©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Copper/Topa Quinone-containing Histamine Oxidase from Arthrobacter globiformis
MOLECULAR CLONING AND SEQUENCING, OVERPRODUCTION OF PRECURSOR ENZYME, AND GENERATION OF TOPA QUINONE COFACTOR (*)

(Received for publication, October 24, 1994; and in revised form, December 14, 1994)

Yoon-Ho Choi Ryuichi Matsuzaki Toshio Fukui Eiichi Shimizu (1) Takamitsu Yorifuji (1) Hidetoshi Sato (2) Yukihiro Ozaki (2) Katsuyuki Tanizawa (§)

From the  (1)Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, the Department of Bioscience and Biotechnology, Shinshu University, Kamiina, Nagano 399-45, and the (2)Department of Chemistry, School of Science, Kwansei Gakuin University, Nishinomiya, Hyogo 662, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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).


INTRODUCTION

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^1), 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.


EXPERIMENTAL PROCEDURES

Isolation and Sequencing of Peptide Fragments

Histamine oxidase (120 µg) purified from the cell extracts of A. globiformis IFO12137 (ATCC8010) grown on histamine as an inducer (13) was dissolved in 100 mM Tris-HCl buffer (pH 8.5), containing 2 M urea, and digested at 37 °C for 12 h with 2 µg of N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Millipore) or 2 µg of lysyl endopeptidase (Takara Shuzo). After terminating the digestion by heating in boiling water for 5 min, the peptide mixture was separated on a Tosoh high performance liquid chromatography system equipped with a Cosmosil 5C18-AR reverse phase column (Nacalai Tesque) using a solvent system of 0.1% trifluoroacetic acid (A) and 0.095% trifluoroacetic acid containing 90% acetonitrile (B). A 70-min linear gradient from 10 to 70% B was used to elute peptides at a flow rate of 0.5 ml/min with continuous monitoring of the absorbance at 215 nm. The amino-terminal amino acid sequences of the undigested enzyme and 8 peptide fragments thus obtained were determined with an Applied Biosystems Model 477A protein sequencer linked with an Applied Biosystems Model 120A PTH analyzer.

Design of Oligonucleotide Probes

To screen genomic DNA fragments containing the histamine oxidase gene, two oligonucleotide probes (P1: 5`-TCGGCCGGGTGGTCCTCGCGGAAGCCCAGTCC-3` and P2: 5`-GA(AG)CT(GC)AACCC(GC)GA(AG)GC-3`) were synthesized on the basis of the partial amino acid sequences determined as above. To confer the hybridization specificity, one probe (P1) was made relatively long (32-mer), and the extents of degeneracy in both probes were reduced by considering the preferred base usage in the third position of each codon in the phenethylamine oxidase gene of A. globiformis(10) and 2 arbitrarily chosen Arthrobacter genes (xylA and 6-HDNO) registered in the GenBank/EMBL data bank. P1 and P2 corresponded to the sequence from Gln-314 to Val-324 and that from Glu-110 to Ala-115 (both in peptide fragments obtained by trypsin digestion, see Fig. 1), respectively, in the sense strand. The oligonucleotides synthesized with an Applied Biosystems 381A DNA synthesizer were radiolabeled with T(4) polynucleotide kinase and [-P]ATP (>5,000 Ci/mmol, Amersham) according to the standard protocol (14) and were used without further purification (10^7 cpm/µg).


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.



Gene Cloning and Nucleotide Sequencing

Total DNA was isolated from A. globiformis cells as described previously (15) , and about 50 µg of it was digested for 2 h with BamHI (70 units) according to the standard procedure for partial digestion(14) , followed by size fractionation by sucrose density gradient centrifugation. The DNA fragments of about 3-5 kbp were ligated into the BamHI site of plasmid BluescriptII SK(+) (Stratagene) and transformed into E. coli JM109 cells. About 5000 colonies in total grown on agar plates of LB medium containing 50 µg/ml sodium ampicillin were transferred onto nitrocellulose membrane filters. The filters were incubated in the prehybridization solution (6 times SSC, 10 times Denhardt's solution, 0.2% SDS) at 68 °C for 4 h, followed by hybridization with radiolabeled probes P1 and P2 (10 pmol/10 ml) in the hybridization solution (6 times SSC) at 68 °C for 16 h. The filters were washed at room temperature and then at 65 °C in 6 times SSC for 10 min. Twelve positive clones obtained in the first hybridization were picked up from the master plates and subjected to the secondary screening with probes P1 and P2 under the same conditions as above. An about 6.6-kbp plasmid containing a 3.6-kbp insertion (designated as pHAO-1) was isolated from one positive clone obtained finally and purified by polyethylene glycol precipitation(14) . DNA sequence analyses were done in both directions of a 2.5-kbp region containing the entire histamine oxidase gene by the dideoxy chain termination method (16) with an Applied Biosystems 373A DNA sequencer using a Taq dye-terminator cycle sequencing kit (Applied Biosystems) and the above probe P1 as the first sequencing primer and several other primers successively synthesized based on the farthest regions of the determined sequences.

Construction of Expression Plasmid

An about 0.4-kbp DNA fragment encoding the amino-terminal region of the enzyme was amplified by the polymerase chain reaction (17) with pHAO-1 as a template and two synthetic oligonucleotides as primers (P3: 5`-TGCCATGGCCCTTCAGACCA-3`, corresponding to nucleotides -4 to +16 (A of the initiation codon ATG as +1) and containing a new NcoI site (underline) with a codon change from Thr-2 (ACC) to Ala (GCC); and P4: 5`-GTTCCACTGCGGATCTTC-3`, corresponding to nucleotides 360 to 342, complementary). The amplified fragment was digested with NcoI and EcoRI and ligated with the NcoI/EcoRI-digested expression vector pTrc99A (Pharmacia Biotech Inc.). To the resultant plasmid was then inserted the 2.2-kbp EcoRI-BamHI fragment from pHAO-1 encoding the remaining carboxyl-terminal region of the enzyme. After the polymerase chain reaction-amplified region was confirmed for its nucleotide sequence, the final plasmid thus constructed (pTrc99HAO) was transformed into E. coli JM109 cells.

Expression and Purification of Recombinant Histamine Oxidase

Water used in the preparation of culture media and other solutions was obtained from a NANOpure II (Barnstead) system and had resistance greater than 17.6 Mbulletcm. Solutions of chemicals were all passed through Chelex chelating ion exchange filters (Bio-Rex ion exchange membrane, Bio-Rad), and all glassware was washed with 0.1 M sulfuric acid to remove traces of metal ions. E. coli JM109 cells harboring pTrc99HAO were grown at 37 °C in an LB medium supplemented with 50 µg/ml sodium ampicillin. After cultivation until the cell density reached A = 0.8 (about 3 h), isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 0.4 mM to induce the expression of histamine oxidase, and the bacteria were further cultivated at 30 °C for 4 h in the presence or absence of 50 µM CuSO(4). The cells were harvested by centrifugation (5,000 times g) for 10 min.

The cells grown in the absence of CuSO(4) 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(4), buffer A was not supplemented with EDTA and N,N-diethyldithiocarbamate but added with 0.05 mM CuSO(4) until the step of ammonium sulfate fractionation. The resulting lysate was centrifuged at 25,000 times 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.

Enzyme and Protein Assays

In all cases unless otherwise stated, the Cu-deficient inactive enzyme (10-100 µM subunit) was incubated at 25 °C for 30 min with its 5times concentration of CuSO(4) (0.05-0.5 mM) in 50 mM HEPES (pH 6.8), before assaying the activity. The assay for histamine oxidase was carried out at 30 °C in 100 mMN,N-bis(2-hydroxyethyl)glycine buffer (pH 8.0) with 1 mM histamine dihydrochloride as substrate, by monitoring the rate of H(2)O(2) production as described previously(18) . Protein concentration was determined spectrophotometrically at 280 nm using an extinction coefficient of 13.8 for a 10 mg/ml solution of the purified enzyme, estimated from the amino acid composition(19) . For calculation of the concentration of enzyme subunit, a molecular weight of 75,000 was employed. One unit of the histamine oxidase activity is defined as the amount of enzyme that produces 1 µmol of H(2)O(2) per min.

Atomic Absorption Analysis

The copper contents in the enzyme proteins were analyzed with a Nippon Jarrell-Ash AA-880 mark II atomic absorption spectrophotometer (acetylene/air flame) at 324.8 nm.

Derivatization with p-Nitrophenylhydrazine and Isolation of Topa Quinone-containing Peptide

The purified enzyme (about 25 mg, 0.33 µmol of subunit) in 2 ml of 50 mM HEPES (pH 6.8) was first incubated with 0.5 mM CuSO(4) at 25 °C for 30 min to generate the topa quinone cofactor (see below) and then modified at 37 °C by the addition of a 4-fold molar excess of 10 mMp-nitrophenylhydrazine (in 50% methanol) in several portions over a 30-min period. The reaction mixture was repeatedly concentrated by ultrafiltration in an Amicon Centricon-30 cartridge and diluted with 50 mM HEPES (pH 6.8) to remove unreacted p-nitrophenylhydrazine. The derivatized enzyme solution was added with solid urea to 8 M, incubated at 40 °C for 10 min, and diluted with 0.1 M Tris-HCl (pH 8.0) to a final urea concentration of 2 M. Digestion with thermolysin was carried out at 40 °C for 12 h at a protease-to-substrate ratio of 1:50 (w/w) followed by further digestion for 6 h after the second addition of thermolysin.

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 times 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) .

Resonance Raman Spectroscopy

Resonance Raman spectra were obtained with an apparatus consisting of a triple polychromator (Spex 1877c), an intensified photodiode array detector (PAR 1455R-HQ) operated at -20 °C, and a personal computer (NEC 9801) for data collection. The 457.9 nm line from an argon laser (Spectra Physics 2016-05) was used for excitation. The laser power at the sample position was about 30 milliwatts, and a 5 cm spectral slit width was employed. 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.


RESULTS

Cloning and Sequencing of Histamine Oxidase Gene from A. globiformis

On screening of about 5000 E. coli clones carrying A. globiformis genomic DNA fragments (3-5 kbp) with the simultaneous use of two oligonucleotide probes, P1 and P2, synthesized on the basis of the partial amino acid sequences determined, one positive clone containing a 3.6-kbp BamHI fragment was finally isolated (pHAO-1). The crude extract of E. coli JM109 cells transformed with pHAO-1 showed a weak but explicit activity of histamine oxidase, which was undetectable in the control E. coli JM109 cells without the plasmid (data not shown), encouraging us to further analyze pHAO-1. Initial DNA sequencing for pHAO-1 primed with probe P1 revealed a 3`-downstream sequence of some 400 bases, translatable to an amino acid sequence including that of a peptide fragment obtained by lysyl endopeptidase digestion of the enzyme protein (see Fig. 1). Therefore, using several new oligonucleotide primers successively synthesized on the basis of the farthest regions of the established sequences, a region of about 2.5 kbp enough to cover the whole histamine oxidase gene was sequenced in both directions.

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) .

Overexpression in E. coli and Purification of Recombinant Histamine Oxidase

To facilitate the structural identification of the quinone cofactor assumed to be contained in histamine oxidase from A. globiformis(13) using a sufficient amount of the homogeneous enzyme, we then tried to overproduce the recombinant enzyme in E. coli cells. However, initial attempts by placing the cloned gene 3`-downstream the lac promoter in a multicopy plasmid pTV118 or the T7 RNA polymerase promoter in a pET plasmid were unsuccessful; only a very low activity was detected or most of the plasmid-derived protein was produced in an insoluble form, although the recombinant phenethylamine oxidase was expressed efficiently in a soluble form with a pET system(10) . Significant expression of the soluble histamine oxidase was achieved by placing the structural gene 3`-downstream of the trc promoter in the pKK233-2-derived plasmid pTrc99A (23) (see ``Experimental Procedures'' for plasmid construction). In this construction, to manipulate only the region of the open reading frame, we had to introduce a new restriction site (NcoI) at the translational initiation codon, which led to the alteration of the Thr-2 codon (ACC) to that for Ala (GCC). However, the crude extract of E. coli cells transformed with the expression plasmid pTrc99HAO and grown in the presence of CuSO(4) exhibited a high level of the histamine oxidase activity (0.23 unit/mg of protein) (see Table 1). Thus, Ala-2 newly formed at the amino terminus, as identified by direct sequencing of the recombinant enzyme purified (data not shown), had apparently no effect on the enzyme activity.



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-beta-D-galactopyranoside, and further cultivated the cells in the presence or absence of CuSO(4). The two forms thus produced were separately purified in the presence of CuSO(4) 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(r) = 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(m) 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 Cudeficient 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.



Activation by Cupric Ions

We have shown that the Cu-deficient precursor form of phenethylamine oxidase is readily activated by aerobic incubation with excess Cu(11) . Likewise, the Cu-free inactive form of histamine oxidase presently obtained was activated dramatically by incubation with excess Cu for about 30 min to a level even doubly higher than the enzyme expressed and purified in the presence of Cu (see Table 1). When the Cu-incubated enzyme was dialyzed thoroughly against 50 mM HEPES (pH 6.8) containing 1 mM EDTA, followed by further dialysis against buffer alone, to remove the unbound or adventitiously bound Cu, the maximally activated enzyme was found to contain 1.09 mol atom of copper per mol of subunit by atomic absorption analysis. In contrast, the enzyme expressed and purified in the presence of Cu was not much further activated (to 13.5 units/mg), incorporating Cu to less than the stoichiometric level (0.72 mol atom/mol of subunit). These results suggest that the enzyme had suffered some irreversible inactivation during the purification in the presence of Cu and that the partially inactivated enzyme lost the Cu-binding capability.

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(r) = 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(r) of 95,000 (24) has been corrected for M(r) of 82,800 plus 4% carbohydrate(8) ). It is noted that the (max) 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.

Titration with Phenylhydrazine

The quinone compound generated in the Cu-activated enzyme was titrated with the carbonyl reagent phenylhydrazine, reacting with the quinone to form a hydrazine adduct(9, 25) . After substoichiometric amounts of phenylhydrazine were added to an enzyme solution and incubated at 30 °C until the change of the absorption spectrum ceased (for 10-15 min), the spectrum of the enzyme was recorded and also the remaining enzyme activity was assayed. As shown in Fig. 4, the titration with phenylhydrazine was accompanied by the formation of a yellow adduct with an absorption maximum at 438 nm, similar to those observed with bovine serum and yeast amine oxidases(9, 25) . The complete inactivation was obtained when 0.67 mol of phenylhydrazine was added per mol of the enzyme subunit (Fig. 4, inset). The increase in absorbance at 438 nm also reached an end point where the phenylhydrazine:subunit molar ratio was 0.62:1. These results suggest that less than one quinone compound has been generated per enzyme subunit by incubation with Cu. Alternatively, one quinone has been indeed produced in each subunit, but the quinones in the dimeric enzyme may have reduced reactivity to phenylhydrazine if one of the two is modified, as reported for bovine and pig plasma amine oxidases with the quinone cofactor showing half-site reactivity(26, 27) .


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 (bullet) and percent remaining activity (circle) are shown in the inset.



Identification and Localization of Topa Quinone

Following the general method established for isolation of the topa quinone-containing peptide from various amine oxidases(3, 4, 5, 6) , the Cu-activated enzyme was first derivatized with p-nitrophenylhydrazine and then digested completely with thermolysin as described under ``Experimental Procedures.'' As observed in the previous studies(3, 5, 6, 9, 11) , a single yellow peptide was eluted on the reversed phase liquid chromatography of the thermolysin digest (Fig. 5). Automated Edman degradation of this p-nitrophenylhydrazone-containing peptide further purified with an analytical reversed phase column identified its sequence as Val-Gly-Asn-X-Asp-Tyr-Gly, where X is an unidentifiable residue. This sequence corresponds to that from Val-399 to Gly-405, and the unidentifiable residue X is found at the position corresponding to Tyr-402 in the deduced primary structure (Fig. 1). Thus, the quinone cofactor is derived from Tyr-402 occurring in the conserved Asn-Tyr-Asp sequence.


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 (max) 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.




DISCUSSION

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(E) 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(K)) from Klebsiella aerogenes(21) , although the nucleotide sequence and the product of another E. coli gene named maoX, which is located close to maoA(E), 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(K))(21) , 29% with the monoamine oxidase of E. coli (product of maoA(E))(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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D38508[GenBank].

§
To whom correspondence and reprint requests should be addressed. Tel.: 81-6-879-8462; Fax: 81-6-876-4194.

(^1)
The abbreviations used are: topa, (2,4,5-trihydroxyphenyl)-Lalanine (also known as 6-hydroxydopa); kb(p), kilobase (pairs).


ACKNOWLEDGEMENTS

We thank Prof. S. Suzuki, Faculty of Science, Osaka University, for the atomic absorption analysis and Dr. S. Yamaguchi, Institute of Scientific and Industrial Research, Osaka University, for the synthesis of the topa quinone hydantoin model compound.


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