(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--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.
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--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.
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 S-dATP was employed for DNA sequencing. Additional DNA
sequencing was also carried out by the Molecular Genetics Facility at
the University of Georgia.
Quantitation of HO
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 H
O
concentration was determined. A standard curve for scopoletin
quantitated H
O
was constructed using a
H
O
solution whose concentration had been
verified with 4-aminoantipyrine(14) .
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 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).
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 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.
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--D-glucopyranoside.
Figure 4:
Molecular weight determination for
native M. xanthus protoporphyrinogen oxidase. Details are in
the text. The molecular weight markers shown are cytochrome c,
-globulin (intact and light chain), and bovine serum albumin. The
elution position of protoporphyrinogen oxidase is shown in the box.
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 of 1.6 µM and k
5.2 min
and it
generates 3 mol of H
O
/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.
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()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.
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 are consumed per porphyrinogen substrate (21) and above
we document that three H
O
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.