). Lane 4 contains molecular
weight markers and lane 8 contains a sample of crude
solubilized enzyme (2 µg). The molecular weight markers were:
myosin, phosphorylase b, bovine serum albumin, ovalbumin,
carbonic anhydrase, and trypsin inhibitor.
Properties of the Purified Enzyme
The
visible/ultraviolet spectrum of purified M. xanthus protoporphyrinogen oxidase is shown in Fig. 3. The spectra
clearly is consistent with the presence of FAD and the amount of FAD
was determined to be 0.4-0.5 FAD per subunit. Growth of the
cultures with 1 µM riboflavin added during the final 2 h
of induction did not cause any alteration in the ratio of FAD to
enzyme, but did increase the amount of enzyme recovered by
approximately 4-fold. Five percent trichloroacetic acid precipitates
the protein and releases free flavin into solution. Visible
spectroscopy shows a typical FAD/FMN spectrum and FAD was identified by
its pH-dependent change in fluorescence as described
previously(15) . In the spectrum of the isolated enzyme there
is a small feature at around 410 nm which is attributable to a small
amount (less than 0.05 mol/mol enzyme) of residual enzyme product
(protoporphyrin). Based upon the derived amino acid sequence, the
extinction coefficient at 275 nm is 21,700 M/cm. Gel
filtration on a Sephacel S-300 column (25 2 cm) in the presence
of 20 mM sodium phosphate, pH 7.4, 0.2% n-octyl--D-glucopyranoside resulted in the
elution of a single protein peak with an estimated molecular weight of
100,000 (Fig. 4). Metal analysis did not demonstrate the
presence of any redox active metal (data not shown).
-D-glucopyranoside.
-globulin (intact and light chain), and bovine serum albumin. The
elution position of protoporphyrinogen oxidase is shown in the box.
of 1.6 µM and k 5.2 min and it
generates 3 mol of HO/1 mol of porphyrinogen
(average of six determinations was 3.0 ± 0.3).
Coproporphyrinogen III is not a substrate for this enzyme. Addition of
FAD to reaction mixtures had no detectable effect upon enzyme activity.
)and Mycoplasma genitalium(
)genomes. It now seems clear
that among bacteria two distinct protoporphyrinogen-oxidizing systems
are found; the FAD-containing, oxygen-dependent homodimer enzyme, and
the multisubunit, respiratory chain-linked enzyme system as typified by E. coli and D. gigas(7) . Limited data
suggest that anaerobes or facultative organisms may possess the
multisubunit enzyme that is obligatorily linked to the cell's
respiratory chain(3, 6, 7) , whereas strict
aerobes possess an oxygen dependent protoporphyrinogen
oxidase(3, 5, 8) . Of these enzymes only the
protein from B. subtilis has been cloned and
expressed(5, 8) . This enzyme was found to have both
sequence and catalytic similarities to the eukaryotic enzymes although
it differed significantly in that its substrate specificity was much
broader and it was not inhibited by the diphenyl ether herbicide,
acifluorfen. Since this second property may be a desirable one to clone
into selected crop plants, it was of interest to determine if
acifluorfen resistance is a general property of all bacterial oxygen
dependent protoporphyrinogen oxidases and, if so, to identify the
structural feature that imparts this property. are consumed per porphyrinogen substrate (21) and above
we document that three HO are produced. Studies
by others on crude enzyme extracts did not detect the in vitro accumulation of a tetra or dihydro porphyrin intermediate. If the
enzyme contains only a single FAD and no additional redox active
cofactors or metals, then the reaction must proceed in three distinct
steps unless residue side chains such as tyrosine are involved. Among
currently published sequences there is only one conserved tyrosine
residue. With the ability to produce and purify this enzyme in
milligram quantities as well as the possibility to carry out
site-directed mutagenesis on the cloned enzyme, we should now be able
to determine the sequence of catalytic events in the oxygen dependent
conversion of the porphyrinogen to porphyrin.
)) accession nos.
U32799 [GenBank]and
L42023[GenBank].) accession nos. U39704 [GenBank]and L43967[GenBank].
We thank Dr. L. J. Shimkets for the gift of plasmid
pLJS43.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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