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INTRODUCTION |
Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes
the formation of 4-hydroxyproline in collagens and more than 10 other
proteins with collagen-like sequences. This cotranslational and
posttranslational modification plays a central role in the synthesis of
all collagens because the 4-hydroxyproline residues are essential for
formation of the collagen triple helix at body temperature (for
reviews, see Refs. 1-3). The vertebrate type I and type II enzymes are
(
(I))2
2 and
(
(II))2
2 tetramers, respectively, in
which the
subunit is identical to protein-disulfide isomerase (EC
5.3.4.1; PDI)1 and has PDI
activity even when present in the tetramer (2-6).
An
subunit of prolyl 4-hydroxylase has also been cloned from the
nematode Caenorhabditis elegans (7). This forms an active enzyme in insect cell coexpression experiments with both the C. elegans and human PDI polypeptides, but surprisingly, the enzymes containing the subunit are 
dimers (7, 8). The C. elegans PDI polypeptide also forms an active prolyl 4-hydroxylase
with the human
subunits, but these enzymes are
2
2 tetramers (8). Assembly of a tetramer
or dimer must therefore depend on the properties of the
subunit. It
is currently unknown whether other nonvertebrate prolyl
4-hydroxylases are likewise dimers rather than tetramers.
Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate,
O2, and ascorbate (2, 3). A search for conserved amino acid
residues within sequences of several 2-oxoglutarate dioxygenases and
prolyl 4-hydroxylase
subunits from various species, together with
extensive site-directed mutagenesis studies on the human
(I)
subunit, have identified five amino acid residues that are critical at
the cosubstrate binding sites of prolyl 4-hydroxylases (4, 5, 7,
9-11). Three of these, His-412, Asp-414, and His-483 (numbered
according to the human
(I) subunit), are involved in binding of the
Fe2+ atom, whereas Lys-493 binds the C-5 carboxyl group of
2-oxoglutarate (10, 11). His-501 is an additional critical residue,
probably involved in both the binding of the C-1 carboxyl group of
2-oxoglutarate to the Fe2+ atom and the decarboxylation of
this cosubstrate (11).
The aim of the present study was to clone the
subunit of prolyl
4-hydroxylase from Drosophila melanogaster and to
characterize the corresponding enzyme. Special emphasis was given to
comparison of the cDNA-derived amino acid sequence of the
Drosophila
subunit with those of the human
(I) and
(II) subunits and the C. elegans
subunit, to identify
conserved and nonconserved residues and regions, and to the question of
whether the Drosophila enzyme is an
2
2 tetramer or an 
dimer. The amino
acid sequence of the Drosophila
subunit was found to
show several distinct differences as compared with those of the other
species studied, and the Drosophila enzyme was found to have
some unique catalytic properties that appear related to these differences.
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MATERIALS AND METHODS |
Isolation of cDNA Clones--
A sequence homology search in
FlyBase indicated the presence of a 179-nucleotide-long sequence
(DM59D7T, FlyBase ID no. FBgn0015713) homologous to those of the
vertebrate prolyl 4-hydroxylase
subunits. PCR primers PKA5
(5'-AAATGGAGGTTCCACGGCAG-3') and PKA3 (5'-CCGATCCCATAGTTTGCCAC-CTG-3') were synthesized based on this sequence and used to obtain a 160-bp PCR
product from a cDNA pool generated from D. melanogaster
mRNA. The purified PCR product was 32P-labeled and used
to screen a D. melanogaster larva
gt10 cDNA library
(CLONTECH). This screening yielded seven positive
clones among 600,000 recombinants, two of which, termed D
2 and
D
6, were characterized in detail.
Nucleotide Sequencing and Sequence Analysis--
The nucleotide
sequences were determined by the dideoxynucleotide chain termination
method (12) with the Sequenase enzyme (U. S. Biochemical Corp.) in an
automated DNA sequencer (Applied Biosystems). Vector-specific or
sequence-specific primers (Pharmacia) were used. DNASIS and PROSIS
(version 6.0) (Pharmacia) were used to analyze the nucleotide and amino
acid sequence data. The cleavage site of the signal pepide was
predicted using the computational parameters of von Hejne (13).
Construction of Baculovirus Transfer Vectors and Generation of
Recombinant Baculoviruses--
An expression construct for the
Drosophila
subunit was obtained by digesting the D
2
clone with EcoRI. This fragment, which covers the whole
coding region, 62 bp of the 5'-untranslated region and 12 bp of the
3'-untranslated region, was ligated to the transfer vector pVL1392
(14).
Arginine 490 (codon CGT) was converted to histidine (CAT) or serine
(AGT) to produce the transfer vectors for the recombinant baculoviruses
D
H and D
S, respectively. The mutagenesis steps were performed in
a pSP72 vector (Promega) containing the D
2 clone, using a PCR-based
method (QuickChange site-directed mutagenesis kit; Stratagene).
To construct a baculovirus transfer vector for the
Drosophila PDI polypeptide, a PCR product containing 9 bp of
the 5'-untranslated region, the whole coding region, and 227 bp of the
3'-untranslated region, was amplified using sequence-specific (15)
primers DRO1 (5'-GTGACCGCAATGAAATTCCTG-3') and DRO4
(5'-GCAAAAAGCTTCGATGGCTAC-3') from a cDNA pool generated from
D. melanogaster mRNA, and the fragment was ligated to
the SmaI site of the pVL1393.
Spodoptera frugiperda--
Sf9 insect cells (Invitrogen)
were cultured as monolayers in TNM-FH medium (Sigma) supplemented with
10% fetal bovine serum (Bioclear) at 27 °C. The recombinant
baculovirus transfer vectors were cotransfected into Sf9 cells
(Invitrogen) with a modified Autographa californica nuclear
polyhedrosis virus DNA (BaculoGold, PharMingen) by calcium phosphate
precipitation (16). The resultant viral pools were collected 4 days
later, plaque-purified, and amplified (16). The recombinant viruses
coding for the Drosophila
subunit, its R490H and R490S
mutants, and the Drosophila PDI polypeptide were termed
D
, D
H, D
S, and D
. Other recombinant baculoviruses used were
human
(I), C. elegans
, human PDI, and C. elegans PDI coding for the corresponding prolyl 4-hydroxylase subunits (7, 8, 17).
Expression and Analysis of Recombinant Proteins--
Sf9
insect cells were cultured as described above, either as monolayers or
in suspension in spinner flasks (Techne Laboratories, Princeton, NJ),
and were infected at a multiplicity of 5. For the production of an
enzyme tetramer/dimer, the D
, D
H, D
S, human
(I), or
C. elegans
, and the human, C. elegans, or
D. melanogaster PDI viruses were used in a ratio 1:1. The
cells were harvested 3 days after infection, washed with a solution of
0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in a solution of 0.1 M glycine, 0.1 M NaCl, 10 µM dithiothreitol, 0.1% Triton X-100, and 0.01 M Tris, pH 7.8, and centrifuged at
10,000 × g for 20 min at 4 °C. The resulting
supernatants were analyzed by 8% SDS-PAGE or nondenaturing 8% PAGE
and assayed for enzyme activity.
Northern Blot Analysis--
Two µg of poly(A)+
RNAs from embryonic, larval, and adult Drosophila
(CLONTECH) were run on a 1% formaldehyde gel with
molecular weight standards (Promega). The poly(A)+ RNAs
were then transferred onto a nitrocellulose filter, and the filter was
hybridized under stringent conditions with 32P-labeled
D
2 clone covering the whole coding region of the
Drosophila
subunit. Autoradiography time was 9 days.
Other Assays--
Prolyl 4-hydroxylase activity was assayed by a
method based on the hydroxylation-coupled decarboxylation of
2-oxo-[1-14C]glutarate as described previously (18),
except that in the assays involving the Drosophila enzyme
the 2-oxoglutarate concentration was increased from 0.1 to 0.2 µmol/ml and the (Pro-Pro-Gly)10·9H2O peptide substrate concentration from 0.1 to 1.5 mg/ml. The same method
was used for measuring the uncoupled decarboxylation activity, except
that the peptide substrate was omitted and the amount of enzyme was
increased about 10-fold. Km values were determined by varying the concentration of one substrate in the presence of fixed
concentrations of the second, whereas the concentrations of the other
substrates were held constant (19). The Triton X-100-soluble fraction
of the insect cell homogenate was used as the enzyme source for the
Km determinations.
The amounts of recombinant proteins were compared by densitometry of
Coomassie Brilliant Blue-stained bands in nondenaturing PAGE using
a BioImage instrument (BioImage, Millipore).
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RESULTS |
Isolation of cDNA Clones--
The first cDNA clone was a
160-bp PCR product obtained from a D. melanogaster cDNA
pool with primers PK5 and PK3, which were based on a 179-nucleotide
sequence DM59D7T present in FlyBase. This PCR product was used to
screen a D. melanogaster larva
gt10 cDNA library, and
one of the 7 positive clones obtained, D
2, was found to cover 62 bp
of the 5'-untranslated sequence, the whole coding region and 12 bp of
the 3'-untranslated sequence of the corresponding Drosophila
subunit mRNA. D
6 continued 201 bp downward from the 3'-end
of D
2, and thus a total of 213 bp of the 3'-untranslated sequence
was characterized. This 3'-untranslated region does not contain the
canonical polyadenylation signal AATAAA, but it does contain a GATAAA
sequence, which is accompanied 21 bp downstream by a possible poly(A)
tail of 4 nucleotides at which the clone ends (these cDNA sequences
are not shown but have been deposited in the GenBank/EMBL Data Bank
with accession number AF096284).
Amino Acid Sequence of the Drosophila
Subunit and Its
Comparison with Those of the Human
(I) and
(II) Subunits and the
C. elegans
Subunit--
The cDNA clone encodes a 535-amino
acid polypeptide. A putative signal peptide is present at its N
terminus, the most likely first amino acid of the processed
subunit
being glutamate, based on the computational parameters of von Hejne
(13) and comparison with the N terminus of the processed human
(II)
subunit (5). Thus the length of the signal peptide is probably 19 amino
acids and that of the processed
subunit 516 amino acids (Fig.
1).

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Fig. 1.
Comparison of the amino acid sequence of
the subunit of Drosophila
prolyl 4-hydroxylase with those of the human
(I), human (II), and
C. elegans subunits. Numbering of
amino acids begins with the first residue in the processed
polypeptides. Drosophila is indicated by D, human
(I) and human (II) by H(I) and
H(II), respectively, and C. elegans by
C. Gaps (· ·) were introduced for maximal alignment of
the polypeptides. Positions of cysteine residues (·) and potential
Asn glycosylation sites ( ) in any of the polypeptides are indicated
above all the sequences. The residues required for the binding of the
Fe2+ atom and the C-5 carboxyl group of 2-oxoglutarate as
well as the residue that is probably involved in the binding of the C-1
carboxyl group of 2-oxoglutarate and the decarboxylation of this
cosubstrate are also indicated (asterisk). White
letters on a black background indicate identity, and
black letters on a gray background indicate
similarity. The following are groups of similar amino acids: G, A, S;
A, V; V, I, L, M; I, L, M, F, Y, W; L, K, H; D, E, Q, N; and S, T, E,
N.
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The Drosophila
subunit is very similar in size to the
human
(I) and
(II) subunits which have 517 (20) and 514 (4) residues, respectively (Fig. 1), whereas the C. elegans
subunit (7) is longer, 542 amino acids, mainly because of a 32-residue C-terminal extension (Fig. 1). The Drosophila
subunit
also has a C-terminal extension as compared with the vertebrate
(I)
and
(II) subunits (3), but this extension is much shorter than in
the C. elegans
subunit, being only 10 residues (Fig. 1). The Drosophila
subunit sequence has several minor
deletions and insertions as compared with the other
subunit
sequences, the longest deletion, between residues 225 and 226, being 9 amino acids (Fig. 1).
The overall amino acid sequence identities between the
Drosophila
subunit (excluding its 10-residue C-terminal
extension) and the human
(I) and
(II) subunits are 34 and 35%,
respectively, whereas its identity to the C. elegans
subunit is 31%. The identity is highest within the catalytically
important (2, 3) C-terminal region, residues 385-502 being 51 and 52%
identical to those in the human
(I) and
(II) subunits,
respectively, and 46% identical to those in the C. elegans
subunit. All three residues that bind the Fe2+ atom and
the lysine that binds the C-5 carboxyl group of the 2-oxoglutarate (11)
are conserved, these amino acids being His-402, Asp-404, His-472, and
Lys-482 in the Drosophila polypeptide. Surprisingly, however, the fifth critical residue, an additional histidine that is
probably involved in both the binding of the C-1 carboxyl group of the
2-oxoglutarate to the Fe2+ atom and the decarboxylation of
this cosubstrate (11), is replaced by Arg-490. The recently identified
peptide substrate binding domain (21), corresponding to residues
139-229 in the Drosophila
subunit, contains a 9-residue
deletion close to its C-terminal end (between residues 225 and 226),
whereas residues 139-225 show only 31, 36, and 23% identity to those
in the human
(I) and
(II) subunits and the C. elegans
subunit, respectively.
The 32-amino acid sequence of residues 230-261 shows a particularly
low degree of identity to those in the other
subunits, namely 3.1, 6.3, and 12.5% when compared with those of the human
(I) and
(II) subunits and the C. elegans
subunit (Fig. 1). The sequence of residues 27-60 represents another region of low identity, namely 5.9, 11.8, and 2.9% to the same subunits.
The Drosophila
subunit has three potential attachment
sites for asparagine-linked oligosaccharide units, but the positions of
these sites are different from those of the two sites (4, 20) present
in the human
(I) and
(II) subunits (Fig. 1). The five cysteine
residues present in the vertebrate
(I) subunits (2-5, 20) and the
C. elegans
subunit (7) are all conserved in the
Drosophila
subunit, whereas the vertebrate
(II)
subunits (4, 5) contain an additional cysteine not present in the other
subunits.
The Drosophila
Subunit Forms Prolyl 4-Hydroxylase
2
2 Tetramers with the Drosophila and
Human PDI Polypeptides--
A recombinant baculovirus encoding the
Drosophila
subunit was generated and used to infect
S. frugiperda insect cells. The cells were harvested 72 h after infection, homogenized in a buffer containing Triton X-100, and
centrifuged. The cell pellet was solubilized in 1% SDS, and the 0.1%
Triton X-100 soluble and 1% SDS soluble proteins were analyzed by 8%
SDS-PAGE under reducing conditions followed by Coomassie Blue staining.
Most of the insect cell proteins were soluble in the Triton X-100
containing buffer (details not shown). As with prolyl 4-hydroxylase
subunits from other sources, the recombinant polypeptide formed
insoluble aggregates, and efficient extraction of the recombinant
Drosophila
subunit required the use of 1% SDS. The
polypeptide was found in the form of three main bands (Fig.
2, lane 2) with
mobilities slightly less than those of the three human
(I) subunit
bands (Fig. 2, lane 1). Digestion of the sample
with endoglycosidase H produced a single band with a mobility slightly
higher than that of the lowest of the three bands present in the
untreated sample (Fig. 2, lane 3), suggesting
that the original polypeptide was present in tri-, di-, and
monoglycosylated forms. This agrees with the presence of three
potential N-glycosylation sites in the Drosophila
subunit (Fig. 1) and indicates that all three sites are used, at
least in insect cells.

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Fig. 2.
SDS-PAGE analysis of expression of the
Drosophila prolyl 4-hydroxylase subunit in insect cells and carbohydrate moieties after
endoglycosidase H treatment under reducing conditions. Lanes
1, 2, and 4 show 1% SDS-soluble polypeptides from
expressions with baculoviruses coding for the human (I),
Drosophila , and C. elegans subunits, and
lane 3 shows the SDS-soluble polypeptides from
expression with the Drosophila subunit baculovirus
incubated in the presence of endoglycosidase H. The samples were
analyzed by 8% SDS-PAGE and Coomassie Blue staining. Arrows
indicate migration of the tri- and monoglycosylated forms of the
Drosophila subunit.
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To produce a Drosophila prolyl 4-hydroxylase tetramer or
dimer, a cDNA coding for the Drosophila PDI polypeptide
was prepared by PCR using primers based on the published sequence (15).
A recombinant baculovirus encoding the Drosophila PDI
polypeptide was then generated and used to infect insect cells together
with the virus coding for the Drosophila
subunit. The
cells were harvested 72 h after infection, and Triton
X-100-soluble proteins of the cell homogenate were analyzed by
nondenaturing 8% PAGE. A Coomassie Blue-stained band with a mobility
slightly higher than that of the enzyme tetramer produced by
coinfection of insect cells with recombinant viruses coding for the
(I) subunit of human prolyl 4-hydroxylase and the human PDI
polypeptide (Fig. 3A,
lane 1) was seen in samples coinfected with the
two types of Drosophila virus (Fig. 3A,
lane 2), whereas no such band was seen in a
corresponding sample from cells infected with the Drosophila
subunit virus alone (not shown). The mobility of this
Drosophila prolyl 4-hydroxylase band is distinctly different
from that produced by coinfecting insect cells with baculoviruses
coding for the C. elegans
subunit and the human PDI
polypeptide (Fig. 3A, lane 5), shown
previously (7) to represent an 
dimer. The data thus strongly
suggest that the Drosophila prolyl 4-hydroxylase is an
2
2 tetramer.

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Fig. 3.
Analysis by PAGE under nondenaturing
conditions of prolyl 4-hydroxylase tetramer formation by the
Drosophila subunit with
Drosophila or human PDI and by the human
(I) subunit with Drosophila PDI
when coexpressed in insect cells. Lanes 1-5, Triton
X-100-soluble samples are from cells coinfected with viruses coding for
human (I) and human PDI (lane 1), Drosophila
and Drosophila PDI (lane 2),
Drosophila and human PDI (lane 3), human
(I) and Drosophila PDI (lane 4), and C. elegans and human PDI (lane 5). The samples were
electrophoresed on 8% PAGE under nondenaturing conditions and analyzed
by Coomassie Blue staining (A) or by Western blotting using
an anti-human PDI polyclonal antibody (B). Arrows
marked with T indicate migration of the tetramers; the
arrow marked with D, migration of a dimer; and
the arrow marked with PDI, migration of free PDI.
The prominent band migrating about 75% of the lane distance in
A represents a viral protein, and the faint bands below it
represent the nonassembled Drosophila (lanes 2 and 4) or human (lanes 1, 3, and 5)
PDI polypeptides.
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No band corresponding to an enzyme tetramer or dimer could be seen in
Coomassie Blue-stained samples from cells coexpressing the
Drosophila
subunit and the human PDI polypeptide (Fig.
3A, lane 3), but formation of a small
amount of a hybrid enzyme tetramer could be demonstrated by Western
blotting with an antibody to the human PDI polypeptide (Fig.
3B, lane 3). Coexpression of the human
subunit with the Drosophila PDI polypeptide produced an enzyme tetramer that could easily be seen by Coomassie Blue staining (Fig. 3A, lane 4).
Catalytic Properties of the Drosophila Prolyl 4-Hydroxylase
Tetramer--
Prolyl 4-hydroxylase activity of the Triton
X-100-soluble proteins was measured by a method based on the
hydroxylation-coupled decarboxylation of
2-oxo-[1-14C]glutarate (18). When the specific activity
of the Drosophila enzyme was compared with that of the human
type I enzyme, the Coomassie Blue-stained bands in nondenaturing PAGE
corresponding to these enzyme tetramers were first studied by
densitometry, and the activity levels obtained were corrected (10, 11)
for the differences in the amounts of the two types of enzyme. Initial prolyl 4-hydroxylase activity assays performed under standard conditions (18) suggested that the specific activity of the Drosophila enzyme was very low, but when the values were
corrected for saturating concentrations of 2-oxoglutarate and the
(Pro-Pro-Gly)10 peptide substrate (see below), the specific
activity of the Drosophila enzyme increased to about
one-third of that of the human type I enzyme (details not shown). The
rate of uncoupled 2-oxoglutarate decarboxylation, i.e.
decarboxylation observed in the absence of the peptide substrate, was
3.7% of the rate of the complete reaction (details not shown, see also
Table II below). This percentage is markedly higher than the value of
0.7% measured for the human type I enzyme (11).
The Km values of the Drosophila enzyme
for Fe2+ and ascorbate were very similar to those of the
human type I and type II enzymes, whereas the Km for
2-oxoglutarate was about 4-fold (Table
I). A major difference was found in the
Km for the (Pro-Pro-Gly)10 peptide
substrate, because that of the Drosophila enzyme was about
12 times that of the human type I enzyme and 3 times that of the human
type II enzyme (Table I). The Drosophila enzyme resembled
the vertebrate type II enzymes in being inhibited by
poly(L-proline) only at high concentrations, the
Ki for poly(L-proline)
Mr 7,000 being about 30-fold relative to that
obtained for the human type I enzyme and that for
poly-(L-proline) Mr 44,000 about
150-fold (Table I). These Ki values of the
Drosophila enzyme are not as high as those of the human type
II enzyme, however, because the latter values are still about 5-fold
and 7-fold higher than the Drosophila enzyme values for
poly-(L-proline) Mr 7,000 and 44,000, respectively (Table I).
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Table I
Km values of the Drosophila and human type I and type II prolyl
4-hydroxylases for cosubstrates and the (Pro-Pro-Gly)10
substrate and Ki values for poly-(L-proline)
The values are given as mean ± S.D.
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Catalytic Properties of
R490H and
R490S Mutant Enzyme
Tetramers--
Arg-490 in the Drosophila
subunit was
converted to either histidine or serine, and baculoviruses coding for
the mutant
subunits were used to infect insect cells. The cells
were harvested and homogenized as above, and the 1% SDS-soluble
proteins were analyzed by 8% SDS-PAGE under reducing conditions
followed by Coomassie Blue staining. The expression levels of both
types of mutant
subunit per µg of protein were found to be
similar to that of the wild-type polypeptide (Fig.
4A).

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Fig. 4.
Analysis by SDS-PAGE under reducing
conditions (A) and PAGE under nondenaturing conditions
(B) of the expression of Drosophila
Arg-490 mutants in insect cells and their tetramer
formation. Lanes 1-3 show 1% SDS-soluble proteins
from expressions with baculoviruses coding for Drosophila
, Drosophila R490H, and Drosophila R490S
run on 8% SDS-PAGE under reducing conditions and analyzed by Coomassie
Blue staining (A). Arrows indicate migration of
the tri-, di-, and monoglycosylated polypeptides. Triton X-100-soluble
proteins of the coexpressions of Drosophila and R490H
and of R490S and Drosophila PDI viruses were analyzed on
8% PAGE under nondenaturing conditions with Coomassie Blue staining
(B). The arrow marked with T indicates
migration of the tetramers.
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The mutant
subunits were then coexpressed in insect cells with the
Drosophila PDI polypeptide, and the cells were harvested 72 h after infection. Proteins soluble in a Triton X-100 buffer were analyzed by nondenaturing 8% PAGE and assayed for prolyl 4-hydroxylase activity. The Coomassie Blue-stained bands corresponding to the enzyme tetramer (Fig. 4B) were studied by
densitometry in each experiment, and these values were used to correct
the enzyme activity levels for differences in the amounts of the
various types of enzyme tetramer, as described previously (10, 11).
The specific activity of the R490H mutant enzyme tetramer was found to
be about 90% that of the wild-type enzyme, whereas the specific
activity of the R490S enzyme tetramer was only about 30% (Table
II). The rate of uncoupled
2-oxoglutarate decarboxylation as a percentage of the rate of the
complete reaction was lower with the R490H mutant enzyme than with the
wild-type enzyme, whereas the R490S enzyme gave a very high percentage
(Table II). The Km of the R490H mutant enzyme for
2-oxoglutarate was similar to that of the wild-type enzyme, whereas
that of the R490S enzyme was about 4-fold that of the wild-type enzyme
(Table II).
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Table II
Prolyl 4-hydroxylase activity, rate of uncoupled decarboxylation of
2-oxoglutarate and Km for 2-oxoglutarate of the Arg-490
mutant Drosophila enzymes
The values are given in dpm/50 µl of Triton X-100-soluble cell
protein, mean ± S.D. The statistical significances of the values
obtained with the mutant enzymes were calculated versus the
wild-type enzyme.
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Expression of the Drosophila Prolyl 4-Hydroxylase
Subunit
mRNA at Different Developmental Stages--
Northern analysis of
poly(A)+ RNA from embryonal, larval, and adult
Drosophila gave a distinct hybridization signal only with the larval RNA (Fig. 5). The size of the
mRNA was about 1.9 kilobases, which agrees well with the size of
the 1883 nucleotides characterized by cloning and sequencing.

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Fig. 5.
Northern blot analysis of
Drosophila poly(A)+ RNAs. Lanes
1-3 show 2 µg of embryonic, larval, and adult
Drosophila poly(A)+ RNA, respectively,
hybridized with the 32P-labeled probe covering the coding
region of the Drosophila prolyl 4-hydroxylase subunit.
The arrow indicates the 1.9-kilobase band from the larval
mRNA. Molecular weight markers are indicated in kilobases.
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DISCUSSION |
The data reported here indicate that the
subunit of
Drosophila prolyl 4-hydroxylase shows about 30-35% amino
acid sequence identity with the two types of human and the C. elegans
subunit. The expression level of the mRNA for the
Drosophila
subunit was much higher at the larval stage
than the embryonic or adult Drosophila. Our data further
indicate that the Drosophila enzyme resembles the vertebrate
enzymes (2-4) rather than the C. elegans prolyl
4-hydroxylase 
dimer (7, 8) in that it is an
2
2 tetramer.
The Drosophila
subunit was found to have several
distinct amino acid sequence differences from those of other species in both its catalytic and peptide substrate binding domains. These differences appear to be related to certain unique catalytic properties of the Drosophila enzyme. The most distinct of these
differences was that the histidine likely to assist in the binding of
the C-1 carboxyl group of 2-oxoglutarate to the Fe2+ atom,
and also in the decarboxylation of this cosubstrate (11), was replaced
by Arg-490. Site-directed mutagenesis studies on the human
(I)
subunit have indicated that replacement of this histidine by other
amino acids, including arginine, leads to a 2-3-fold increase in the
Km for 2-oxoglutarate, a marked decrease in the
reaction velocity, and a marked increase in the rate of uncoupled
2-oxoglutarate decarboxylation as a percentage of the rate of the
complete reaction (11). The present data on the wild-type
Drosophila enzyme are similar to those obtained with the
histidine to arginine mutant human type I enzyme, in that the
Km of the former for 2-oxoglutarate was about 4-fold
that of the wild-type human enzyme, the reaction velocity was lower,
and the percentage of uncoupled decarboxylation was about 5-fold.
Mutation of Arg-490 to histidine in the Drosophila
subunit reduced the percentage of uncoupled decarboxylation but did not
increase the reaction velocity or reduce the Km for
2-oxoglutarate, whereas mutation of Arg-490 to serine gave a much lower
reaction velocity, a much higher percentage of the uncoupled reaction,
and an increased Km for 2-oxoglutarate relative to
those determined with the wild-type Drosophila enzyme. The
data obtained with the R490S mutant Drosophila enzyme agree with those reported for the corresponding human
(I) subunit mutant (11), whereas the data obtained with the R490H Drosophila
mutant resemble those expected on the basis of the human mutants (11) only in that the rate of uncoupled 2-oxoglutarate decarboxylation was
lower than with the wild-type Drosophila enzyme. Thus, other residues in the Drosophila
subunit must influence the
environment around residue 490 so that the arginine in this position is
much more acceptable than in the human
(I) subunit.
The finding that the lysine that binds the C-5 carboxyl group of
2-oxoglutarate (11) is conserved in the Drosophila
subunit is of interest, as the corresponding position in all other
2-oxoglutarate dioxygenases sequenced so far, including lysyl
hydroxylase (22-25), is occupied by an arginine (9, 26). It thus seems
that this feature is specific to prolyl 4-hydroxylases from various species.
The relatively low degree of identity between the Drosophila
subunit and the other
subunits within the peptide binding domain agrees with the finding that the Km of the
Drosophila enzyme for the (Pro-Pro-Gly)10
peptide substrate is higher than that of any vertebrate prolyl
4-hydroxylase tetramer studied (Table I and Ref. 3) or of the C. elegans enzyme dimer (7, 8). It is currently unknown whether this
difference is related to possible differences between the sequences of
the susceptible prolines in the Drosophila collagens
(27-29) and the human collagen substrates. The actual residues in the
peptide binding domain that are critical for the binding of
(Pro-Pro-Gly)10 and poly-(L-proline) are
likewise unknown. The high Km of the
Drosophila enzyme for (Pro-Pro-Gly)10 and the
high Ki values for poly-(L-proline) indicate that the peptide binding properties of the
Drosophila enzyme are more closely related to those of the
vertebrate type II enzymes (4, 5) and the C. elegans enzyme
(7, 8) than to those of the vertebrate type I enzyme (3).
A 32-amino acid residue region very close to the C-terminal end
of the peptide binding domain was found to have a particularly low
degree of identity between the Drosophila
subunit and
those from other species. No function is currently known for this
region, but the low degree of identity suggests that it may represent a
variable interdomain sequence. It is also currently unknown whether the
catalytic domain begins around residue 262, the C-terminal end of this
variable region, or whether there is an additional domain with an
unknown function between this region and the catalytic domain, as the
highly conserved C-terminal region begins only around residue 385.
The formation of only very small amounts of an enzyme tetramer between
the Drosophila
subunit and the human PDI polypeptide is
of interest, as the C. elegans
subunit readily forms an
active enzyme dimer with the human PDI polypeptide (7) and as the human
(I) subunit readily formed an enzyme tetramer with the Drosophila PDI polypeptide. Thus the Drosophila
subunit must have some structural feature that acts against
tetramer formation with human PDI, and the Drosophila PDI
polypeptide must have, in addition to structural features that allow
prolyl 4-hdyroxylase formation with both the human and
Drosophila
subunits, some specific feature that is not
present in the human PDI polypeptide and that is recognized by the
Drosophila
subunit. Further work will be required to
elucidate these features, as no specific data are available at present
on sequences in these polypeptides that are critical for prolyl
4-hydroxylase assembly.