Vol. 182, No. 3, 1992 February 14, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 981-986
IMPORTANCE OF HISTIDINE RESIDUE 25 OF RAT HEME OXYGENASE FOR ITS CATALYTIC ACTIVITY Kazunobu Ishikawa, Michihiko S&o*, Mariko Ito, and Tadashi Yoshida Department of Biochemistry and*Department of Molecular and Pathological Biochemistry, Yamagata University School of Medicine, Yamagata 990-23, JAPAN Received
December
26,
1991
Summary: A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction. 0 1992 Academic Press,Inc.
Microsomal heme oxygenase catalyzes oxidative degradation of heme to biliverdin with NADPH-cytochrome P-450 reductase, which functions as an electron donor (1). Heme oxygenase is not a hemeprotein, but the substrate-enzyme complex formed by its stoichiometric binding with ferric heme behaves as a kind of hemeprotein (2, 3).
The spectral resemblance of the heme-heme oxygenase
complex to hemoglobin suggests that the heme is linked to histidine residues of the heme oxygenase protein through both the fifth and sixth coordination positions of its iron atom.
The finding that
diethylpyrocarbonate,
a modifier of histidine
residues, strongly inhibits the heme oxygenase activity also supports the possibility that a histidine residue plays a critical role in the heme oxygenase reaction (4). On the basis of these findings we expected that substitution of histidine residues essential for heme binding would eliminate heme oxygenase activity. cDNAs for rat (5), human (6), mouse (7), and chicken (8) heme oxygenase have been cloned. From the sequence homology among these cDNAs, His25, 84, 119, and 132 of the rat enzyme have been proposed to be heme-binding ligands in heme To examine these possibilities, we carried out site-directed oxygenase. mutagenesis at each of these four residues and determined the activities of the mutated enzymes. MATERIALS AND METHODS restriction Chemicals and materials The following compounds were used: endonucleases, DNA polymerase I (Klenow fragment), Tth DNA polymerase, T4 ooo6-291x/92
981
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
polynucleotide kinase, sequencing primers for pTVl18N, P8(5’-AGCGGATAACAATTT CACACAGGAAAC-3’) and P7(5’-CGCCAGGGTTTTCCCAGTCACGAC-3’), and calf intestinal alkaline phosphatase from Toyobo; phosphorylated linker of XbaI (10 mer) from New England Biolabs; T DNA ligase from Boehringer; Sequenase version 2.0 from US Biochem.; prokaryo%1c expression vector, pKK233-2, from Pharmacia; E. coli strain JM109 from Stratagene; isopropyl B-D-thiogalactopyranoside and expression vector pTV118N from Takara; ampicillin, chlorhemin and bovine serum albumin from Sigma; nitrocellulose membranes from Schleicher & Schiill, and peroxidase-conjugated sheep IgG against rabbit IgG from Cappel. Oligonucleotides were synthesized with an Applied Biosystems model 381A DNA synthesizer and purified in a Hitachi 638-30 HPLC system. The procedures used for preparing native heme oxygenase, a 28-kDa tryptic peptide of heme oxygenase, NADPH-cytochrome P-450 reductase, biliverdin reductase and polyclonal antibodies for heme oxygenase were as described in our recent papers (9, IO). Construction of expression plasmid carrying a truncated rat heme oxygenase cDNA: For an other purpose we have made pKK-ARHO, which carries a truncated cDNA for rat heme oxygenase lacking a coding sequence of a membraneassociated, C-terminal region. In the present study we used a pTV118N construct carrying the same coding sequence as pKK-ARHO, because primers for pTVl18N (Pi’ and P8) used for the polymerase chain reaction and sequencing are commercially available. The method for construction of pKK-ARHO was similar to that for construction of pKK-RHO (IO). Briefly, pKK233-2 was linearized by digestion with Ncol and the single stranded ends were filled in by treatment with Klenow fragment followed by dephosphorylation with calf intestinal alkaline phosphatase. A cDNA clone encoding the rat heme oxygenase gene, pRHO1 (51, was cut with XhoI and SpeI. The resulting DNA fragment (XhoI/SpeI, -59/782) was filled in with Klenow fragment, ligated with IO mer-XbaI linker so that TAG in the linker functioned as a stop codon, and then cleaved with XbaI. The resulting fragment was filled in and then partially cut with TaqI. This DNA fragment (TaqI/XbaI, 18/793) was used as a truncated cDNA. A synthetic 14-residue nucleotide (5’GAGCGCCCACAGCT3’) was phosphorylated with T4 polynucleotide kitiase and then annealed with a synthetic 16-residue nucleotide (3’CTCGCGGGTGTCGAGC5’) to form a linker DNA bridging the gap between the NcoI site of pKK233-2 and TaqI site of the truncated cDNA. Finally, the three DNA fragments were ligated to construct pKK-ARHO. For preparation of the pTV118N construct (pTV-ARHO), pKK-ARHO was partially cleaved with NcoI and HindIII. The resulting DNA fragment (NcoI/HindIII, -l/807) was ligated with pTV118N plasmid which had been pretreated with NcoI and HindIII. pTV-ARHO encodes a truncated, 30-kDa heme oxygenase consisting of amino acid residues 1 to 261 of the native enzyme with two replacements; namely, serine residues 262 and 263 of the native enzyme are replaced by arginine and leucine, respectively. Mutagenesis of a truncated cDNA for rat heme oxygenase by the polymerase chain reaction: Template DNA for site-specific mutagenesis was prepared by digestion of pTV-ARHO with PvuII. The resultant DNA fragment (1060 bp) contained the whole coding region of the truncated heme oxygenase and restriction sites for NcoI and Hind111 near the 5’ and 3’ end, respectively. Two contiguous regions of the template were amplified in two separate polymerase chain reactions. Table I shows the mutagenic and flanking primers used for each site-directed mutagenesis reaction. The primers used for amplification of the 5’-side region were P8 and the flanking primer. The mutagenic primer and P7 were used for the 3’-side region. The amplification reaction was performed in 100 ul of reaction mixture containing 0.5 ug of template DNA, 20 pmol of each primer, 0.2 mM dNTPs, and 2.5 units of Tth polymerase. The reaction was cycled 25 times in a HYBAID thermal reactor HB-TR 1. The amplified DNA fragments were purified with polyacrylamide gel electrophoresis and then the fragments of the 5’ and 3’ regions were cleaved with NcoI and HindIII, respectively. After phosphorylation of the 5’-end, the DNA fragments were ligated to pTV118N which had been pretreated with NcoI and HindIII. The resulting mutant plasmids were transfected into E. coli strain JM109. The complete nucleotide sequences of the inserts were 982
Vol.
182,
No.
3, 1992
BIOCHEMICAL
Table I.
Mutation
Summary used for
Flanking
AND
BIOPHYSICAL
of mutagenic site-directed
RESEARCH
and flanking mutagenesis
primer
primers
Mutagenic
H25A
COMMUNICATIONS
primer
5’-6&AGGAGGTGGCCATCCGTGC;23’ 3’-?TCCGGAACTTCCTCCGGTG&5’
H84A
220 3’-ATACGGGGCGAGATGAAGGGA-5’
5 ‘;$‘AGGAGCTGGCCCGAAGGGC?3
H119A
325 3’-GGTCGGTGTGTCGTGATGCAT-5’
5’;;$AGCGTCTCGCCGAGGTGGG;P-63’
H132A
364 3’-CCTCCATGAGTAGGACTCGAC-5’
385 5’&TGGTGGCCGCCGCATATACC-3’
The numbering of nucleic acids begins with the A of the ATG of rat heme oxygenase. The CAC codon for histidine alanine. Mutant codons are underlined.
confirmed
by
Other
the
procedures:
E. coli, immunoblot from the soluble recent papers (9,
’
405
initiation methionine codon was converted to GCC for
dideoxynucleotide chain-termination method (11). Other methods, including expression of heme oxygenase in analysis, assay of heme oxygenase, and extraction of biliverdin fraction of cultured E. coli, were the same as described in our 10).
RESULTS AND DISCUSSION Expression
in B. coli
sequence heme
for
coli of
region
heme
(10).
proteins,
we A
soluble
(lane
fraction truncated
We
observed
that
had
biliverdin
to
heme 1
in in had
heme
in
with often
oxygenase 1). presence
activity
for
oxygenase
heme in
an
place
heme as
a
heme
few
degraded
breakdown
in
soluble,
30-kDa
(Table
heme
system.
E.
coli. protein
activity and
of
found
that
Therefore,
II).
to
membrane-bound
oxygenase
full-length
to
ability
reductase of
system
of
retain
oxygenase
reductase
the
binds
C-terminal
to
that
rat
that
form
P-450
than
the
of
found
added
expressed
P-450
a
degraded
was
easier
measured of
a
the coding
expressing
membrane-associating,
soluble
was We
in form
cultivation,
30-kDa
a truncated,
Fig. the
is
succeeded
enzymes,
about
lacking
full-length
during
their
concert
proteins
express
that lost
of
we active,
soluble
mass
of heme oxygenase
Recently
a catalytically
these
soluble
tried
fraction
soluble
to
truncated
coli
segment:
a molecular
of
cDNA
as
Among
heme
treatment
coli
oxygenase
with
decompose
the
E.
appeared.
oxygenase
As
in
membranes
forms
E.
a hydrophobic
oxygenase
E.
of a truncated
enzyme
in
we the
in the this use
present
study.
Expression histidines To
determine
and activity are
conserved which
of mutated among
histidine
the residues
heme oxygenase: rat,
human, are
983
As
mouse
essential
and for
described chick
heme
heme oxygenase
above, oxygenases. activity,
four
Vol.
182, No. 3. 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,‘i’:,:;.
Expressions of a truncated wild-type heme oxygenase and its mutants in . Soluble fractrons (100 ug as protein) of E. coli harvested 6 hr after the addition of isopropyl 8 -D-galactopyranoside were used. Lane 1, truncated wild-type heme oxygenase; lane 2, H25A; lane 3, H84A; lane 4, H119A; lane 5, H132A.
we
prepared
pTV118N
in which these histidine residues were replaced
constructs
individually by an alanine residue by site-directed mutagenesis. Bacteria
harboring
these
express proteins reacting Western blot oxygenase. demonstrated that
mutated
with
recombinants
polyclonal
analyses
of
the
were
tested
for
antibodies against rat soluble
fractions
of
ability
liver E. coli
to
heme cells
all the mutated enzymes except H132A (His132 + Ala) were
expressed to similar extents to the wild-type enzyme (Fig. 1).
The low expression
of the H132A mutant was confirmed by immunoblot analysis of whole sonicates of E. coli expressed
cells (data not shown), eliminating the possibility that the H132A protein The reason for its low expression was sequestered in inclusion bodies.
is unknown. The activities
of equal amounts of protein of the respective soluble fractions for heme breakdown were compared (Table II). The mutant heme oxygenases H84A and H119A exhibited 41 and 39%, respectively, of the wild heme oxygenase activity.
In contrast, the H25A mutant showed almost no activity,
His25 is essential for
the heme oxygenase reaction.
suggesting that
The H132A mutant also
exhibited extremely low activity, but it was difficult to draw any conclusion from this finding about the role of H132 because expression of the H132A mutant protein was so low.
Table
Wild-type H25A H84A H119A H132A
II.
Comparison wild-type
of heme oxygenase activities and mutant enzyme Heme oxygenase activity nmole bilirubin formed (mg protein) -‘h-r 66.2 0.5 27.1 26.1 1.0
984
of
% 100 20 41 39 1
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Wild-type
500
400
WAVELENGTH
600
700
(t-am)
Fie. 2. Absorption spectra of chloroform extracts from soluble fractions of E. coli expressingtruncated wild-type hemeoxygenaseand its mutants.
Extraction
of biliverdiu from the soluble fraction
of cultured B- COli:
Recently
we reported that the full-length heme oxygenase expressed in a. coli cells actually degraded endogenous heme and so the E. coli cells accumulated a significant amount of biliverdin ( 10). Therefore we treated the soluble fractions of the cultured cells with chloroform under acidic conditions to extract and
recorded the absorption spectra of the extracts
(Fig. 2).
biliverdin
The extracts of
cells expressing H25A or H132A showed no significant absorption in the range of 600-700 nm, whereas the extracts of cells expressing enzymatically active heme oxygenases showed the characteristic spectrum of biliverdin. These spectral studies and those on the activities
of the mutant enzymes
suggest that His25 plays an essential role in the heme oxygenase reaction. Spectral examinations with the purified H25A mutant protein and heme are needed to determine whether His25 functions as a heme-binding ligand.
The activities
of
the two mutants H84A and H119A for heme breakdown were only about 40% of that of the wild-type enzyme. These low activities suggest that substitution of His84 or His119 influences the structure of the interaction of the enzyme with P-450 reductase.
active center and/or the The amino acid sequence
corresponding to residues 126 to 154 of the rat heme oxygenase is conserved in the other three species examined so far (8). Therefore, His132 is likely to play a key role in substrate binding. But in the present study we could not confirm this because expression of the H132A mutant in E. coli was very low. To investigate this possibility, we are currently purifying the H132A protein from the supernatant of E.
coli
expressing the H132A mutant. 985
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS
This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 02680150 and 03670137) from the Ministry of Education, Science and Culture of Japan. REFERENCES
1. Tenhunen, R., Marver, H.S., and Schmid, R. (1969) J. Biol. Chem. 244, 63886394. 2. Yoshida, T., and Kikuchi, G. (19781 J. Biol. Chem. 253, 4224-4229. 3. Yoshida, T., and Kikuchi, G. (1979) J. Biol. Chem. 254, 4487-4491. 4. Yoshinaga, T., Sassa, S., and Kappas, A. (1982) J. Biol. Chem. 257, 7794-7802. 5. Shibahara, S., MUller, R.M., Taguchi, H., and Yoshida, T. (1985) Proc. Natl. Acad. Sci. USA 82, 7865-7869. 6. Yoshida, T., Biro, P., Cohen, T., Muller, R.M., and Shibahara, S. (1988) Eur. J. Biochem. 171, 457-461. 7. Kageyama, H., Hiwasa, T., Tokunaga, K., and Sakiyama, S. (1988) Cancer Res. 48, 4795-4798. 8. Evans, C-O., Healey, J.F., Greene, Y., and Bonkovsky, H.L. (1991) Biochem. J. 273, 659-666. 9. Yoshida, T., Ishikawa, K., and Sato, M. (1991) Eur. J. Biochem. 199, 729-733. 10. Ishikawa, K., Sato, M., and Yoshida, T. (1991) Eur. J. Biochem. 202, 161-165. 11. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 981-986
IMPORTANCE OF HISTIDINE RESIDUE 25 OF RAT HEME OXYGENASE FOR ITS CATALYTIC ACTIVITY Kazunobu Ishikawa, Michihiko S&o*, Mariko Ito, and Tadashi Yoshida Department of Biochemistry and*Department of Molecular and Pathological Biochemistry, Yamagata University School of Medicine, Yamagata 990-23, JAPAN Received
December
26,
1991
Summary: A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction. 0 1992 Academic Press,Inc.
Microsomal heme oxygenase catalyzes oxidative degradation of heme to biliverdin with NADPH-cytochrome P-450 reductase, which functions as an electron donor (1). Heme oxygenase is not a hemeprotein, but the substrate-enzyme complex formed by its stoichiometric binding with ferric heme behaves as a kind of hemeprotein (2, 3).
The spectral resemblance of the heme-heme oxygenase
complex to hemoglobin suggests that the heme is linked to histidine residues of the heme oxygenase protein through both the fifth and sixth coordination positions of its iron atom.
The finding that
diethylpyrocarbonate,
a modifier of histidine
residues, strongly inhibits the heme oxygenase activity also supports the possibility that a histidine residue plays a critical role in the heme oxygenase reaction (4). On the basis of these findings we expected that substitution of histidine residues essential for heme binding would eliminate heme oxygenase activity. cDNAs for rat (5), human (6), mouse (7), and chicken (8) heme oxygenase have been cloned. From the sequence homology among these cDNAs, His25, 84, 119, and 132 of the rat enzyme have been proposed to be heme-binding ligands in heme To examine these possibilities, we carried out site-directed oxygenase. mutagenesis at each of these four residues and determined the activities of the mutated enzymes. MATERIALS AND METHODS restriction Chemicals and materials The following compounds were used: endonucleases, DNA polymerase I (Klenow fragment), Tth DNA polymerase, T4 ooo6-291x/92
981
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
polynucleotide kinase, sequencing primers for pTVl18N, P8(5’-AGCGGATAACAATTT CACACAGGAAAC-3’) and P7(5’-CGCCAGGGTTTTCCCAGTCACGAC-3’), and calf intestinal alkaline phosphatase from Toyobo; phosphorylated linker of XbaI (10 mer) from New England Biolabs; T DNA ligase from Boehringer; Sequenase version 2.0 from US Biochem.; prokaryo%1c expression vector, pKK233-2, from Pharmacia; E. coli strain JM109 from Stratagene; isopropyl B-D-thiogalactopyranoside and expression vector pTV118N from Takara; ampicillin, chlorhemin and bovine serum albumin from Sigma; nitrocellulose membranes from Schleicher & Schiill, and peroxidase-conjugated sheep IgG against rabbit IgG from Cappel. Oligonucleotides were synthesized with an Applied Biosystems model 381A DNA synthesizer and purified in a Hitachi 638-30 HPLC system. The procedures used for preparing native heme oxygenase, a 28-kDa tryptic peptide of heme oxygenase, NADPH-cytochrome P-450 reductase, biliverdin reductase and polyclonal antibodies for heme oxygenase were as described in our recent papers (9, IO). Construction of expression plasmid carrying a truncated rat heme oxygenase cDNA: For an other purpose we have made pKK-ARHO, which carries a truncated cDNA for rat heme oxygenase lacking a coding sequence of a membraneassociated, C-terminal region. In the present study we used a pTV118N construct carrying the same coding sequence as pKK-ARHO, because primers for pTVl18N (Pi’ and P8) used for the polymerase chain reaction and sequencing are commercially available. The method for construction of pKK-ARHO was similar to that for construction of pKK-RHO (IO). Briefly, pKK233-2 was linearized by digestion with Ncol and the single stranded ends were filled in by treatment with Klenow fragment followed by dephosphorylation with calf intestinal alkaline phosphatase. A cDNA clone encoding the rat heme oxygenase gene, pRHO1 (51, was cut with XhoI and SpeI. The resulting DNA fragment (XhoI/SpeI, -59/782) was filled in with Klenow fragment, ligated with IO mer-XbaI linker so that TAG in the linker functioned as a stop codon, and then cleaved with XbaI. The resulting fragment was filled in and then partially cut with TaqI. This DNA fragment (TaqI/XbaI, 18/793) was used as a truncated cDNA. A synthetic 14-residue nucleotide (5’GAGCGCCCACAGCT3’) was phosphorylated with T4 polynucleotide kitiase and then annealed with a synthetic 16-residue nucleotide (3’CTCGCGGGTGTCGAGC5’) to form a linker DNA bridging the gap between the NcoI site of pKK233-2 and TaqI site of the truncated cDNA. Finally, the three DNA fragments were ligated to construct pKK-ARHO. For preparation of the pTV118N construct (pTV-ARHO), pKK-ARHO was partially cleaved with NcoI and HindIII. The resulting DNA fragment (NcoI/HindIII, -l/807) was ligated with pTV118N plasmid which had been pretreated with NcoI and HindIII. pTV-ARHO encodes a truncated, 30-kDa heme oxygenase consisting of amino acid residues 1 to 261 of the native enzyme with two replacements; namely, serine residues 262 and 263 of the native enzyme are replaced by arginine and leucine, respectively. Mutagenesis of a truncated cDNA for rat heme oxygenase by the polymerase chain reaction: Template DNA for site-specific mutagenesis was prepared by digestion of pTV-ARHO with PvuII. The resultant DNA fragment (1060 bp) contained the whole coding region of the truncated heme oxygenase and restriction sites for NcoI and Hind111 near the 5’ and 3’ end, respectively. Two contiguous regions of the template were amplified in two separate polymerase chain reactions. Table I shows the mutagenic and flanking primers used for each site-directed mutagenesis reaction. The primers used for amplification of the 5’-side region were P8 and the flanking primer. The mutagenic primer and P7 were used for the 3’-side region. The amplification reaction was performed in 100 ul of reaction mixture containing 0.5 ug of template DNA, 20 pmol of each primer, 0.2 mM dNTPs, and 2.5 units of Tth polymerase. The reaction was cycled 25 times in a HYBAID thermal reactor HB-TR 1. The amplified DNA fragments were purified with polyacrylamide gel electrophoresis and then the fragments of the 5’ and 3’ regions were cleaved with NcoI and HindIII, respectively. After phosphorylation of the 5’-end, the DNA fragments were ligated to pTV118N which had been pretreated with NcoI and HindIII. The resulting mutant plasmids were transfected into E. coli strain JM109. The complete nucleotide sequences of the inserts were 982
Vol.
182,
No.
3, 1992
BIOCHEMICAL
Table I.
Mutation
Summary used for
Flanking
AND
BIOPHYSICAL
of mutagenic site-directed
RESEARCH
and flanking mutagenesis
primer
primers
Mutagenic
H25A
COMMUNICATIONS
primer
5’-6&AGGAGGTGGCCATCCGTGC;23’ 3’-?TCCGGAACTTCCTCCGGTG&5’
H84A
220 3’-ATACGGGGCGAGATGAAGGGA-5’
5 ‘;$‘AGGAGCTGGCCCGAAGGGC?3
H119A
325 3’-GGTCGGTGTGTCGTGATGCAT-5’
5’;;$AGCGTCTCGCCGAGGTGGG;P-63’
H132A
364 3’-CCTCCATGAGTAGGACTCGAC-5’
385 5’&TGGTGGCCGCCGCATATACC-3’
The numbering of nucleic acids begins with the A of the ATG of rat heme oxygenase. The CAC codon for histidine alanine. Mutant codons are underlined.
confirmed
by
Other
the
procedures:
E. coli, immunoblot from the soluble recent papers (9,
’
405
initiation methionine codon was converted to GCC for
dideoxynucleotide chain-termination method (11). Other methods, including expression of heme oxygenase in analysis, assay of heme oxygenase, and extraction of biliverdin fraction of cultured E. coli, were the same as described in our 10).
RESULTS AND DISCUSSION Expression
in B. coli
sequence heme
for
coli of
region
heme
(10).
proteins,
we A
soluble
(lane
fraction truncated
We
observed
that
had
biliverdin
to
heme 1
in in had
heme
in
with often
oxygenase 1). presence
activity
for
oxygenase
heme in
an
place
heme as
a
heme
few
degraded
breakdown
in
soluble,
30-kDa
(Table
heme
system.
E.
coli. protein
activity and
of
found
that
Therefore,
II).
to
membrane-bound
oxygenase
full-length
to
ability
reductase of
system
of
retain
oxygenase
reductase
the
binds
C-terminal
to
that
rat
that
form
P-450
than
the
of
found
added
expressed
P-450
a
degraded
was
easier
measured of
a
the coding
expressing
membrane-associating,
soluble
was We
in form
cultivation,
30-kDa
a truncated,
Fig. the
is
succeeded
enzymes,
about
lacking
full-length
during
their
concert
proteins
express
that lost
of
we active,
soluble
mass
of heme oxygenase
Recently
a catalytically
these
soluble
tried
fraction
soluble
to
truncated
coli
segment:
a molecular
of
cDNA
as
Among
heme
treatment
coli
oxygenase
with
decompose
the
E.
appeared.
oxygenase
As
in
membranes
forms
E.
a hydrophobic
oxygenase
E.
of a truncated
enzyme
in
we the
in the this use
present
study.
Expression histidines To
determine
and activity are
conserved which
of mutated among
histidine
the residues
heme oxygenase: rat,
human, are
983
As
mouse
essential
and for
described chick
heme
heme oxygenase
above, oxygenases. activity,
four
Vol.
182, No. 3. 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,‘i’:,:;.
Expressions of a truncated wild-type heme oxygenase and its mutants in . Soluble fractrons (100 ug as protein) of E. coli harvested 6 hr after the addition of isopropyl 8 -D-galactopyranoside were used. Lane 1, truncated wild-type heme oxygenase; lane 2, H25A; lane 3, H84A; lane 4, H119A; lane 5, H132A.
we
prepared
pTV118N
in which these histidine residues were replaced
constructs
individually by an alanine residue by site-directed mutagenesis. Bacteria
harboring
these
express proteins reacting Western blot oxygenase. demonstrated that
mutated
with
recombinants
polyclonal
analyses
of
the
were
tested
for
antibodies against rat soluble
fractions
of
ability
liver E. coli
to
heme cells
all the mutated enzymes except H132A (His132 + Ala) were
expressed to similar extents to the wild-type enzyme (Fig. 1).
The low expression
of the H132A mutant was confirmed by immunoblot analysis of whole sonicates of E. coli expressed
cells (data not shown), eliminating the possibility that the H132A protein The reason for its low expression was sequestered in inclusion bodies.
is unknown. The activities
of equal amounts of protein of the respective soluble fractions for heme breakdown were compared (Table II). The mutant heme oxygenases H84A and H119A exhibited 41 and 39%, respectively, of the wild heme oxygenase activity.
In contrast, the H25A mutant showed almost no activity,
His25 is essential for
the heme oxygenase reaction.
suggesting that
The H132A mutant also
exhibited extremely low activity, but it was difficult to draw any conclusion from this finding about the role of H132 because expression of the H132A mutant protein was so low.
Table
Wild-type H25A H84A H119A H132A
II.
Comparison wild-type
of heme oxygenase activities and mutant enzyme Heme oxygenase activity nmole bilirubin formed (mg protein) -‘h-r 66.2 0.5 27.1 26.1 1.0
984
of
% 100 20 41 39 1
Vol.
182,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Wild-type
500
400
WAVELENGTH
600
700
(t-am)
Fie. 2. Absorption spectra of chloroform extracts from soluble fractions of E. coli expressingtruncated wild-type hemeoxygenaseand its mutants.
Extraction
of biliverdiu from the soluble fraction
of cultured B- COli:
Recently
we reported that the full-length heme oxygenase expressed in a. coli cells actually degraded endogenous heme and so the E. coli cells accumulated a significant amount of biliverdin ( 10). Therefore we treated the soluble fractions of the cultured cells with chloroform under acidic conditions to extract and
recorded the absorption spectra of the extracts
(Fig. 2).
biliverdin
The extracts of
cells expressing H25A or H132A showed no significant absorption in the range of 600-700 nm, whereas the extracts of cells expressing enzymatically active heme oxygenases showed the characteristic spectrum of biliverdin. These spectral studies and those on the activities
of the mutant enzymes
suggest that His25 plays an essential role in the heme oxygenase reaction. Spectral examinations with the purified H25A mutant protein and heme are needed to determine whether His25 functions as a heme-binding ligand.
The activities
of
the two mutants H84A and H119A for heme breakdown were only about 40% of that of the wild-type enzyme. These low activities suggest that substitution of His84 or His119 influences the structure of the interaction of the enzyme with P-450 reductase.
active center and/or the The amino acid sequence
corresponding to residues 126 to 154 of the rat heme oxygenase is conserved in the other three species examined so far (8). Therefore, His132 is likely to play a key role in substrate binding. But in the present study we could not confirm this because expression of the H132A mutant in E. coli was very low. To investigate this possibility, we are currently purifying the H132A protein from the supernatant of E.
coli
expressing the H132A mutant. 985
Vol.
182, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS
This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 02680150 and 03670137) from the Ministry of Education, Science and Culture of Japan. REFERENCES
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