(Received for publication, October 24, 1994; and in revised form, December 14, 1994)
From the
The gene coding for histamine oxidase has been cloned and sequenced from a Coryneform bacterium Arthrobacter globiformis. The deduced amino acid sequence consists of 684 residues with a calculated molecular mass of 75,109 daltons and shows a high overall identity (58%) with that of phenethylamine oxidase derived from the same bacterial strain. Although the sequence similarities are rather low when compared with copper amine oxidases from other organisms, the consensus Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the quinone cofactor (the quinone of 2,4,5-trihydroxyphenylalanine, topa) covalently bound to this class of enzymes, is also conserved in the histamine oxidase sequence.
To identify the quinone cofactor, an overexpression plasmid has been constructed for the recombinant histamine oxidase. The inactive enzyme purified from the transformed Escherichia coli cells grown in a copper-depleted medium gained maximal activity upon stoichiometric binding of cupric ions. Concomitantly with the enzyme activation by copper, a brownish pink compound was generated in the enzyme, which was identified as the quinone of topa by absorption and resonance Raman spectroscopies of the p-nitrophenylhydrazine-derivatized enzyme and found at the position corresponding to the precursor Tyr (Tyr-402). Therefore, the copper-dependent autoxidation of a specific tyrosyl residue operates on the formation of the topa quinone cofactor in this enzyme, as recently demonstrated with the precursor form of phenethylamine oxidase (Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., and Tanizawa, K.(1994) FEBS Lett. 351, 360-364).
Copper-containing amine oxidases (EC 1.4.3.6) are generally
composed of two identical subunits with a molecular weight ranging from
70,000 to 105,000 and catalyze the oxidation of various biogenic
primary amines to the corresponding aldehydes, ammonia, and hydrogen
peroxide(1, 2) . Besides copper, the enzymes were also
known to contain a covalently bound organic cofactor with a carbonyl
function, whose structure in the bovine plasma enzyme has recently been
identified as the quinone of 6-hydroxydopa
(2,4,5-trihydroxyphenylalanine; topa), residing in the
polypeptide chain (3) . This novel cofactor has thereafter been
shown to occur ubiquitously in the enzymes of both eukaryotes (4, 5, 6) and prokaryotes (4, 7) . Comparison of the sequences of the topa
quinone-containing peptides isolated proteolytically with those deduced
from the nucleotide sequences of the coding genes has revealed a
tyrosine codon in the position of topa quinone(5, 8) ,
implying that the cofactor is generated co- or post-translationally
from a specific tyrosyl residue occurring in the highly conserved
sequence, Asn-Tyr-Asp/Glu(6) .
However, the mechanism by which the topa quinone cofactor is generated in vivo has been hitherto unknown, although participation of an external system with a tyrosine hydroxylase-like or tyrosinase-like enzyme has been suggested together with a possibility of self-processing assisted by copper bound close to the active site of the enzymes(5, 9) . More recently we have demonstrated, using the copper/topa quinone-less precursor form of the recombinant phenethylamine oxidase from Arthrobacter globiformis(10) , the topa quinone generation by copper-dependent autoxidation of a specific tyrosyl residue(11) . It was thus elucidated that the post-translational conversion of the precursor Tyr to topa quinone requires, at least in vitro, no external enzymic systems except for the prosthetic metal ion.
The Coryneform bacterium A. globiformis produces two copper amine oxidases, phenethylamine oxidase and histamine oxidase, when grown on phenethylamine and histamine as an inducer, respectively(12, 13) . However, genetic and structural differences of the two enzymes are unknown. The purpose of the present investigation is to elucidate the structural relationship between phenethylamine and histamine oxidases induced separately in the same bacterial strain and to examine whether the copper-dependent topa quinone generation is common to histamine oxidase. We have cloned and sequenced the gene of the inducible histamine oxidase from A. globiformis and overproduced the recombinant enzyme in Escherichia coli cells. Despite the fact that the deduced primary structure of the enzyme is considerably similar to that of phenethylamine oxidase cloned and sequenced previously(10) , the two enzymes share no immunochemical cross-reactivities. Furthermore, the copper-free inactive histamine oxidase, like the precursor form of phenethylamine oxidase(11) , can be markedly activated by incubation with cupric ions, and the topa quinone cofactor is indeed generated at the position corresponding to the conserved Tyr (Tyr-402), corroborating the copper-dependent autoxidation of a specific tyrosyl residue as a common mechanism for the biogenesis of the topa quinone cofactor in bacterial amine oxidases.
Figure 1: Nucleotide sequence and deduced amino acid sequence of histamine oxidase from A. globiformis. Nucleotides are numbered in the 5` to 3` direction beginning at the first base of the initiator ATG codon, and the nucleotides on the 5` side of base 1 are numbered with a negative sign. The deduced amino acid sequence (numbered from Met at the amino terminus) is shown below the nucleotide sequence using single-letter code. The amino-terminal and 8 peptide sequences determined protein-chemically are underlined. The consensus sequence Asn-Tyr-Asp is boxed, and the precursor Tyr-402 to topa quinone is circled (see text). A potential ribosome-binding site (underline) and a stem-loop structure possibly serving as a transcriptional terminator (arrows) are also shown.
The cells grown in the
absence of CuSO were suspended in 50 mM potassium
phosphate buffer (pH 6.8) containing 1 mM concentration each
of EDTA and N,N-diethyldithiocarbamate (buffer A),
supplemented with 0.1 mg/ml each of phenylmethylsulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone, and then
disrupted at 4 °C by ultrasonic disintegration. For the cells grown
in the presence of CuSO
, buffer A was not supplemented with
EDTA and N,N-diethyldithiocarbamate but added with
0.05 mM CuSO
until the step of ammonium sulfate
fractionation. The resulting lysate was centrifuged at 25,000
g for 30 min, and the supernatant solution was fractionated
with ammonium sulfate (20-50% saturation). The precipitates by
50% saturation of ammonium sulfate were dissolved in a minimum volume
of buffer A and dialyzed overnight against 2 liters of buffer A. The
enzyme solution was applied to a DEAE-Toyopearl column (70 ml) equipped
on a Pharmacia fast protein liquid chromatography system and
equilibrated with buffer A. The column was washed thoroughly with
buffer A, and the bound proteins were eluted with a 120-min linear
gradient of 0.1-0.25 M KCl in the same buffer at a flow
rate of 2 ml/min. The active fractions were pooled and concentrated by
ultrafiltration through a UK-10 membrane (Advantec). The enzyme
solution was then added with solid ammonium sulfate to 20% saturation
and applied to a Phenyl-Toyopearl column (25 ml) pre-equilibrated with
22% saturated ammonium sulfate in buffer A. The bound proteins were
eluted with an 80-min linear gradient of 22-0% saturation of
ammonium sulfate at a flow rate of 1.5 ml/min. The enzyme eluted at
about 9% saturation of ammonium sulfate was pooled, concentrated by
ultrafiltration, and dialyzed against buffer A. Finally, the enzyme was
purified by twice repeating the anion exchange chromatography with a
Resource Q (Pharmacia) column (6 ml), first eluted by a 60-min linear
gradient of 0-0.3 M NaCl in 30 mM potassium
phosphate buffer (pH 6.8), and second eluted by a 60-min linear
gradient of 0-0.15 M NaCl in 50 mM sodium
acetate buffer (pH 5.0), both containing 1 mM EDTA and 1
mMN,N-diethyldithiocarbamate for
purification of the Cu
-deficient inactive enzyme. The
enzyme thus purified to homogeneity as judged by SDS-polyacrylamide gel
electrophoresis was dialyzed thoroughly against 50 mM HEPES
(pH 6.8) and stored at -20 °C until use.
The peptide mixture was separated on a
Tosoh high performance liquid chromatography system equipped with a
preparative reverse phase column (Cosmosil 5C18-300, 0.8
25 cm) using a solvent system of 0.32% (v/v) triethylamine acetate (pH
7.0) (A) and 0.32% triethylamine acetate containing 90% acetonitrile
(B), as reported previously(6, 9) . A 60-min linear
gradient from 0 to 60% B was used to elute peptides at a flow rate of
0.5 ml/min with continuous monitoring of the absorbance at 215 nm
(peptide absorbance) and at 380 nm
(cofactor-p-nitrophenylhydrazone absorbance). The p-nitrophenylhydrazone of the topa quinone peptide was further
purified using an analytical column (Vydac C18) with a shallower
gradient of acetonitrile concentration. The sequence of the peptide
fragment purified was determined with a gas-liquid phase protein
sequencer. The p-nitrophenylhydrazone of the topa quinone
hydantoin model compound (3) was synthesized and purified
according to the previously published
procedures(6, 20) .
A single
open reading frame starting with an initiation codon, ATG, and
terminating in a nonsense codon, TGA, at 2053 bases 3`-downstream from
the 1st base of the ATG codon was found (Fig. 1). The coding
region was also located and oriented on the basis of the amino-terminal
amino acid sequence determined for the A. globiformis enzyme,
although the amino-terminal Met had been removed in the enzyme.
Furthermore, all the partial amino acid sequences determined
protein-chemically were found in the deduced sequence (Fig. 1, underlines). A potential ribosome-binding site (AGGAAG) was
identified 5 bp 5`-upstream from the start codon. A stem-loop structure
with an 8-base pairing stem and a 14-base loop (-29.5 kcal
(approximately -123 kJ) mol) was identified 28
bp 3`-downstream from the translational stop codon, which may serve as
a transcriptional terminator (Fig. 1). All these features
support the view that the open reading frame found encodes the
polypeptide of histamine oxidase, consisting of 684 amino acid residues
with a calculated molecular mass of 75,109 daltons. The coincidence of
the sequence in the amino-terminal region determined for the purified
protein with that deduced from the nucleotide sequence confirms the
cytoplasmic localization of the enzyme(13) , unlike the enzymes
from Gram-negative bacteria occurring in the periplasmic space of the
cell membrane with a signal peptide(21, 22) . On the
other hand, the internal sequence from Asn-401 to Asp-403 agrees with
the conserved Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the
precursor to the topa quinone cofactor covalently bound to copper amine
oxidases from various
sources(5, 6, 8, 9) .
We previously found that the production of
an active quinone-containing form of the recombinant phenethylamine
oxidase is markedly dependent on the presence of Cu ions in the culture medium for the transformed E. coli cells and that an inactive Cu
-deficient
precursor form is produced by cultivation in the
Cu
-depleted medium(10) . Therefore, to
prepare both of the Cu
-containing active and
Cu
-deficient inactive forms of histamine oxidase, we
first cultivated E. coli JM109 cells carrying
pTrc99HAO in a Cu
-depleted medium, then
induced the enzyme expression by addition of
isopropyl-1-thio-
-D-galactopyranoside, and further
cultivated the cells in the presence or absence of CuSO
.
The two forms thus produced were separately purified in the presence of
CuSO
and a strong Cu
-chelating agent, N,N-diethyldithiocarbamate, respectively. The results
of those purification runs are summarized in Table 1; in the
purification of the inactive form, the purification steps were
monitored by SDS-polyacrylamide gel electrophoresis or by measuring the
enzyme activity after incubation with excess Cu
as
described below. The active and inactive enzymes, both purified to
>98% homogeneity on SDS-polyacrylamide gel electrophoresis (not
shown), had specific activities of 9.0 and 0.059 units/mg of protein
and contained 0.56 and <0.01 mol atom of Cu
/mol of
enzyme subunit (M
= 75,000) when measured
by atomic absorption analysis, respectively. Thus, the inactive enzyme
is almost free from the bound copper. The
Cu
-containing active enzyme concentrated to >10
mg/ml exhibited a brownish pink color with an absorption maximum around
500 nm, whereas the Cu
-free inactive enzyme was
colorless (Fig. 2).
Figure 2:
Absorption spectra of the purified
Cu-free inactive enzyme and its
Cu
-activated form. The spectra were taken with
protein samples of 16.7 mg/ml for the Cu
-free
inactive enzyme(- - -) and 18.1 mg/ml for the
maximally activated enzyme (-), prepared in 50 mM HEPES (pH 6.8).
The Cu-containing
active enzyme oxidized histamine as the most preferred substrate with a K
value of 0.06 mM, being consistent with
the finding that the enzyme is inducibly produced by A. globiformis grown on histamine and probably participates in the degradation of
histamine as a carbon and nitrogen source(2, 13) .
Other amines including phenethylamine (relative activity, 72% of
histamine), tyramine (69%), tryptamine (13%), putrescine (8.1%), and
benzylamine (0.5%), each at 0.1 mM, also served as substrate
for the enzyme, although the former two caused very strong substrate
inhibition above 0.2 mM (data not shown).
Both the
Cu-containing active and Cu
-free
inactive enzymes reacted with the rabbit antiserum raised against the
purified histamine oxidase (Cu
-containing active
form) in an Ouchterlony double diffusion analysis to form precipitation
lines completely fused with each other (Fig. 3). However, they
did not react with the antiserum raised against the purified
phenethylamine oxidase (Cu
-containing active form)
from A. globiformis(11) . Similarly, both the
Cu
-containing active and
Cu
-deficient inactive forms of phenethylamine oxidase (11) reacted with the antiserum against the same enzyme but not
with the antiserum against histamine oxidase. These results indicate
that the Cu
-containing active and
Cu
deficient inactive forms of either enzyme have the
same antigenic structures on the protein surface, whereas the two
enzymes of the same bacterial strain are immunochemically distinct from
each other, even though they show sequence identities of about 60% as
described below.
Figure 3:
Ouchterlony double diffusion analysis of
the recombinant histamine and phenethylamine oxidases. Center wells were filled with the rabbit antisera (15 µl each) raised
against the purified recombinant phenethylamine oxidase (left)
and histamine oxidase (right). Peripheral wells were
filled with the enzyme solutions (about 20 µg of each protein): well 1, the Cu-containing active
phenethylamine oxidase; well 2, the
Cu
-deficient inactive phenethylamine oxidase; well 3, the Cu
-containing active histamine
oxidase; well 4, the Cu
-free inactive
histamine oxidase.
The
Cu-dependent activation of the
Cu
-free inactive enzyme was accompanied by brownish
pink coloring of the enzyme solution with an absorption maximum at
about 500 nm (Fig. 2), which is attributable to the formation of
a quinone compound by analogy with the Cu
-dependent
topa quinone generation in phenethylamine oxidase(11) . On
anaerobic incubation of the enzyme with Cu
, the
spectral change did not take place at all, in agreement with the
requirement of the dissolved oxygen for the formation of the quinone
cofactor(11) . The molar absorption coefficient (
) of the quinone formed in the
fully activated enzyme was calculated to be about 1,600 M
cm
at 498 nm in 50
mM HEPES (pH 6.8), assuming that each polypeptide (M
= 75,000) contains 1 mol of the quinone.
This value is consistent with those of phenethylamine oxidase (
= 1,400 M
cm
at
475 nm) (11) and bovine serum amine oxidase (
=
1,700 M
cm
at 476 nm;
of 1,900 M
cm
reported for subunit M
of 95,000 (24) has been corrected for M
of 82,800
plus 4% carbohydrate(8) ). It is noted that the
of the quinone in histamine oxidase is red-shifted by about 22 nm
as compared with those of phenethylamine oxidase and bovine serum
enzyme. The quinone contents in various preparations of histamine
oxidase calculated from 498 nm absorption with the
value roughly corresponded with the
enzyme activities and copper contents (data not shown), suggesting the
necessity of copper binding for proper generation of the quinone
cofactor.
Figure 4:
Titration of the
Cu-activated histamine oxidase with phenylhydrazine.
The maximally activated enzyme (13.3 µM subunit) dissolved
in 50 mM HEPES (pH 6.8) was titrated with a freshly prepared
solution (1 mM) of phenylhydrazine to final concentrations of
0, 3.0, 4.5, 6.0, 7.0, 8.0, 9.0, and 12.0 µM,
respectively. Each spectrum was recorded after a 10-15-min
incubation at 30 °C following each addition, and the remaining
activity was determined with histamine as substrate. The arrow indicates the direction of spectral change. The titration curves
for spectral change at 438 nm (
) and percent remaining activity
(
) are shown in the inset.
Figure 5:
Isolation of quinone-containing peptide
from a thermolytic digest of the p-nitrophenylhydrazine-derivatized,
Cu-activated enzyme. The chromatographic conditions
are described in the text. The absorbances at 215 nm (peptide
absorbance) (A) and at 380 nm (p-nitrophenylhydrazone
absorbance) (B) were continuously monitored. The peak of the
quinone-containing peptide is indicated with arrows.
Both the p-nitrophenylhydrazine-derivatized enzyme and the
quinone-containing peptide obtained by thermolysin digestion showed an
absorption maximum at about 460 nm at neutral pH and at about 580 nm at
alkaline pH (in 2 M KOH) (Fig. 6). Essentially
identical spectra were obtained with the p-nitrophenylhydrazone of the topa quinone hydantoin model
compound with an absorption maximum at 455 nm at neutral pH and at 572
nm at alkaline pH. The p-nitrophenylhydrazone of the
Cu-free inactive enzyme had no absorption in the
visible wavelength region (not shown). The pH-dependent changes in
of about 120 nm have been shown to be
characteristic to the topa quinone derivative, the p-nitrophenylhydrazones of pyridoxal phosphate and
pyrroloquinoline quinone revealing quite different spectral
behavior(6) .
Figure 6:
Visible absorption spectra of p-nitrophenylhydrazone adducts. Spectra of the p-nitrophenylhydrazone derivatives of the
Cu-activated enzyme (28 µM subunit) (A), the quinone-containing peptide obtained from thermolytic
digests (arbitrary concentration) (B), and the topa quinone
hydantoin model compound (C) were measured in 50 mM HEPES (pH 6.8) (-) or in 1 M KOH (-
- -).
In the last experiment to unequivocally
identify the structure of the Cu-generated quinone
cofactor, resonance Raman spectra were measured for the p-nitrophenylhydrazones of the Cu
-activated
enzyme, the quinone-containing peptide, and the model hydantoin. As
shown in Fig. 7, the relative intensity of signals and their
peak positions can be virtually superimposed for the three samples,
establishing the presence of topa quinone in the
Cu
-activated enzyme. We thus conclude that the topa
quinone cofactor in histamine oxidase is produced by
Cu
-dependent autoxidation of a specific tyrosyl
residue (Tyr-402), as has been shown first with phenethylamine oxidase
from the same bacterial strain(11) .
Figure 7:
Resonance Raman spectra for the p-nitrophenylhydrazone of the Cu-activated
enzyme (A), the quinone-containing peptide (B), and
the topa quinone hydantoin model compound (C). Spectra were
obtained upon excitation by the 457.9 nm line from an argon laser with
a laser power of about 30 milliwatts at the sample position. Frequency
calibration was based on the Raman spectra of acetone, indene, and
ethyl acetate, and estimated frequency errors were ±2
cm
for well-resolved bands; baseline drifts due to
the intense fluorescence by the chromophore have been uncorrected. The
structure of the p-nitrophenylhydrazone of topa quinone
hydantoin is also shown.
In this study we have cloned and sequenced an A.
globiformis gene encoding histamine oxidase, which is produced by
the bacterium grown on histamine as an inducer(13) . Although
this bacterium produces another copper amine oxidase, phenethylamine
oxidase, when grown on phenethylamine as an inducer(12) , our
previous (10) and present studies clearly show that the two
amine oxidases are the distinct gene products without immunochemical
cross-reactivities and showing different substrate specificities.
Combined with the recent reports on cloning and sequencing of the amine
oxidase genes from various
sources(8, 21, 22, 28, 29, 30, 31) ,
it is therefore assumed that a single species of various types of
organisms possesses multiple structural genes for the amine oxidases.
Indeed, two genes designated maoxI and maoxII, which
are greater than 99% homologous in the open reading frames but quite
different in the 5`- and 3`-flanking regions, were previously isolated
from a Gram-positive methylotrophic bacterium Arthrobacter strain P1, although maoxII alone was shown to encode
methylamine oxidase(28) . Similar duplication of the structural
genes has been reported for monoamine oxidase of E. coli K-12(22) . The product of a gene named maoA was identified as monoamine oxidase acting on tyramine and
phenethylamine as preferred substrate and showing a high sequence
similarity with the same enzyme (product of maoA
)
from Klebsiella aerogenes(21) , although the
nucleotide sequence and the product of another E. coli gene
named maoX, which is located close to maoA
, remain unknown(22) .
Furthermore, eukaryotic organisms also have at least two genes for copper amine oxidase; a methylotrophic yeast Hansenula polymorpha produces both methylamine oxidase (9) and benzylamine oxidase (5) in the presence of a single type of amine inducer in cultures of the yeast, although only the gene for the former enzyme has been cloned and sequenced(29) . In mammals as well, two classes of copper amine oxidases are known; the cellular amine oxidase, also called diamine oxidase (or histaminase) (cf.(2) ), being distributed in tissues such as kidney and placenta and probably involved in the regulation of histamine and polyamine levels, has been shown recently to be identical with the amiloride-binding protein(8, 32) , whose cDNA was cloned and sequenced previously(30) . The gene for the serum amine oxidase, probably participating in the control of the level of circulating biogenic amines such as dopamine and phenethylamine, has also been cloned recently from a liver cDNA library (8) .
The complete
sequences of 9 copper/topa quinone-containing amine oxidases were
compared based on the Clustal analysis made previously for the
sequences of 4 enzymes(28) . The residues totally conserved in
the 9 sequences aligned (not shown) were counted up to only about 30
scattering throughout the whole region, whereas pairwise comparisons
using the sequence of the A. globiformis histamine oxidase
(this study) as a reference gave identity scores of 58% with the
phenethylamine oxidase of A. globiformis(10) , 46%
with the methylamine oxidase of Arthrobacter P1 (product of maoxII)(28) , 30% with the tyramine oxidase of K.
aerogenes (product of maoA)(21) , 29%
with the monoamine oxidase of E. coli (product of maoA
)(22) , 33% with the methylamine
oxidase of Hansenula polymorpha(29) , 25% with the
bovine serum amine oxidase(8) , 22% with the human kidney
amiloride-binding protein (identical with the cellular amine
oxidase)(30) , and 22% with the lentil (Lens
culinaris) seedling diamine oxidase(31) . Therefore, the
overall percent similarity between histamine oxidase of A.
globiformis and each of the other enzymes is not very high, except
for the significantly high similarity between histamine and
phenethylamine oxidases from the same bacterial strain.
However, a part of the alignments, in particular the middle-to-carboxyl-terminal portion of each sequence containing the consensus sequence Asn-Tyr-Asp/Glu for topa quinone(6) , is contiguously homologous (Fig. 8), as pointed out previously(8, 22, 28, 32) . Besides the consensus sequence, a histidine 25-30 residues from Asn of the consensus sequence toward the amino terminus and 2 histidines in a His-X-His motif 46-58 residues from Asp/Glu of the consensus sequence toward the carboxyl terminus are conserved in all alignments and hence are most likely the 3 histidines proposed as ligands to copper in the amine oxidases (33, 34, 35) . Further inspection of this region revealed other totally conserved residues; 1 Arg, 1 Tyr, 3 Asp, 5 Gly, 1 Glu, 1 Pro, 1 Thr, and 1 Asn (Fig. 8). Charged or hydrogen bond-forming residues among these could serve as an acid/base catalyst postulated from kinetic studies (25, 36, 37) or those interacting with the quinone cofactor (38) , while others may have structural roles in constituting the active site. Site-specific mutagenesis of these conserved residues including the 3 histidines of copper ligands and each residue in the consensus Asn-Tyr-Asp/Glu sequence would provide valuable information on the structure-function relationship of the copper amine oxidases, as exemplified by our recent studies revealing that the replacement by Phe of the precursor Tyr to topa quinone of phenethylamine oxidase results in the complete loss of enzymatic activity and the inability of the topa quinone formation(10, 11) .
Figure 8: Sequence comparison near the active site of prokaryotic and eukaryotic copper amine oxidases. The sequences were aligned by introducing gaps (hyphens) to maximize identities. Numbers of the first and last residues are referred to the reported sequences including the signal peptide, if any. AgHAO, histamine oxidase of A. globiformis (this study); AgPEAO, phenethylamine oxidase from A. globiformis(10) ; APMAO, methylamine oxidase from Arthrobacter P1(28) ; EcMAO, monoamine oxidase from E. coli(22) ; KaTAO, tyramine oxidase from K. aerogenes(21) ; HpMAO, methylamine oxidase from H. polymorpha(29) ; BSAO, bovine serum amine oxidase(8) ; HKABP, human kidney amiloride-binding protein (30) ; LSDAO, lentil seedling diamine oxidase(31) . Residues totally conserved among the aligned sequences are shown in reverse, and the consensus sequence for topa quinone and conserved histidines possibly involved in copper binding are indicated with asterisks.
Finally but most importantly, we have shown here that the recombinant histamine oxidase overproduced in E. coli cells grown in a copper-depleted medium is the copper/topa quinone-less precursor. The inactive precursor enzyme purified to homogeneity can be activated by later incubation with cupric ions, and the copper-reconstituted active enzyme contains the topa quinone cofactor at the position corresponding to Tyr-402 occurring in the conserved Asn-Tyr-Asp sequence. These results thus corroborate the copper-dependent autoxidation of a specific tyrosyl residue as a common mechanism for the formation of the topa quinone cofactor in bacterial amine oxidases, as first demonstrated with the precursor form of phenethylamine oxidase(11) . To generalize this autoxidation mechanism for the biogenesis of topa quinone, it should be important to examine whether the formation of the cofactor in copper amine oxidases from eukaryotic organisms is also copper-dependent and requires no external enzymatic systems. In an effort along similar lines, Cai and Klinman (9) have recently succeeded in heterologous expression of an active, topa quinone-containing methylamine oxidase of H. polymorpha in a different yeast Saccharomyces cerevisiae, which itself is unable to metabolize amines and appears to lack the ability to produce any endogenous amine oxidases, pointing toward a self-processing mechanism for topa quinone.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D38508[GenBank].