From the Department of Microbiology, Department of Biochemistry and
Molecular Biology, University of Georgia, Athens, Georgia
30602-7229
A large number of FAD-containing proteins have
previously been shown to contain a signature sequence that is referred
to as the dinucleotide binding motif. Protoporphyrinogen oxidase (PPO), the penultimate enzyme of the heme biosynthetic pathway, is an FAD-containing protein that catalyzes the six electron oxidation of
protoporphyrinogen IX. Sequence analysis demonstrates the presence of
the dinucleotide binding motif at the amino-terminal end of the
protein. Analysis of the current data base reveals that PPO has
significant sequence similarities to mammalian monoamine oxidases (MAO)
A and B, as well as to bacterial and plant phytoene desaturases (PHD).
Previously MAOs have been shown to contain FAD, but there are no
publications demonstrating the presence of FAD in purified PHDs. We
have carried out the expression and purification of PHD from the
bacterium Myxococcus xanthus and demonstrate the presence of noncovalently bound FAD. Sequence analysis demonstrate that PPO is
closely related to bacterial PHDs and more distantly to plant PHDs and
animal MAOs. Interestingly bacterial MAOs are no more closely related
to PPOs, PHDs, and animal MAO's than they are to the unrelated
Pseudomonas phenyl hydroxylase. All of the related
sequences contain not only the basic putative dinucleotide binding
motif that is found frequently for FAD-binding proteins, but they also
have high similarity in an approximately 60-residue long region that
extends beyond the dinucleotide motif. This region is not found among
any other proteins in the current data base and, therefore, we propose
that this region is a signature motif for a superfamily of
FAD-containing enzymes that is comprised of PPOs, animal MAOs, and
PHDs.
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INTRODUCTION |
The ability to synthesize tetrapyrroles is one that is found
almost universally in living organisms. One of the most abundant of the
biological tetrapyrroles synthesized by Bacteria and Eucarya is
protoporphyrin IX or one of its derivatives. Within organisms that
possess the ability to synthesize protoporphyrin there appear to exist
two mechanisms to catalyze the six electron oxidation of
protoporphyrinogen to protoporphyrin. For facultative or anaerobic bacteria there exists a multienzyme complex that is linked to the
cell's respiratory chain (1-4), whereas aerobic organisms possess a
single protein that utilizes molecular oxygen as terminal electron
acceptor (5-8). Currently both systems are named protoporphyrinogen oxidase (PPO)1 although it is
now clear that the biochemical properties of the proteins and the
cofactor involvements are distinct.
The oxygen-dependent PPO (EC 1.3.3.4) has been
cloned, sequenced, expressed, and purified from both bacterial and
mammalian sources (6-12), and it has been demonstrated that this
protein is an FAD-containing homodimer with a subunit molecular weight of approximately 50,000. This enzyme is of considerable interest for
several reasons. In plants this protein is the target of a large class
of commercial herbicides (13, 14), and in man a genetic deficiency of
the enzyme results in the disorder variegate porphyria (12, 15-18). In
addition, PPO is interesting from a biochemical standpoint since it is
an enzyme with only a single FAD, and yet it catalyzes a six electron
oxidation of protoporphyrinogen IX to protoporphyrin IX (11).
As one approach to learn more about this enzyme data base searches of
Swiss Prot release 32, TIGR, and GenBankTM were carried out
to determine if PPO was unique or if it is similar to another class of
enzymes that are better characterized structurally. The analysis
presented below demonstrates that there exists significant sequence
similarities between PPO, monoamine oxidases (MAO), and phytoene
desaturases (PHD). Although data have previously been presented
demonstrating that PPO (7, 10-12) and MAO (see Ref. 19) contain FAD,
no such information was available for a PHD. Herein we report the
expression, purification, and demonstration that the bacterial PHD from
Myxococcus xanthus contains an FAD. The finding of
similarity between PPOs and PHDs is of interest since these two enzymes
are the known targets of photoactive herbicides. Data presented also
suggest that PPO, MAO, and PHD are members of a superfamily of
FAD-containing proteins that possess a unique fingerprint motif
spanning approximately 60 amino acid residues.
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MATERIALS AND METHODS |
Expression Plasmid Construction--
The plasmid pMAR140,
containing the coding region for M. xanthus phytoene
desaturase, was a gift of Dr. L. J. Shimkets. Primers were
designed to carry out the polymerase chain reaction of the gene
introducing convenient restriction sites for cloning into the
histidine-tagged expression vector pTrcHisC (Invitrogen). Specifically, the sense primer was 5'-
GTTAAGGCTAGCAGTGCATCGACACAGGGCAGG-3', where an
NheI site, shown in bold, was introduced for cloning purposes. The coding region of the phytoene desaturase gene,
corresponding to the first 7 amino acids after the 1st methionine in
the sequence, are underlined. The antisense primer was
5'-GAAATCAAGCTTGTCACGCGGCCACCCCTTCCAG-3', in which a HindIII site, shown in bold, was introduced
for cloning purposes. The 3'-end of the gene, including the TGA stop
codon, is underlined. The polymerase chain reaction conditions were as follows: 3 min 95° (1 min 95°, 1 min 62°, 2 min 72°) × 30, 7 min 72°, using 100 µM of each primer and Taq
polymerase (Fisher Biotech). The resulting polymerase chain reaction
product was cloned into the pGEMT vector (Promega) to facilitate
cleavage by restriction enzymes, and the resulting plasmid digested
with NheI and HindIII to release the phytoene
desaturase DNA. The DNA fragment was then cloned into the
NheI/HindIII site of pTrcHisC. The resulting
plasmid was named pPDH.
Expression of PDH--
A 1-liter culture of Escherichia
coli JM109 containing pPDH was grown at 37 °C overnight with
shaking in Circlegrow (Bio101, Inc) supplemented with 100 µg/ml
ampicillin. The cells were harvested and resuspended in 60 ml
resuspension buffer (50 mM sodium phosphate, pH 8.0, 0.2%
w/v
-octylglucoside). Additional
-octylglucoside was added to
bring the final concentration to 1.0%, and 10 µg/ml phenylmethylsulfonyl fluoride was added. The cells were sonicated 4 × 30 s on high power at 4 °C, and then centrifuged
100,000 × g at 4 °C for 30 min. The resulting
supernatant was loaded onto a Talon (CLONTECH)
column that was formed from 3.5 ml of a 50% slurry that had been
equilibrated with resuspension buffer. The column was then washed with
20 ml of resuspension buffer, 20 ml of resuspension buffer containing
300 mM NaCl, and then 30 ml of resuspension buffer
containing 20 mM imidazole. The purified protein was eluted
in resuspension buffer containing 300 mM imidazole, and 0.5 ml fractions were collected.
Data Base Analysis--
An initial search of the Swiss Prot data
base was done using BLASTP and Wordsearch (20) with the sequence of
human protoporphyrinogen oxidase (7) as a probe. Elimination of all
sequences lacking an obvious putative dinucleotide binding motif (21)
yielded 54 unique protein sequences. Sequence comparisons were carried out by using GCG PileUp (20) using the program's default values for
gap creation and extension penalties. From this initial list those
sequences with an amino-terminal dinucleotide binding motif, such as
PPO possesses, were selected for further study. These 32 sequences
along with the PPO sequence for Deinococcus radiodurans (from TIGR) were submitted to a second PileUp before being
phylogenetically sorted using GCG Growtree. All known PPO sequences
were included in this analysis. Phenol hydroxylase of
Pseudomonas was included in this analysis as an outlying,
nonrelated sequence to root the trees. This hydroxylase is an
FAD-containing protein, but it lacks the dinucleotide binding motif
that the other proteins in this analysis possess. Manipulations of
sequence length (i.e. truncation of amino or
carboxyl-terminal segments to obtain similar protein sizes) had no
significant effect on the overall clustering, but for the analysis
displayed, the amino-terminal ends (upstream from the dinucleotide
binding motif) were truncated from the bacterial MAO sequences. Both
Jukes-Cantor and Kimura distance corrections were employed and
Growtrees for both neighbor-joining and unweighted pairs group method
using arithmetic averages were created.
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RESULTS AND DISCUSSION |
After the cloning, expression, and purification of PPO (6, 7,
9-12) an analysis of the current data base was performed to identify
any related proteins. The analysis yielded a number of interesting
results. First of these was the similarity of PPO to both MAO A and MAO
B, and PHD. Previously we had reported that PPO had physical properties
similar to mammalian MAOs so a close association between PPOs and MAOs
was anticipated (7, 11). Among the similarities are that both proteins
are nuclear encoded, contain FAD as a cofactor, have a dinucleotide
binding motif near the amino terminus, are homodimers with similar
subunit molecular sizes, and are mitochondrial membrane-associated
proteins that lack a typical amino-terminal targeting sequence. Two
major differences between the enzymes are that MAOs catalyze a single
two-electron redox step and contain a covalently bound FAD (see Ref.
19), whereas the overall reaction catalyzed by protoporphyrinogen
oxidase is a six-electron oxidation (Scheme
I) and the protein contains a
noncovalently bound FAD (see Ref. 11).
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To facilitate presentation of over 30 protein sequences and to
determine the amount of relatedness of other proteins, a Growtree analysis was performed. As described above, four different analyses were performed: two corrected neighbor-joining, and two corrected unweighted pairs group method using arithmetic averages. Although some
variations in relative distances existed among the four trees, all
produced the same clustering pattern. The data revealed that two
distinct classes of enzymes have structural homology to PPO: animal MAO
and PHD (dehydrogenases) (Fig. 1). The
pattern places all PPOs in a cluster most closely linked to a cluster
of bacterial PHD and more distantly related to mammalian MAO (A and B
forms) and plant PHD.

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Fig. 1.
Growtree analysis. The analysis shown is
for all published sequences of protoporphyrinogen oxidases
(Ppo), monoamine oxidases (Mao (a,
b, c)), phytoene desaturase (Phytdeh),
hydroxyneurosporine desaturase (Hndeh), methoxyneurosporine
desaturase (Mndeh) and phenol hydroxylase
(phen_Hydrox). Species names are clearly identified except
for P32614, which is a yeast unidentified open reading frame. The
analysis shown is for Kimura corrected unweighted pairs group method
using arithmetic averages.
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The finding of a close linkage between PPO and PHDs was not anticipated
since published reports on PHDs had not demonstrated the presence of
FAD. The genes for PHD have been cloned and sequenced from a number of
plants and bacteria (see Refs. 22 and 23). The plant PHDs (referred to
as pds type PHDs) catalyze the oxidation of phytoene to
carotene, a
four-electron oxidation that may utilize molecular oxygen, whereas
bacterial PHDs (referred to as CrtI type PHDs) convert phytoene all the
way to lycopene (an eight-electron oxidation), or to neurosporene (a
six-electron oxidation) in reactions that are not stimulated by
molecular oxygen (see Ref. 23). The protein from the bacteria
Rhodobacter sphaeroides (24) and R. capsulatus
(25), which both form neurosporene as product, have been cloned and
expressed. The expressed R. capsulatus PHD has been purified
to near homogeneity although purification yields were quite low (25).
These enzymes are membrane associated and difficult to purify and
assay. Because of these factors there are no definitive studies on the
enzyme mechanism. Enzymatic studies on the CrtI protein of R. spheroides and R. capsulatus do, however, demonstrate
that the enzyme catalyzes three desaturations of phytoene to yield
neurosporene (Scheme I). In this regard it is similar to PPO, which
also carries out a six-electron oxidation. Purified PHD of R. capsulatus was shown to be stimulated by FAD but not NAD(P) (25).
Because of the predicted sequence similarity of PHDs to PPOs and MAOs,
and the lack of information on PHD, we chose to express and
characterize a bacterial PHD from Myxococcus xanthus, which
catalyzes a reaction similar to the R. capsulatus enzyme, to
determine if its physical properties are similar to those of PPOs and
MAOs.
The expression of M. xanthus PHD as a histidine-tagged
protein allowed for rapid purification of the protein when we used a
procedure that our laboratory has worked out to purify PPOs. The
molecular weight of purified PHD is 60,000 as determined by SDS gel
electrophoresis (Fig. 2), which is in
agreement with the predicted size of 58,600. The calculated extinction
coefficient for the apoprotein is 57,000 M
1
at 278 nm and the pI is 9.7. The protein as purified contains noncovalently bound 0.5 flavin/monomer of PHD (Fig.
3), and this was identified as FAD by the
pH-dependent fluorescence (26). No redox active metals were
found associated with PHD. As isolated from the expression system in
E. coli, PHD contains a small and variable amount of
porphyrin. This amount of bound porphyrin represents a few percent of
the total PHD present, but because of the presence of porphyrin and the
similarities with PPO, PHD was assayed for PPO activity. PHD was found
to have no significant PPO activity, and the PHD expression vector did
not complement E. coli SAS38X cells, which lack PPO activity
(27) (data not shown).

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Fig. 2.
SDS gel electrophoresis of purified M. xanthus phytoene desaturase. The gel photograph shows molecular
weight markers on the left side, solubilized cell extract,
and purified histidine-tagged phytoene desaturase (10 µg).
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Fig. 3.
UV/visible spectrum of purified M. xanthus
phytoene desaturase and acid-extracted flavin. The
upper figure shows the spectrum of 20 µM
purified enzyme. The inset is an enlargement of the visible
spectrum. The lower figure is the spectrum of 5%
trichloroacetic acid extracted flavin from the purified enzyme. The
concentration of the extracted flavin fraction is one-half of that
shown for the enzyme in the upper figure.
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Based upon protein size, amino acid sequence homology (including the
presence of the highly conserved amino-terminal dinucleotide binding
motif), the presence of FAD as a cofactor, and the similarity in the
reactions catalyzed, it would appear that phytoene desaturase might
catalyze an oxygen-dependent oxidation of phytoene.
However, the observations that carotenoid biosynthesis in
photosynthetic bacteria is maximal during anaerobic photosynthetic
growth (28) and that mRNA levels for this enzyme in R. sphaeroides are decreased under aerobic conditions (24) but are
unchanged under the same conditions in R. capsulata (29) do
raise questions that may be addressed experimentally now that the
protein can be expressed and purified.
As shown in the Growtree analysis (Fig. 1), mammalian MAOs cluster
together as would be expected, but, interestingly, the bacterial MAOs
are quite distinct phylogenetically. In fact the bacterial MAOs appear
no more closely linked to animal MAOs than to phenol hydroxylase. One
point of interest is the presence of an unidentified yeast open reading
frame (P32614) in this analysis. Yeast PPO is identified and is present
in this analysis, and yeast have been reported to not produce MAO, so
the identity of this putative protein will be of interest when it is
identified. Sequences for plant phytoene desaturases were also found in
the present analysis even though it has previously been reported that
these enzymes are biochemically distinct from the microbial phytoene desaturases (see Refs. 23 and 30). Whereas it is clear from sequence
analysis that these two groups of enzymes are distinct, it appears that
they all are members of a protein superfamily.
Examination of the putative dinucleotide binding motif sequences
(Fig. 4) clearly shows that significant
identity and close homologies exist among all of the enzymes and that
this identity extends past the requisite Glu (or Asp) (21). When a
Growtree analysis of this approximately 60-residue long segment is
carried out using the same parameters described above the cluster
pattern found in the tree is very similar to that obtained when one
uses the complete protein sequences (Fig. 1). The only significant change is that the sequence for Myxococcus
hydroxyneurosporine dehydrogenase becomes clustered with
protoporphyrinogen oxidase sequences. Such findings suggest that
members of this superfamily arose via divergence from a common ancestor
and did not acquire the dinucleotide motif from a gene fusion event.
One interesting feature of the alignment examination is that a
consensus sequence exists that includes the dinucleotide binding motif
and spans approximately 60 residues (Fig. 4). The consensus sequence
found is:
U4G(G/A)GUXGL(X2)(A/S)(X2)L(X6-12)UX(L/V)UE(X4)UGG(X9-13)(G/V)(X3)(D/E)XG, where X stands for any residue and U stands for a
hydrophobic residue. This suggests that either the actual dinucleotide
binding motif region is larger than previously considered (21) or that the currently demonstrated consensus sequence may represent a signature motif for this superfamily of proteins. Examination of the
sequences of a variety of proteins other than PPO, MAO, and PHDs that
have been shown to possess a dinucleotide binding motif indicates that
the additional region of identity is not present (data not shown) and,
therefore, this consensus sequence is a fingerprint motif for this
particular superfamily of enzymes. Assigning a role to this motif is
not easy without protein structure information but it is possible to
reasonably rule out some possibilities based upon what we currently
know about these three types of enzymes. It does appear clear that the
motif region would not be involved specifically in interactions with
molecular oxygen since not all PHDs utilize oxygen directly as an
electron acceptor. A role in dinucleotide binding likewise seems
unlikely since a large number of proteins are known to possess the
currently identified dinucleotide binding motif (21) and do not
contain the additional residues reported here, and a role for the
motif in substrate recognition is unanticipated considering the wide
variety of substrates utilized by the three enzymes. Perhaps then the
answer lies simply in a structural role that links these proteins
temporally in evolution.

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Fig. 4.
Pileup alignment of the proposed superfamily
fingerprint motif. The dinucleotide binding motif of Wierenga
et al. (21) is underlined, and the conserved
glutamate is marked (·). The sequence of the proposed fingerprint for
this superfamily is:
U4G(G/A)GUXGL(X2)(A/S)(X2)L(X6-12)UX(L/V)UE(X4)UGG(X9-13)(G/V)(X3)(D/E)XG
where U is a hydrophobic residue and X is any
residue. Note the almost complete identity in this region for all plant
phytoene desaturases. Abbreviations are as in the legend to Fig. 1
except that Dbm stands for dinucleotide binding motif.
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One point of interest concerning PPO and PHD are that these two enzymes
are both sites of action of plant herbicides. PPOs are the target of
diphenyl ether type of herbicides (13, 14). These compounds kill plants
via a light-dependent destruction of plant cells that have
accumulated free protoporphyrin. PHDs are the target of photobleaching
herbicides that act by inhibiting PHD, and thus carotenoid synthesis
(31). Inhibition of PHD allows the build up of colorless saturated
carotenoid precursor that absorb in the UV and generates singlet
oxygen. These classes of herbicides comprise two of the largest groups
of currently utilized plant herbicides, but at the present time their
exact mode of action is unknown. It has been suggested that diphenyl
ether herbicides may be substrate analogs for PPO but lack of purified
PHD preparations has slowed research on these enzymes.
We acknowledge the helpful suggestions of
W. B. Whitman of the Department of Microbiology and M. Weise of
the Department of Genetics at the University of Georgia.