FEMS Microbiology Letters 189 (2000) 183^187 www.fems-microbiology.org

Puri¢cation and characterization of histamine dehydrogenase from Nocardioides simplex IFO 12069

Jahan Ara Siddiqui, Syed Mohammed Shoeb *, Shigeo Takayama, Eiichi Shimizu,

Takamitsu Yorifuji Downloaded from https://academic.oup.com/femsle/article/189/2/183/523654 by guest on 23 September 2021

Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano-ken 399-4598, Japan

Received 19 May 2000; received in revised form 19 June 2000; accepted 19 June 2000

Abstract

Histamine dehydrogenase from Nocardioides simplex IFO 12069 was purified to homogeneity. The had a molecular mass of 170 kDa and was suggested to be a dimer of subunits that had a molecular mass of 84 kDa. The enzyme showed highest activity toward histamine and produced in its to imidazole acetaldehyde. The Km and Vmax values for histamine were 0.075 mM and 4.76 Wmol min31 mg31, respectively. The enzyme was sensitive to the carbonyl reagent iproniazid and a structurally similar compound, tryptophan. The enzyme showed absorption maxima at 442 and 280 nm. Reduction with histamine under anaerobic conditions resulted in a different absorption maximum at 360 nm instead of 442 nm. The enzyme was most active at pH 8.5 in Tris^HCl buffer and most stable at pH 7.0 in potassium phosphate buffer. The E1% value of the enzyme was 8.6 at 280 nm. ß 2000 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Keywords: Histamine dehydrogenase; Nocardioides simplex; Absorption spectrum; Quinone

1. Introduction mine oxidase from Arthrobacter globiformis IFO 12137 [7,8]. Histamine oxidase catalyzes the oxidation of hista- Amine oxidases and dehydrogenases are known to be mine to imidazole acetaldehyde and ammonia with con- that catalyze the oxidative deamination of comitant formation of hydrogen peroxide. We found a amines. Several amine dehydrogenases have been reported histamine-degrading bacterial enzyme, histamine dehydro- mostly in Gram-negative bacteria. Amine dehydrogenase genase, in Nocardioides simplex which catalyzes the oxida- (EC 1.4.2) from Pseudomonas sp. [1], -containing tion of histamine to imidazole acetaldehyde and ammonia. amine dehydrogenase (EC 1.4.99.3) from Pseudomonas pu- Nocardioform bacteria are Gram-positive, have high gua- tida [2], spermidine dehydrogenase (EC 1.5.99) from Pseu- nine and cytosine contents in their DNA, and form a domonas aeruginosa and Citrobacter freundii [3], aromatic phylogenetic group of actinomycetes-related bacteria. amine dehydrogenase (EC 1.4.99) from Alcaligenes faecalis This paper describes the puri¢cation to homogeneity of [4] and methylamine dehydrogenase (EC 1.4.99.3) of Pseu- histamine dehydrogenase from Nocardioides simplex and domonas sp. J [5] have been reported. The discovery of its molecular and catalytic properties. pyrroloquinoline quinone in 1979 has attracted strong in- terest in novel quinone cofactors for their biological roles in cycling enzymes [6]. 2. Materials and methods 4-(2-Aminoethyl)-imidazole, a biogenic amine known as histamine, is generally formed by enzymatic decarboxyla- 2.1. Chemicals tion of L-histidine. Histamine metabolism of animal origin has been studied widely, but studies on bacterial degrada- Histamine dihydrochloride was purchased from Sigma tion have been restricted to only copper-containing hista- Chemical Co. (St. Louis, MO, USA). Other reagents were purchased from Nacalai Tesque (Kyoto, Japan), Toyobo Co. (Osaka, Japan), Aldrich Chemical Co. (Milwaukee, * Corresponding author. Tel.: +81 (265) 77-1614; WI, USA), Boehringer Mannheim (Mannheim, Germany) Fax: +81 (265) 77-1629; E-mail: [email protected] and Pharmacia Biotech (NJ, USA).

0378-1097 / 00 / $20.00 ß 2000 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII: S0378-1097(00)00277-9

FEMSLE 9501 26-7-00 184 J.A. Siddiqui et al. / FEMS Microbiology Letters 189 (2000) 183^187

2.2. Microorganism and culture conditions oughly dialyzed with the bu¡er and then put on a column (5.3 cm2U50 cm) of DEAE Toyopearl 650M (Tosoh N. simplex IFO 12069 was obtained from the Institute Corp., Tokyo, Japan) equilibrated with the bu¡er contain- for Fermentation, Osaka (Juso Nishinocho, Osaka, Ja- ing 0.15 M KCl. The enzyme was eluted with a linear pan). The medium for the test of histamine as nitrogen gradient of bu¡er containing 0.18^0.35 M KCl. Fractions source contained 0.1% (w/v) of the histamine, 0.2% (w/v) 31^39 were pooled (270 ml). The enzyme preparation con-

K2HPO4, 0.1% (w/v) KH2PO4, 0.01% (w/v) MgSO4W7H2O, taining 52 mg protein and 85 units of activity was diluted 0.02% (w/v) NaCl, 0.01% (w/v) yeast extract and 0.2% (w/ with 115 ml of bu¡er and then subjected to a second v) glycerol; the pH was adjusted to 7.0. Substrates other DEAE Toyopearl treatment with a di¡erent column size than histamine as a nitrogen source were used at a rate of of 5.3 cm2U29 cm. Pooled fractions (39^60) containing 41 0.1%. Cells used for enzyme puri¢cation were grown in 15 l mg of protein and 71 units of enzyme activity were sub- of peptone^glycerol medium at 30³C for 10 h with aera- jected to two consecutive steps of hydrophobic chromatog-

tion, and then histamine dihydrochloride was added to the raphy on butyl Toyopearl. Columns of butyl Toyopearl Downloaded from https://academic.oup.com/femsle/article/189/2/183/523654 by guest on 23 September 2021 culture at a concentration of 0.05% (w/v). After further (4.9 cm2U20 cm and 2.3 cm2U20 cm) were equilibrated cultivation for 12 h, the cells were harvested by continuous with 50 mM bu¡er containing 0.8 M ammonium sulfate. centrifugation at 4³C, washed once with 0.85% (w/v) The enzyme was eluted by a reverse gradient of 0.8^0 M NaCl, and stored at 320³C. A 15-l culture yielded about ammonium sulfate with the bu¡er. Fractions (48^58) from 35 g of wet cells. the second butyl Toyopearl were pooled and contained 11 mg of protein and 40 units of enzyme activity. The pooled 2.3. Preparation of cell extracts enzyme preparation was concentrated by ultra¢ltration to about 2 ml and then charged to a Toyopearl HW-55 Cells were thawed in 50.0 mM potassium phosphate (Tosoh Corp., Tokyo, Japan) column (0.79 cm2U123 cm) bu¡er pH 7.0 and then disrupted with a Kaijo Denki of molecular sieve chromatography equilibrated with bu¡- 19-kHz sonicator at 5^10³C. The sonicate was centrifuged er containing 0.1 M KCl. The enzyme was eluted with at 15 000Ug at 4³C for 20 min, and then the supernatant the equilibration bu¡er, and fractions 46^65 (19 ml) was dialyzed thoroughly against the bu¡er at 4³C. were stored in a freezer at 320³C mixed with 50% glyc- erol. 2.4. Enzyme assay condition 2.6. Gel electrophoresis and gel ¢ltration The activity of histamine dehydrogenase was determined spectrophotometrically according to Shinagawa et al. [5] Polyacrylamide gel electrophoresis (PAGE) was done by with some modi¢cations. The initial rate of histamine ox- the method of Davis [9] with 7.2% (w/v) polyacrylamide idation was measured by reading the decreasing intensity gels. PAGE in the presence of SDS was done with the of 2,6-dichlorophenolindophenol (DCPIP) at 600 nm in bu¡er system of Laemmli [10]. Protein bands in gels the presence of phenazine methosulfate (PMS) at 30³C. were located with Coomassie brilliant blue G-250. The The reaction mixture contained 100 mM of Tris^HCl bu¡- molecular mass of histamine dehydrogenase was deter- er pH 8.5, 500 WM of PMS, 50 WM of DCPIP, 1 mM of mined by gel ¢ltration on a Toyopearl HW-55 column histamine dihydrochloride and the enzyme solution, in a by the method of Reinland [11]. total volume of 0.5 ml. One unit of enzyme activity was de¢ned as the amount of enzyme catalyzing the oxidation 2.7. Analytical methods of 1 Wmol of histamine per minute. Protein in the crude extracts was measured by the 2.5. Puri¢cation of histamine dehydrogenase method of Lowry et al. [12] and that in the chromato- graphic fractions was measured by the absorption at All manipulations were done at 4³C in 20.0 mM potas- 280 nm using an absorption coe¤cient of 10.0. Enzyme sium phosphate bu¡er (pH 7.0) unless stated otherwise. activity was measured with a Hitachi model 100-40 spec- All centrifugation were done at 15 000Ug for 30 min. trophotometer. Histamine concentration in the culture The cell-free crude extract prepared from 68 g (wet weight) broth was measured colorimetrically by the method of cells of N. simplex IFO 12069 grown on histamine con- Dubins [13]. Weight-based protein concentration was tained 3743 mg of protein and 96 units of histamine de- measured by weighing the dry weight of the protein in hydrogenase activity. Neutralized polyethyleneimine (400 the enzyme solution and by the refractometric method mg in 40 ml) was added gradually to the cell-free extract. of Shimizu et al. [14]. The absorption spectrum was The precipitate that formed was removed by centrifuga- taken with a Hitachi U-3210 spectrophotometer. The cop- tion, and the protein in the supernatant was fractionated per content in the enzyme protein was measured with a with ammonium sulfate. The 40^60% saturation fraction, Shimadzu ICPS-1000TR sequential plasma spectropho- which contained most of the enzyme activity, was thor- tometer.

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Fig. 1. PAGE and SDS^PAGE of the histamine dehydrogenase. A: Sin- gle band of the homogeneous preparation of histamine dehydrogenase by PAGE. B: SDS^PAGE of the histamine dehydrogenase. Proteins were electrophoresed on a gel slab containing 8% (w/v) polyacrylamide and 0.2% (w/v) SDS. The right and left lanes contain puri¢ed histamine Fig. 2. Absorption spectra of histamine dehydrogenase (oxidized form) dehydrogenase. The center lane contains (from the top) standard pro- in 20 mM potassium phosphate bu¡er, pH 7.0 (a: solid line) and in teins (molecular masses, kDa) as follows: phosphorylase b (94), bovine 20 mM potassium phosphate bu¡er, pH 7.0 containing 5 mM histamine serum albumin (67), egg white ovalbumin (43), bovine erythrocyte car- under anaerobic condition (b: broken line). bonic anhydrase (30), soybean trypsin inhibitor (20.1), bovine milk K-lactalbumin (14). masses, kDa) such as ferritin (450), catalase (240), aldolase (158), bovine serum albumin (66), hen egg albumin (45), 3. Results chymotrypsinogen A (25), and c (12.4) were

used as the standard samples. The subunit Mr of the SDS- 3.1. Puri¢cation of histamine dehydrogenase denatured enzyme was found to be 84 000 (Fig. 1B). These results showed that the enzyme was composed of two The extract of the cells grown on histamine medium identical subunits. showed activity, whereas the extracts of the cells grown on L-asparagine, peptone or histidine medium showed es- 3.3. Absorption spectrum sentially trace to no activity. Histamine dehydrogenase was puri¢ed to homogeneity. A puri¢cation pro¢le of his- The sole absorption maximum of the oxidized enzyme tamine dehydrogenase is shown in Table 1. The enzyme preparation was found in the visible region at 442 nm with was puri¢ed about 154-fold with a yield of 38%. The en- a protein peak at 280 nm (Fig. 2). The reduced enzyme zyme showed a single protein band on PAGE (Fig. 1A); it showed an absorption peak at 360 nm instead of 442 nm. gave a clear single band on SDS^PAGE (Fig. 1B). The enzyme was reduced by the addition of histamine to the enzyme solution under anaerobic conditions. To

3.2. Mr and subunit size achieve the anaerobic condition strictly, the enzyme was placed in a cuvette, then exposed to a gentle £ow of N2 for The Mr of the native enzyme was found to be 170 000 3 min. After addition of histamine at a ¢nal concentration by gel ¢ltration chromatography. Proteins (molecular of 5 mM, the cuvette was sealed with a rubber cap. Two

Table 1 Puri¢cation of the histamine dehydrogenase from N. simplex IFO 12069 Step Total protein (mg) Total activity (U) Speci¢c activity (U mg31) Yield (%) Puri¢cation (fold) Cell-free extract 3743a 96 0.026 100 1 Polyethyleneimine 1909a 111 0.058 116 2 Ammonium sulfate 785a 78 0.099 81 4 First DEAE Toyopearl 52b 85 1.63 86 63 Second DEAE Toyopearl 41b 71 1.73 74 67 First butyl Toyopearl 23b 47 2.04 49 78 Second butyl Toyopearl 11b 40 3.63 42 140 Toyopearl HW-55 9b 36 4.00 38 154 aMeasured by the method of Lowry et al. [12]. b Measured from 280 nm using an E1% value of 10.0.

FEMSLE 9501 26-7-00 186 J.A. Siddiqui et al. / FEMS Microbiology Letters 189 (2000) 183^187 three-directional stopcocks equipped with a stainless steel zyme competitively (data not shown). Diaminopropane needle were injected through the rubber cap for alternate and putrescine inhibited the enzyme at a low rate like cycles of evacuation and £ushing of N2 to the cuvette. that of tryptophan. Such cycling was repeated for about 10 min before assay. The absorption peak of the reduced enzyme was found to 3.5. Electron acceptors be irreversible after exposure for 20 min under aerobic conditions. The availability of electron acceptors for histamine de- hydrogenase activity was examined. The absorbance of 3.4. E¡ects of various compounds on enzyme activity various electron acceptors was assayed at 340 nm for NAD(P), 420 nm for ferricyanide, 450 nm for FAD and The e¡ects of inhibitors such as sulfhydryl and carbonyl FMN, 550 nm for cytochrome c, 585 nm for nitroblue reagents, chelators and metal ions on histamine dehydro- tetrazolium and 600 nm for DCPIP with or without

genase were examined. The enzyme (10 Wg) was incubated PMS. The only oxidation was observed with DCPIP^ Downloaded from https://academic.oup.com/femsle/article/189/2/183/523654 by guest on 23 September 2021 at 30³C for 15 min in 20 mM K^PO4 bu¡er pH 7.0 con- PMS. taining an inhibitor at the concentration indicated in Table 2, and then the enzyme activity was measured under the 3.6. Kinetic and other catalytic properties of histamine standard condition described in Section 2. Among the car- dehydrogenase bonyl reagents, semicarbazide, hydroxylamine, isonicotinic acid hydrazide, phenylhydrazine, hydrazine and iproniazid A homogeneous preparation of histamine dehydroge- were tested. Only iproniazid (isonicotinic acid 2-isopropyl nase showed the highest activity toward histamine. Among hydrazide) inhibited the enzyme drastically. The rates of various amines, only agmatine and putrescine were oxi- inhibition were 5, 25, and 50% at iproniazid concentra- dized at a rate of 30^34% that of histamine. The dry tions of 0.10, 0.20 and 0.50 mM, respectively. The inhib- weight and a refractometric measurement of the protein ited enzyme resulted in no recovery of the activity with the of histamine dehydrogenase gave the same absorption co- addition of higher concentrations of histamine, suggesting e¤cient (E1%) value of 8.6 at 280 nm. The Michaelis con- that iproniazid inhibited the enzyme non-competitively. stants (Km value) and the maximum reaction velocity for Sulfhydryl reagents such as p-chloromercuribenzoate, io- histamine were determined by Lineweaver^Burk plots. Km 31 doacetamide and monoiodoacetate caused a low rate of and Vmax values were 0.075 mM and 4.76 Wmol min inhibition. 5,5P-Dithiobis nitrobenzoic acid and N-ethyl- mg31, respectively. The copper content of the enzyme maleimide had no e¡ect on the enzyme activity. It has was measured, and copper was not found in the enzyme further been shown that EDTA and phenanthroline had solution. The enzyme was most active in Tris^HCl bu¡er, a very low rate of inhibition and that other chelators such pH 8.5, and most stable in potassium phosphate bu¡er, as sodium azide, bathocuproine and diethyldithiocarbamic pH 7.0. When the enzyme was heated at various temper- acid showed essentially no inhibition. Among various met- atures for 10 min in 20 mM potassium phosphate bu¡er, al ions, only MnCl2, CuSO4 and CoCl2 were found to pH 7.0, it was found that the enzyme was stable at a have an inhibitory e¡ect on the enzyme. The metabolic temperature of 60³C. The loss of activity was 10, 50, regulator AMP inhibited the enzyme severely. Compounds and 100% at the heating temperatures of 70, 80, and having structural similarities to that of histamine were 85³C, respectively. tested for inhibition e¡ects. Tryptophan inhibited the en-

4. Discussion

Table 2 E¡ect of various compounds on the enzyme reaction This paper described the properties of histamine dehy- drogenase puri¢ed from N. simplex. The enzyme was Compound Concentration (mM) Inhibition (%) found to have a molecular mass of about 170 000 and None ^ 0 dissociated into two identical subunits on SDS gel electro- Iproniazid 1 100 Monoiodoacetic acid 1 19 phoresis. The enzyme was distinctly di¡erent from other Iodoacetamide 1 14 amine dehydrogenases in its molecular mass and subunit p-Chloromercuribenzoate 1 10 structure. Amine dehydrogenases from Pseudomonas sp. o-Phenanthroline 1 17 [1] and P. putida [15], aromatic amine dehydrogenase [4], CuSO4 156and methylamine dehydrogenase [5] exhibited nonidentical MnCl 131 2 subunit structures and di¡erent molecular masses from CoCl2 126 AMP 1 60 histamine dehydrogenase. Spermidine dehydrogenase Tryptophan 0.20 90 showed a monomeric structure of its subunit [3]. The ab- Diaminopropane 0.20 20 sorption spectra of histamine dehydrogenase were distinct Putrescine 0.20 24 from those of c-type cytochrome-containing dehydroge-

FEMSLE 9501 26-7-00 J.A. Siddiqui et al. / FEMS Microbiology Letters 189 (2000) 183^187 187 nase [1], and hemoprotein amine dehydrogenase [2]. The References visible spectrum of histamine dehydrogenase is somewhat similar to those exhibited by tryptophan tryptophylqui- [1] Shinagawa, E., Matsushita, K., Nakashima, K., Adachi, O. and none-containing aromatic amine dehydrogenase [4], meth- Ameyama, M. (1988) Crystallization and properties of amine dehy- drogenase from Pseudomonas sp. Agric. Biol. Chem. 52, 2255^2263. ylamine dehydrogenase from Pseudomonas sp. J [5], and [2] Durham, D.R. and Perry, J.J. (1978) Puri¢cation and characteriza- methylamine dehydrogenase from Paracoccus denitri¢cans tion of a heme-containing amine dehydrogenase from Pseudomonas [16]. Certain similarities of histamine dehydrogenase to putida. J. Bacteriol. 134, 837^843. aromatic amine and methylamine dehydrogenases [4,16] [3] Hisano, T., Abe, S., Wakashiro, M., Kimura, A. and Murata, K. are particularly noteworthy. None of these enzymes (1990) Microbial spermidine dehydrogenase: Puri¢cation and proper- ties of the enzyme in Pseudomonas aeruginosa and Citrobacter freun- formed semiquinone during reduction with the substrate, dii. J. Ferment. Bioeng. 69, 335^340. and the reduced enzymes are relatively stable to reoxida- [4] Govindaraj, S., Eisenstein, E., Jones, L.H., Sanders-Loehr, J., Chis- tion. toserdov, A.Y., Davidson, V.L. and Edwards, S.L. (1994) Aromatic

It is interesting to note that among carbonyl reagents, amine dehydrogenase, a second tryptophan tryptophylquinone en- Downloaded from https://academic.oup.com/femsle/article/189/2/183/523654 by guest on 23 September 2021 only iproniazid, a inhibitor [17], in- zyme. J. Bacteriol. 176, 2922^2929. [5] Matsumoto, T., Hiraoka, B.Y. and Tobari, J. (1978) Methylamine hibited histamine dehydrogenase. Most of the carbonyl dehydrogenase of Pseudomonas sp. J: isolation and properties of the reagents showed very strong inhibition on various amine subunits. Biochim. Biophys. Acta 522, 303^310. dehydrogenases [1,2,4]. Iproniazid caused perturbation of [6] Salisbury, S.A., Forrest, H.S., Cruse, W.B.T. and Kennard, O. (1979) both the visible and UV spectra of histamine dehydroge- A novel coenzyme from bacterial primary alcohol dehydrogenases. nase, indicating that the binding of this inhibitor a¡ects Nature 280, 843^844. [7] Shimizu, E., Odawara, T., Tanizawa, K. and Yorifuji, T. (1994) His- the cofactor or its environment as well as the protein tamine oxidase, a Cu2‡-quinoprotein enzyme of Arthrobacter globi- structure. In contrast, iproniazid inhibited aromatic amine formis. Biosci. Biotechnol. Biochem. 58, 2118^2120. dehydrogenase [4] a¡ecting only the cofactor or its envi- [8] Choi, Y.-H., Matsuzaki, R., Fukui, T., Shimizu, E., Yorifuji, T., Sato, H., Ozaki, Y. and Tanizawa, K. (1995) Copper topa quinone ronment. Among various metal ions, only MnCl2, CoCl2 containing histamine oxidase from Arthrobacter globiformis. Molec- and CuSO4 showed inhibition. Prolongation of the reac- ular cloning and sequencing, overproduction of precursor enzyme, tion time showed a gradual recovery of the enzyme activ- and generation of topa quinone cofactor. J. Biol. Chem. 270, 4712^ ity. Histamine dehydrogenase had a favorable heat stabil- 4720. ity and retained almost full activity at 60³C as did [9] Davis, B.J. (1964) Disc electrophoresis, II. Method and application to aromatic amine dehydrogenase [4], and thus the enzyme human serum proteins. Ann. NY Acad. Sci. 121, 404^427. is very advantageous for practical use as a diagnostic en- [10] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. zyme. Histamine dehydrogenase had very restricted elec- [11] Reinland, J. (1971) Gel ¢ltration. Methods Enzymol. 22, 287^321. tron acceptor speci¢city. The activity was attained only [12] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) when PMS was coupled to DCPIP. Our preliminary ob- Protein measurement with the Folin phenol reagent. J. Biol. Chem. servation by redox-cycling staining revealed that the en- 193, 265^275. zyme had a quinone cofactor unlike pyrroloquinoline qui- [13] Dubins, D.T. (1960) The assay and characterization of amines by means of 2,4-Dinitro£uorobenzene. J. Biol. Chem. 235, 783^786. none. Characterization of the prosthetic group is under [14] Shimizu, E., Kiguchi, S. and Yorifuji, T. (1989) A simple approxi- investigation and will be published elsewhere. mation of weight-based protein measurement with HPLC apparatus. Agric. Biol. Chem. 53, 2801^2803. [15] Adachi, O., Kubota, T., Hacisalihoglu, A., Toyama, H., Shinagawa, Acknowledgements E., Duine, J.A. and Matsushita, K. (1998) Characterization of qui- nohemoprotein amine dehydrogenase from Pseudomonas putida. Bio- sci. Biotechnol. Biochem. 62, 469^478. The study was supported in part by Grants-in-Aid from [16] Husain, M. and Davidson, V.L. (1987) Redox properties of the qui- the Ministry of Education, Science, Sports, and Culture, noprotein methylamine dehydrogenase from Paracoccus denitri¢cans. Japan. We wish to thank Prof. R. Iriye for his technical Biochemistry 26, 4139^4143. guidance in metal analysis. We thank Prof. D. Karasawa, [17] Smith, T.E., Weissbach, H. and Underfriend, S. (1963) Studies on monoamine oxidase: the mechanism of inhibition of monoamine ox- Prof. M. Hirota and Dr. A. Tabuchi for their valuable idase by iproniazid. Biochemistry 2, 746^751. advice.

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