Journal of Experimental Botany, Vol. 48, No. 316, pp. 1897-1907, November 1997Journal ofExperimentalBotany
Confirmation of the presence of a Cu(ll)/topa quinoneactive site in the amine oxidase from fenugreek seedlings
Marek Sebela1'5, Lenka Luhova1, Ivo Frebort1, Shun Hi rota2, Heinz G. Faulhammer3,
Vaclav Stuzka4 and Pavel Pec1
^Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc,Czech Republic
department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,Nagoya 464-01, Japan
^Laboratory of Biochemistry, University of Bayreuth, Universitatsstrasse 30, 95440 Bayreuth, Germany
^Department of Analytical Chemistry, Faculty of Science, Palacky University, Jr. Svobody 8, 771 46 Olomouc,Czech Republic
Received 18 February 1997; Accepted 30 June 1997
Abstract
Amine oxidase from etiolated seedlings of fenugreek{Thgonella foenum-graecum) has been isolated by apurification procedure involving three chromato-graphic steps. The homogeneous enzyme is of pinkcolour with a visible absorption maximum at 500 nm.The dimeric enzyme (2 x 75 kDa) is a slightly acidicprotein (pi 6.8) containing 8% neutral sugars. W-ter-minal amino acid sequence of the enzyme shows ahigh degree of similarity to other plant and microbialcopper-containing amine oxidases. The best sub-strates of the enzyme are aliphatic diamines and somepolyamines, whereas inhibitors are substrate ana-logues, copper complexing agents, some alkaloids andseveral other compounds. Spectrophotometric titra-tions with phenylhydrazines demonstrated one react-ive carbonyl group per subunit of the enzyme andredox-cyclic quinone staining after native electrophor-esis indicated the presence of a quinone cofactor.Differential pulse polarography showed the existenceof a copper/quinone-containing active site. The reson-ance Raman spectroscopy and the pH-dependent shiftof the absorption spectrum of the enzyme p-nitrophenylhydrazone confirm unambiguously theidentity of the cofactor with topa quinone. EPR spectraof the enzyme are in accordance with those of tetra-gonal cupric complexes as known for other copper-containing amine oxidases. Besides the copper, Mn(ll)
ions were detected that partially occupy another metalsite in the enzyme, but their catalytical importance isunlikely.
Key words: Fenugreek, Thgonella foenum-graecum, amineoxidase, topa quinone.
Introduction
The enzymes belonging to the group of copper containingamine oxidases (EC 1.4.3.6) [amine: O2 oxidoreductase(deaminating)] catalyse the oxidative deamination of bio-genic amines to the corresponding aldehydes and ammo-nia accompanied by a two-electron reduction of molecularoxygen to hydrogen peroxide. Although these enzymeshave been found in bacteria, fungi and various plantsand animals, their actual role in these organisms has notbeen completely clarified up to now, although their effectsin several physiological processes connected with themetabolism of amines and polyamines have been recog-nized (Mclntire and Hartmann, 1992). Copper and topaquinone (Janes et al., 1990), the organic cofactor that isgenerated from a specific tyrosyl residue by a self-oxidating mechanism catalysed by cupric copper(Matsuzaki et al., 1994), mediate the catalytic reactionproceeding by a ping-pong mechanism. Recently, thecrystal structures of amine oxidases from Escherichia coli(Parsons et al., 1995), and pea seedling {Pisum sativum)(Kumar et al., 1996), have been reported.
sTo whom correspondence should be addressed. Fax: +420 68 5221332. E-mail: [email protected]
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
1898 Sebela et al.
Previously, the presence of amine oxidase activity wasfound in seedlings of fenugreek (Trigonella foenum-graecum) and the enzyme was first purified as a yellowcoloured monomer of relatively low specific activity witha molecular mass of 80 kDa (Luhova et al., 1995). Sincethis could have been a novel plant amine oxidase thefenugreek enzyme was studied further. A homogeneouspink-coloured enzyme of high specific activity with adimeric structure in the native state was finally obtainedand its molecular and catalytic properties are describedin this work. The quinone nature of the cofactor and itsidentity with topa quinone was confirmed.
Materials and methods
Chemicals and plant material
2-Hydroxyputrescine and 2-hydroxycadaverine (Macholan,1965), 3-hydroxycadaverine (Macholan, 1972), E-l,4-diamino-2-butene (Macholan et al., 1975), Z-l,4-diamino-butene (Pecet al., 1991), 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone (Machol&n, 1965, 1974), were synthesized. Allother chemicals were commercial products of analytical puritygrade. Seeds of fenugreek (Trigonella foenum-graecum) wereobtained from Research Institute of Forage Crops (Troubskou Brna, Czech Republic).
Enzyme purification
All purification procedures were performed at 0-5 °C and thebuffers contained 1 /J.M CU(II ) , unless stated otherwise.Fenugreek seedlings (1 kg) germinated in the dark for 7 d werehomogenized for 10 min by a Moulinex hand blender in 2 1 of0.1 M potassium phosphate buffer, pH 7.0. Crude homogenatewas filtered through a nylon mesh cloth and centnfuged at5000 g for 60 min. The precipitate was discarded and thesupernatant was fractioned with ammonium sulphate in 30%saturation (176 g I"1), stirred for 30 min and centrifuged at5000 g for 60 min. The supernatant obtained was furtherfractioned with ammonium sulphate in 65% saturation(235 g I"1), stirred for 30 min and centrifuged at 5000 g for60 min. The precipitate was then collected, resuspended in 40 mlof 0.1 M potassium phosphate buffer, pH 7.0, and dialysedagainst the same buffer overnight. The dialysed solution wasrapidly heated up to 55-58 °C and kept at 60 °C for 5 min withstirring. The solution was then cooled down to 4 °C on a water-ice bath, centrifuged at 15 000g for 30 min and dialysedovernight against 20 mM potassium phosphate buffer, pH 7.0.The dialysed solution was loaded on to a DEAE-celluloseSH-23 (Fluka, Buchs, Switzerland) column (2.5x20cm) equi-librated with 20 mM potassium phosphate buffer, pH 7.0.During loading and washing with the above buffer, the eluateof A28o>0.4 was collected. This solution, containing more than90% of the total amine oxidase activity, was then applieddirectly on to a hydroxyapatite column (2.5x20 cm) equilib-rated with 20 mM potassium phosphate, pH 7.0. The columnwas washed with 0.1 and 0.3 M potassium phosphate buffers,pH 7.0, in order to remove impurities and the enzyme waseluted by 0.75 M potassium phosphate buffer, pH 7.0. Fractionsof highest amine oxidase activity were pooled, dialysed overnightagainst 20 mM potassium phosphate buffer, pH 7.0, andconcentrated in an ultrafiltration cell (Amicon, Danvers, MA,USA) equipped with an XM-50 filter. Finally, the enzymesolution was submitted to size-exclusion chromatography on a
Sephacryl S-300 HR (Pharmacia Biotech, Uppsala, Sweden)column (2.5 x 50 cm) and eluted with 20 mM potassium phos-phate buffer, pH 7.0, at a flow rate of 1.25 ml min"1. Fractionswith highest enzymatic activity were pooled and concentratedby ultrafiltration as described above.
Amine oxidases from Aspergillus niger AKU 3302 (Frebortet al., 1996a), and pea seedlings (Wimmerova et al., 1993),were prepared as described previously.
Activity, protein, carbohydrate and quinone assay
Amine oxidase activity was determined using a coupled reactionwith horseradish peroxidase and guaiacol according to Frebortet al. (1989). Kinetic constants were calculated from initialrates using the program GraFit 3.0 (Erithacus Software)obtained from Sigma (St Louis, MO, USA). Protein concentra-tion was determined according to Bradford (1976) with BSAas a standard. The total content of neutral sugars of amineoxidases was determined by the phenol-sulphuric acid methodaccording to Dubois et al. (1956) with D-mannose as a reference.Spectrophotometric NBT/glycinate test and a quinone staningon a nitrocellulose membrane after native electrophoresis wasdone as described by Paz et al. (1991).
Molecular mass determination
Gel permeation chromatography was performed on a Superdex200 HR column (1.0x30 cm; Pharmacia LKB, Uppsala,Sweden) with molecular mass marker proteins as describedpreviously by Fr6bort et al. (1996a).
Electrophoretic methods
SDS-PAGE was performed by a standard method accordingto Laemmli (1970) on a slab polyacrylamide gel (10% T, 2.5%C). Protein samples were denatured with 10% SDS and 0.5 M2-mercaptoethanol at 100°C for 7 min before the application.Molecular mass was evaluated using protein markers (14.4,20.1, 30, 43, 67, and 94 kDa; Pharmacia Biotech, Uppsala,Sweden). Isoelectric focusing was performed on a precastPhastGel IEF 3-9 using a Phast System unit and the broad pikit (pi 3.5-9.3) as a reference (Pharmacia Biotech, Uppsala,Sweden). After electrophoresis, protein bands were visualizedby staining with Coomassie Brilliant Blue G-250.
Protein sequence analysis
N-terminal sequencing was performed by automatic Edmandegradation on a Model 476A protein sequencer (AppliedBiosystems, Foster City, CA, USA).
Spectrophotometric active-site titrations
Samples of the enzyme (about 30 nmol) in 3 ml of 20 mMpotassium phosphate buffer, pH 7.0, were titrated either withsolution of phenylhydrazine hydrochloride (1.56mM) or eth-anolic solution of />-nitrophenylhydrazine (3mM). The solu-tions (fresh) were added in 2 /A steps (10 min intervals) into thecontinuously stirred 1 cm cell thermostated at 30 °C. Afteradding aliquots of the reagents, absorption spectra wererecorded. Finally, 2 .1 aliquots of the reaction mixtures weretaken out for activity assay.
Spectroscopic measurements
EPR spectra of native fenugreek amine oxidase (0.163 mM)and its p-nitrophenylhydrazone (0.323 mM) were recorded ona JES-RE1X EPR spectrometer (JEOL, Tokyo, Japan) in liquidnitrogen at 77 K. The samples were repurified by FPLC onSuperdex HR 200, dialysed against metal-free distilled water
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
and lyophilized. Prior to measurements, the samples weredissolved in 0.1 M potassium phosphate buffer, pH 7.5. Coppercontent was estimated by comparing the double integral of theenzyme spectrum with that of 0.25 mM CuSO4.5H2O and25 mM Na2EDTA in the same buffer. Copper and manganesecontent was also assayed by atomic absorption spectrometryon a Avanta E spectrometer (GBS Scientific Equipment,Dondenong, Australia) using flame atomization.
Resonance Raman spectra of the enzyme p-mXro-phenylhydrazone were recorded as described previously byFreborter al. (19966).
Differential pulse polarography
Differential pulse polarography was performed on a PRG 4apparatus (Tacussel, Villeurbanne, France) according to Sebelaet al. (1996).
Results
Purification of the amine oxidase from fenugreek
As a starting material for the isolation of amine oxidase,7-d-old etiolated seedlings of fenugreek (after removal ofpeels and roots) were used. After homogenization of theseedlings, the crude homogenate was fractionated withammonium sulphate, which at 30% saturation enabledremoval of contaminating proteins, whereas the enzymewas completely precipitated between 30-65% saturation.Then after short heating at 60 °C for 5 min, the totalprotein content decreased to about one-third, whereas thetotal activity of the enzyme diminished only slightly. Thecrude enzyme preparation was further purified by DEAE-cellulose chromatography and collected as a passing frac-tion that was directly loaded on to a hydroxyapatitecolumn. Remaining impurities, mainly proteins of lowermolecular mass, were at last removed by size-exclusionchromatography on a Sephacryl S-300 HR. The finalpreparation had the specific activity 915 nkatmg"1 withputrescine as a substrate. Purification grade was 52-foldand yield approached 42% of the total activity in thecrude extract. The overall purification course is shownin Table 1.
Fenugreek amine oxidase 1899
Molecular properties of the fenugreek amine oxidase
In addition to the sharp protein absorbance maximum at280nm (<F28O = 226 000 M"1 cm"1), the visible spectrumof the enzyme showed a broad absorption peak around500 nm, which confers a pink colour to the enzyme. Theabsorption maximum at 500 nm is characterized by amolar extinction coefficient of 4100 M"1 cm"1.
SDS-PAGE of the enzyme produced a single proteinband corresponding to the molecular mass of 75 kDa, asshown in Fig. la. Determination of the molecular massunder non-denaturing conditions on a Superdex 200 HRshowed 155 kDa, which together with the mass of a singlesubunit deduced from SDS-PAGE, confirms the dimericstructure of the native enzyme. However, a small percent-age of enzymatically active protein appeared as a high
a) b)
kDa
30
20.1
14.4Fig. 1. Electrophoretic properties of fenugreek amine oxidase. (a)SDS—PAGE of the fenugTeek amine oxidase (4.6 ftg), protein stainingwith Coomassie Bnlliant Blue G-250; (b) quinone staining of Aspergillusmger AKU 3302 amine oxidase AO-II (1), AO-1 (2), and (3) fenugreekamine oxidase (10 / g each).
Table 1. Purification of the amine oxidase from etiolated fenugreek seedlings
Purification step
Crude extract(NH4)2SO4
30% saturation(NH4)2SO4
65% sat., dialysisControlled heat
denaturationDEAE-cellulose
chromatographyHydroxyapatite
chromatographySephacryl S-300 HR
chromatography
Volume(ml)
19202070
120
105
190
49
1.5
Total activityUkat)
31.8529.33
24.63
20.07
18.15
16.24
13.38
Total protein(mg)
18201115
610
206
49
25
14.6
Specific activity(nkatmg"1)
17.526.3
40.4
97,4
370.4
649.6
916,4
Purification grade(-fold)
11.5
2.3
5.6
21.2
37.1
52.4
Yield(%)
10093
77
63
57
51
42
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
1900 Sebe/a et al.
molecular weight aggregate of about 300 kDa in theelution profile, probably due to a partial tetramericarrangement of the subunits in the native state. Thenature of this peak was confirmed by measuring theabsorption spectrum using a diode array detector. Similaraggregation was observed with an enzyme preparationthat had been derivatized with /7-nitrophenylhydrazine.
The isoelectric point of the fenugreek amine oxidasewas determined by isoelectric focusing on a polyacryl-amide gel, where a pi value of 6.8 was found. The enzymeis a glycoprotein containing 8% neutral sugars.
In the previous study, the jV-terminus of the fenugreekamine oxidase could not be sequenced after SDS-PAGEand electroblotting on to a polyvinylidene difluoridemembrane (Luhova et al., 1995), therefore the nativeenzyme was used for the sequencing. Samples of theenzyme were adsorbed either to polybrene-coated glassfibre filters or soaked to polyvinylidene difluoride mem-branes which had previously been treated with methanoland polybrene that allowed identification of twenty aminoacids, yielding the sequence VKQPLHFQHPLDPLTKEEFV as shown in Table 2.
Substrate specificity of the fenugreek amine oxidase and its
inhibitors
As can be seen from Table 3, the fenugreek amine oxidaseshows a broad substrate specificity. The best substrateswere aliphatic diamines like cadaverine, putrescine, cysta-mine and 1,6-diaminohexane and polyamine agmatine.On the other hand, ethylenediamine and 1,3-diaminopropane were only weakly oxidized. The polyam-ine spermidine together with hydroxyderivatives of putres-cine and cadaverine and both 1,4-diamino-2-butenes wereconverted with lower efficiency. Very low activity wasobserved towards aromatic monoamines, whereas ali-phatic monoamines n-hexylamine, w-propylamine or isop-ropylamine and amino acids lysine, ornithine and argininewere not oxidized at all. For the best substrates, kineticconstants Km, VmA^ and pH optima, shown in Table 4,were determined. Values of Km in the range of
0.09-0.95 mM and of 0.06-1.10 ga t ing" 1 werefound with pH optima between pH 6.8-7.8 slightlydiffering for individual substrates.
The enzyme was inhibited by carbonyl reagents, some
Table 3. Substrate specificity of the fenugreek amine oxidase
Activities were measured using a coupled reaction with horseradishperoxidase and guaiacol according to Frebort et al. (1989) in 0.1 Mpotassium phosphate buffer, pH 7.0 at 30 °C. The final concentrationof substrates in the reaction mixture was 2 5 mM. The rate of putrescineoxidation was arbitrarily taken as 100%.
substrate analogues, alkaloids, copper complexing agents,lathyrogenes and by several other compounds. The inhibi-tion data obtained are given in Table 5. From the groupof substrate analogues, diaminoketones 1,5-diamino-3-pentanone and 1,4-diamino-2-butanone (A^,~10~8M)were the most potent reversible inhibitors. The carbonylreagent aminoguanidine, which is an analogue of thesubstrate agmatine, also showed a strong inhibition. Theinhibitions by diaminoketones and aminoguanidine werecompetitive. Copper complexing agents, with an exceptionof sodium azide, were all non-competitive inhibitors. K{
values ~10~5M were determined for 2,2'-bipyridyl,diethylenetriamine, 8-hydroxyquinoline, o-phenanthro-line, and potassium cyanide. The inhibition effects ofimidazole and sodium azide were substantially weaker(A ,~ 10~2 M). Sodium azide was the only uncompetitiveinhibitor found. Triethylenetetramine was the strongestinhibitor from copper chelators with a micromolar inhibi-
Table 2. N-terminal amino acid sequences of several plant seedlings copper-containing amine oxidases
/V-terminal amino acid sequence of fenugreek amine oxidase was obtained by automated Edman degradation, for the enzymes from pea and lentilseedlings the sequences were translated from cloned cDNA.
This workTipping and McPherson (1995)Rossi et al. (1992)
" Numbering corresponds to cDNA sequence.
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
Fenugreek amine oxidase 1901
Table 4. Kinetic parameters of the fenugreek amine oxidase for the best substrates
Activity of amine oxidase was measured using a coupled reaction with horseradish peroxidase and guaiacol according to Frebort et aL (1989) in0.1 M potassium phosphate buffer, pH 7 0, under air saturating conditions. For pH optimum determination, 0.1 M potassium phosphate buffers ofpH 5.5-8.5 (pH interval of 0.5) were used. The final substrate concentrations were between 0.1 mM and 2.0 mM. Kinetic constants (A ,, Vm^) w e r e
calculated from initial rates using the program GraFit 3.0.
Table 5. Inhibitors of the fenugreek amino oxidase
The enzyme was preincubated 0-15 min with individual inhibitors, then the reaction was started by adding putrescine (final concentration in therange of 0 1—0.5 mM) and the activity was measured by a coupled reaction with horseradish peroxidase and guaiacol. Inhibition constants werecalculated from double reciprocal plots for three inhibitor concentrations using GraFit 3.0.
Other inhibitorsHydroxylamineAcetone oximeBenzamide oximePargyline
0.0200.0251.1"
1.35.7
172589
1600038
10000
210150
5.00.16'
1.5"2 8002 8002000
00
15
55
100
10000
00
015
151000
ccc
NCNCNCNCNCNCNC
uccccNC
NCNCNCC
"Apparent value, irreversible inhibition.
tion constant. Two of the alkaloids tested, cinchonineand L-lobeline, were competitive inhibitors of the enzymewith K, values of 0.15 and 0.21 mM, respectively. Thefenugreek alkaloid trigonelline did not inhibit the enzyme./3-Aminopropionitrile was a competitive inhibitor with amicromolar inhibition constant, aminoacetonitrile inhib-ited non-competitively (K{ = 0.2 ^M). Hydroxylamine wasan efficient non-competitive inhibitor (A^~10~6M),whereas acetone oxime and benzamide oxime showed
weaker effects (A ,weak inhibition (K{
10 3 M ) . Pargyline caused only a10"3 M).
Characterization of the active site of the fenugreek amineoxidase
The occurrence of topa quinone as the cofactor of bovineserum (Janes et aL, 1990), pea seedlings (Janes et al.,1992), and others has been confirmed unambiguously.
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
1902 Sebe/aetal.
On the other hand, the identity of the cofactor of thefenugreek amine oxidase was not clear at all from theprevious work (Luhova et al., 1995). Therefore, a simpletest for the presence of a quinone cofactor, based on itsability to generate a formazan dye from nitroblue tetrazol-ium and glycine under alkaline conditions, was performedwith the fenugreek enzyme, Aspergillus niger amine oxid-ase AO-I as a quinoprotein standard, and bovine serumalbumin as a blank. Absorption maxima at 530 nm wereobserved with absorbances of 2.04 and 0.56 for theAspergillus (2.97 mg ml"1) and fenugreek amine oxidase(0.83 mg ml"1), respectively. Using the molecular massof subunit of 75 kDa for both enzymes, the calculatedmolar extinction coefficients 51.5 and 50.6 mM"1 cm"1
are in very good agreement. These results suggest thatthe structure of the fenugreek enzyme contains one qui-none moiety per subunit as well as the Aspergillus enzyme.After electrophoresis on a non-denaturing polyacrylamidegel and electroblotting to a nitrocellulose membrane, theenzymes gave positive quinone staining as shown inFig. lb.
Phenylhydrazines, which are potent inhibitors ofcopper-containing amine oxidases, react irreversibly withthe carbonyl group of the quinonic cofactor. Because ofthe formation of a coloured Schiff's base, the reactioncan be applied for spectrophotometric titration of theactive site. Titrating the fenugreek amine oxidase withphenylhydrazine, the ratio of 0.73 mol of the reagent permol of the enzyme subunit was found with a molarabsorption coefficient of coloured phenylhydrazone of30.8 mM"1 cm"1 per enzyme subunit at 442 nm. Thetitration with />-nitrophenylhydrazine revealed the stoichi-ometry of 1.08 mol of the reagent per mol of the enzymesubunit with a molar absorption coefficient of30.1 mM"1 cm"1 per subunit at 463 nm. In both cases,the enzyme activity was completely inhibited at the pointof equivalence obtained from inflection of the increase inabsorbance. Absorption maximum at 463 nm in pH 7.0of the enzyme />-nitrophenylhydrazone shifted to 585 nmin 0.5 M NaOH. The existence of this spectral shift hasbeen published for />-nitrophenylhydrazones of severalcopper-containing amine oxidases (Janes et al., 1992),and can be considered as indirect proof of the presenceof topa quinone. The /?-nitrophenylhydrazone of a modelcompound, 2-hydroxy-5-methyl-l,4-benzoquinone, pro-vided similar spectral characteristics showing an absorp-tion maximum at 470 nm in pH 7.0 and at 610 nm in0.5 M NaOH.
Resonance Raman spectroscopy with an excitationlaser at 457.9 nm has been frequently used for confirma-tion of the topa quinone structure in the intact enzymeby comparing a phenyLhydrazine or /7-nitrophenylhydra-zine derivatized enzyme with a derivatized model com-pound (Brown et al., 1991). Resonance Raman spectraof/7-nitrophenylhydrazones of the fenugreek amine oxid-
ase and the model compound (2-hydroxy-5-methyl-l,4-benzoquinone) are presented in Fig. 2. Resonance Ramanspectrum of non-derivatized topa quinone (prepared byair oxidation of 2,4,5-trihydroxyphenylalanine) is shownfor comparison. Two intense peaks of /7-nitrophenylhy-drazone v(N = N) around 1600 and 1330 cm"1 in thespectra of ^-nitrophenylhydrazones of the enzyme and2-hydroxy-5-methyl-l,4-benzoquinone replace the v(C =O) peak at 1676 cm"1 of topa quinone. Other peaks ofthe enzyme />-nitrophenylhydrazone at 1620, 1437, 1402,1264, and 1168cm"1 correspond well with the peaks offree topa quinone at 1633, 1433, 1395, 1254, and1190 cm"1.
All known copper-containing amine oxidases are char-acterized by Type 2 Cu-EPR spectra (Mclntire andHartmann, 1992). The results obtained with the nativefenugreek amine oxidase and its ^-nitrophenylhydrazoneare presented in Fig. 3. Both measured spectra are inaccordance with characteristic spectra of Cu(II) in atetragonal coordination, with a g± of 2.05-2.07, a gx
of 2.28-2.30 and an \AX\ of 15-16 mT. The coppercontent in the enzyme and its /?-nitrophenylhydrazonederivative was estimated by comparing the double integralof the spectra with that of 0.25 mM CuSO4.5H2O and25 mM Na2EDTA in the same buffer. On the basis ofthis approach, the copper content was 1.9 mol mol"1 ofnative enzyme and 1.0 mol mol"1 of />-nitrophenylhy-drazone of the enzyme. Besides the copper signal, Mn(II)ions were clearly detected in both spectra. Five signals ofthe typical manganese sixtet that fit in the range of themagnetic field measured withg value of 2.01 and \A\ of9.3 mT were found. Using atomic absorption spectro-metry the copper and manganese contents 2.0 and0.2 mol mol"1 of native enzyme, respectively, weredetermined.
It has been shown that using differential pulse polaro-graphy, copper-containing amine oxidase from pea seed-lings provides current signals that could be assigned tothe reduction of the copper and topa quinone bound inthe active site (Sebela et al., 1996). Native fenugreekamine oxidase in 50 mM potassium phosphate buffer,pH 7.0, provided current maxima at —1.23, —0.55 and— 0.07 V as shown in Fig. 4, which increased about twotimes after short-time proteolysis of the enzyme bysubtilisin. The first peak at — 1.23 V was absolutelydominant and together with the second (at —0.55 V)showed a good dependence on sample concentration upto 10
Discussion
Previous purification technique of the enzyme from fenu-greek (Luhova et al., 1995), included a precipitation ofcontaminating proteins in the crude homogenate by riv-anol lactate. However, the decoloration of the intensely
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
Fenugreek amine oxidase 1903
(i)
1700 1600 1500 1400 1300Raman shift (cm1)
1200 1100 1100 1000 900 800 700Raman shift (cnv1)
600 500
Fig. 2. Resonance Raman spectra of the /7-nitrophenylhydrazone of fenugreek amine oxidase and model compounds in 10 mM potassium phosphatebuffer, pH 7.0. The spectra of enzyme />-nitrophenylhydrazone (1) and 2-hydroxy-5-methyl-l,4-benzoquinone (2) were excited at 457.9 nm and thespectrum of non-derivatized topa quinone (3) at 488.0 nm.
yellow supernatant by charcoal after the precipitationwas very difficult and had to be repeated several times.Instead, this new purification method uses a two-stepfractionation of the enzyme from crude extracts by ammo-nium sulphate, followed by a controlled heat denaturationprior to chromatography on DEAE-cellulose, hydroxya-patite and Sephacryl S-300 HR. The homogeneity of thefinal preparation was verified by SDS-PAGE and FPLCon a Superdex 200 HR column. It was also confirmed byunambiguous results of N-terminal sequencing of thenative enzyme. The specific activity with substrate putres-cine of 915 nkat mg"1 for homogeneous enzyme is almost10-fold higher than with the previous preparation(Luhova et al., 1995), and comparable to other plantamine oxidases (Padiglia et al., 1991; Wimmerova et al.,1993).
The molecular properties of the fenugreek amine oxid-ase are very similar to those of other copper-containingamine oxidases, especially to the plant ones. The homo-geneous enzyme is pink-coloured due to a broad absorp-tion peak centred around 500 nm with a molar absorptioncoefficient 4100 M"1 cm"1 comparable to that of enzymesfrom lentil and pea seedlings (Medda et al., 1995), andbacterial amine oxidase from Escherichia coli (Steinebachet al., 1996). The occurrence of native fenugreek amine
oxidase as a dimer with a molecular mass of 155 kDa,which can be dissociated into 75 kDa subunits onSDS-PAGE is in agreement with reports for other plantamine oxidases (Medda et al., 1995). Previous speculationabout a monomeric existence of native fenugreek amineoxidase with a molecular mass of 80 kDa for the subunit(Luhova et al., 1995), thus seems to be incorrect. Sincethis value roughly corresponds to the molecular mass ofthe enzyme subunit determined by us now, it is likely thatthe enzyme structure was damaged due to the purificationmethod used at that time. Evidently, there was somenegative effect of the rivanol-acridine dye used in theprevious procedure for the precipitation of ballast pro-teins. This assumption is supported by the fact that theenzyme prepared earlier was yellow-coloured and thecofactor, presumably reduced, did not react with p-nitrophenylhydrazine (Luhova et al., 1995).
The amine oxidase from fenugreek is a slightly acidicprotein and its isoelectric point is close to the pi valuesdetermined for other plant copper-containing amineoxidases, particularly to that of pea seedling amine oxid-ase (Medda et al., (1995), and in accordance with resultsdescribed in the previous work by Luhova et al. (1995).Similar to other copper-containing amine oxidases, theenzyme is glycoprotein, although its carbohydrate content
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
1904 Setoe/aetal.
260 280 300 320
Magnetic field (mT)
340
Fig. 3. EPR spectra of the fenugreek amine oxidase. (1) Native enzyme(0.163 raM), (2) the enzyme ^-nitrophenylhydrazone (0.323 mM).Experimental conditions: microwave power 10 mW, frequency9 234 GHz, modulation 0.63 mT. Spectral charactenstics: Cu(II) signal,(1) g± = 2.056, g, = 2296, \A,\ = 15.3 mT; (2) g± = 2.063, g, = 2.285,M|| = 15.5mT; Mn(II) signal (marked by the arrows), g = 2 01, \A\ =9.3 mT. Spectra are offset for clarity.
Potential (V)
Fig. 4. Differential pulse polarograms of 5 /im native pea seedlingsamine oxidase (1), 5 ^M native fenugreek enzyme (2) and 25 fiM topaquinone (3) recorded in 50 mM potassium phosphate buffer, pH 7.0.
(8%) is about one-third lower than that reported forother plant copper-containing amine oxidases (12-14%)(Medda et al, 1995). Protein sequence analysis of thefenugreek amine oxidase resulted in the identification ofthe first twenty N-terminal amino acids. This part of theprimary structure shows a high degree of identity to thatof pea and lentil seedling amine oxidases (80% and 70%identity in a 16 and 14 amino acid overlap, respectively)and the enzymes of microbial origin, but differs consider-ably from mammalian enzymes (Frebort and Adachi,1995). Since the identification of amino acids in therespective individual cycles of Edman degradation wasunambiguous with only one amino acid per cycle detected,the results can also be taken as a further confirmation ofthe identity of the two enzyme subunits.
As well as other copper-containing amine oxidases, theenzyme from fenugreek seedlings shows broad substratespecificity. However, from a series of amines tested, onlya few can be considered as efficient substrates. In accord-ance with the enzymes of plant origin, diamines andpolyamines are readily converted. Monoamines, whichare the major substrates of mammalian and microbialamine oxidases, are also oxidized, but at a much lowerrate. The enzyme is sensitive to a broad spectrum ofinhibitors of plant copper-containing amine oxidases(Medda et al, 1995; Luhova et al., 1996). The mostpotent inhibitors are substrate analogues such as diamino-ketones, compounds which inhibit pea seedling amineoxidase as well (Skyvova and Macholan, 1970), andwhich are oxidized very slowly compared to the usualsubstrates. A considerable inhibition was also observedwith copper complexing agents that inhibit pea amineoxidase (Hill and Mann, 1962; Pec and Frebort, 1992).The remarkable difference in the type and extent ofinhibition of two monovalent anions, e.g. cyanide andazide, cannot easily be explained by the simple removalof copper from the enzyme. Whereas cyanide is a potentnon-competitive inhibitor of the enzyme, azide inhibitsvery weakly and in an uncompetitive manner. It is likelythat cyanide reacts with Cu(I) of Cu(I)/topa-semiquinone complex, which is an intermediate in theactive site of copper-containing amine oxidases duringthe catalytic cycle of the substrate reduced enzyme(Dooley et al., 1991). The enzyme is sensitive to lathyrog-enes, which are known as strong inhibitors of lysyl oxidase(Narayan et al, 1972; Tang et al, 1983), and plasmaamine oxidase (Maycock et al, 1975; Raimondi et al,1985). j9-Aminopropionitrile showed competitive inhibi-tion, however, it was not oxidized like a substrate asobserved for the diaminoketones. On the other hand,non-competitive inhibition of the enzyme by aminoace-tonitrile may suggest a possible reaction with the enzyme-bound copper. The enzyme was also competitively inhib-ited by some alkaloids, which do not carry a free primaryamino group. Similar to the amine oxidase from pea
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
seedlings (Luhova et al., 1996), this inhibition might becaused by binding to a hydrophobic pocket in the vicinityof the cofactor, thus blocking the active site. From thegroup of other inhibitors tested, a strong inhibition wasmeasured for hydroxylamine, but the effects of its derivat-ives (oxime and amide oxime) were substantially weaker.Finally, pargyline, a typical potent inhibitor of flavin-containing amine oxidases (Chuang et al., 1974), wasfound to be a very weak inhibitor.
The presence of the quinone cofactor was confirmedby quinone staining, furthermore spectrophotometrictitrations with phenylhydrazines provided additionalinformation that the enzyme contains one reactive car-bonyl group per subunit. Although the titration withphenylhydrazine revealed a stoichiometry of only 1.5 molof the reagent per mol enzyme (that is similar to otherreports in the literature (Janes and Klinman, 1991;Steinebach et al., 1995, 1996), the reaction of the enzymewith /7-nitrophenylhydrazine showed a more accuratemolar ratio close to two. There have been many controver-sial results on the reactivity of cofactor carbonyl groupsin copper-containing amine oxidases. However, it is gener-ally accepted that there are two reacting sites per dimer,although in some cases the reactivity with phenylhydra-zines is limited to only one of them because of stericalhindrance due to structural changes induced by thephenylhydrazone formed at the first site (Morpurgo et al.,1992; Frebort et al., 1995). The molar absorption coeffi-cients of the phenylhydrazone (at 442 nm) and the p-nitrophenylhydrazone (at 463 nm) of the amine oxidasefrom fenugreek are similar to those published for othercopper-containing amine oxidases (Medda et al., 1995;Janes and Klinman, 1991; Fr6bort et al., 1994; Steinebachet al., 1995, 1996). It has been shown, that the pH shiftof the absorption maximum of the^-nitrophenylhydrazinederivative of a copper-containing amine oxidase can betaken as an indirect proof for the presence of topaquinone as the cofactor (Janes et al., 1992). This shift,previously confirmed for several enzymes of this class,was also observed with the /?-nitrophenylhydrazone ofthe fenugreek amine oxidase. Ultimate evidence for thepresence of topa quinone in the active site was obtainedby resonance Raman spectroscopy. This establishedmethod can be looked at as a reliable and sensitivetechnique for the identification of topa quinone in copper-containing amine oxidases. As it has been reported for anumber of enzymes, Raman spectra of their p-nitrophenylhydrazones correspond to those of p-nitrophenylhydrazine derivatives of topa quinone modelcompounds (Brown et al., 1991). On the basis of thisstudy's experimental findings, the conclusion was there-fore drawn that topa quinone is indeed the cofactor ofthe fenugreek enzyme.
Recent differential pulse polarographic studies on peaseedlings amine oxidase have shown that this method can
Fenugreek amine oxidase 1905
serve for the identification of copper/quinone-containingactive sites in the enzyme (Sebela et al., 1996). In accord-ance with pea seedling amine oxidase, native fenugreekenzyme provided characteristic current signals of thereduction of the enzyme-bound copper ions at —1.2Vand, as well, of the quinone cofactor at —0.6 V. Thethird distinct signal (at —0.1 V) is probably caused byfree Cu(II) ions in the hydrated form which were liberatedfrom the enzyme (Sebela et al., 1996). The enhancementof the signal of the enzyme cofactor after a short-timeproteolysis by subtilisin and its similarity with the signalof free topa quinone allow confirmation of the identityof the cofactor as topa quinone.
The EPR spectroscopic parameters of native fenugreekamine oxidase and its /?-nitrophenylhydrazine are consist-ent with the presence of Cu(II) in a tetragonalco-ordination with axial symmetry as published for otheramine oxidases (Dooley et al., 1991; Matsuzaki et al.,1995; Steinebach et al., 1996). Estimation of the coppercontent of the native enzyme and the results of spectro-photometric titrations indicate the existence of oneCu(II)/topa quinone site in each subunit of the dimericenzyme. The c. 50% lower content of Cu(II) in theenzyme /7-nitrophenylhydrazone is somewhat unexpectedand could be attributed only to a release of Cu(II) fromthe active site of the derivatized enzyme. The observationof signals of Mn(II) ions in the EPR spectra of bothenzyme and and its /7-nitrophenylhydrazone was, fromthe beginning, somewhat surprising and it was thoughtto be an impurity in the samples. However, the signalpersisted even in enzyme samples that were repeatedlypurified by FPLC using metal-free buffers. The explana-tion came with the recently published crystal structure ofpea seedling amine oxidase which revealed the presenceof a second metal site close to the protein surface whichmight be occupied by manganese (Kumar et al., 1996).This site is highly conserved among amine oxidases fromvarious sources and it also exists in the E. coli amineoxidase where it is thought to be occupied by Ca(II)(Parsons et al., 1995). In the past, Mn(II) signals werefound in the amine oxidase from human placenta, whichwas originally reported as a Cu(II)Mn(II) metalloprotein(Crabbe et al., 1976). Considering the close sequencesimilarity of Fabaceae amine oxidases, the EPR dataprovide experimental evidence for the existence of anothermetal site in the fenugreek enzyme. From the resultsobtained by atomic absorption spectrometry, however,only about 10% of these sites is ocupied by Mn(II) inthe native enzyme. Remaining sites may be occupied byCa(II) (as in E. coli) or other bivalent cationts. Thereforeany role of manganese in the catalytic cycle of the amineoxidase is unlikely.
Following from the above discussion, the recently foundplant amine oxidase from fenugreek seedlings can berapidly purified using the improved purification protocol,
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
1906 3ebe/a etal.
resulting in a high yield of homogeneous enzyme withhigh specific activity. On the basis of previous results(Luhova et ai, 1995), a novel monomeric amine oxidasein fenugreek was being sought, but detailed molecularand kinetic studies revealed its close relationship to otherknown plant amine oxidases and confirmed that theenzyme belongs to the ubiquitous class of copper/topaquinone-containing amine oxidases. The broad specificityof amine oxidase can be exploited for industrial produc-tion of food ingredients, e.g. vanillin from vanillylamineas described recently for the amine oxidase fromAspergillus niger (Yoshida et al., 1997). For such apurpose, the plant amine oxidase looks even more promis-ing due to its high specific activity (10-50 times higherthan for microbial enzymes) and relative thermal stability.
Acknowledgements
The authors thank Professor Lumir Macholan from MasarykUniversity in Brno for his sincere encouragement throughoutthis study, Professor Jan K.aS from Institute of ChemicalTechnology in Prague and Professor Mathias Sprinzl fromUniversity of Bayreuth for arranging the amino acid sequenceanalysis in the frame of TEMPUS II S JEP 07599-94 project,Dr Marie Studniikova from Masaryk University in Brno forkind help with the polarographic measurements, ProfessorOsamu Yamauchi and Mr Masaaki Endo from NagoyaUniversity for help with the EPR spectroscopy, Professor TeizoKitagawa from Institute of Molecular Science in Okazaki forarranging Raman spectroscopy measurements, Professor OsaoAdachi from Yamaguchi University for his kind support andMrs Jana Piknova from the Department of Biochemistry,Faculty of Science, Palacky University in Olomouc, for hertechnical assistance. This work was supported by grants 303/94from the Ministry of Education and 203/97/0097 from theGrant Agency, Czech Republic, and IG 31803005 from theFaculty of Science, Palacky University in Olomouc.
References
Bradford MM. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Analytical Biochemistry72, 248-54.
Brown DE, McGuirl M, Dooley DM, Janes SM, Mu D, KlinmanJP. 1991. The organic functional group in copper-containingamine oxidases. Journal of Biological Chemistry 266, 4049-51.
Chuang HYK, Patek DR, Hellerman L. 1974. Mitochondrialmonoamine oxidase. Inactivation by pargyline. Adductformation. Journal of Biological Chemistry 249, 2381—4.
Crabbe MJ, Waight RD, Bardsley WG, Barker RW, Kelly ID,Knowles PF. 1976. Human placental diamine oxidase.Improved purification and characterization of a copper- andmanganese-containing amine oxidase with novel substratespecificity. Biochemical Journal 155, 679-87.
Dooley DM, McGuirl MA, Brown DE, Turowski PN, MclntireWS, Knowles PF. 1991. A Cu(I)-semiquinone state insubstrate-reduced amine oxidases. Nature 349, 262-4.
Dubois M, Gilles KA, Hamilton JK, Rebes PA, Smith F. 1956.Colorimetric method for determination of sugars and relatedsubstances. Analytical Chemistry 28, 350-6.
Frebort I, Adachi O. 1995. Copper/quinone-containing amineoxidases, an exciting class of ubiquitos enzymes. Journal ofFermentation and Bioengineering 80, 625-32.
Frebort I, Haviger A, Pec P. 1989. Employment of guaiacol forthe determination of activities of enzymes generating hydro-gen peroxide and for the determination of glucose in bloodand urine. Biologia (Bratislava) 44, 729-37.
Frebort I, Pec P, Luhova L, Matsushita K, Toyama H, AdachiO. 1994. Active-site covalent modifications of quinoproteinamine oxidases from Aspergillus niger. European Journal ofBiochemistry 225, 959-65.
Frebort I, PeC P, Luhova L, Toyama H, Matsushita K, HirotaS, Kitagawa T, Ueno T, Asano Y, Kato Y, Adachi O. 1996*.Two amine oxidases from Aspergillus niger AKU 3302contain topa quinone as the cofactor: unusual cofactor linkto the glutamyl residue occurs only at one of the enzymes.Biochimica et Biophysica Ada 1295, 59-72.
Frebort I, Tamaki H, Ishida H, Pef P, Luhova L, Tsuno H,Halata M, Asano Y, Kato Y, Matsushita K, Toyama H,Kumagai H, Adachi O. 1996a. Two distinct quinoproteinamine oxidases are induced by rt-butylamine in the myceliaof Aspergillus niger AKU 3302. Purification, characterization,cDNA cloning and sequencing. European Journal ofBiochemistry 237, 255-65.
Frebort I, Toyama H, Matsushita K, Adachi O. 1995. Half-sitereactivity with /7-nitrophenylhydrazine and subunit separationof the dimeric copper-containing amine oxidase fromAspergillus niger. Biochemistry and Molecular BiologyInternational 36, 1207-16.
Hill JM, Mann PJG. 1962. The inhibition of pea-seedlingdiamine oxidase by chelating agents. Biochemical Journal85, 198-207.
Janes SM, Klinman JP. 1991. An investigation of bovine serumamine oxidase active site stoichiometry: evidence for anaminotransferase mechanism involving two carbonyl cofac-tors per enzyme dimer. Biochemistry 30, 4599-605.
Janes SM, Mu D, Wemmer D, Smith AJ, Kaur S, Maltby D,Burlingame AL, Klinman JP. 1990. A new redox cofactor ineukaryotic enzymes: 6-hydroxydopa at the active site ofbovine serum amine oxidase. Science 248, 981-7.
Janes SM, Pakic MM, Seaman CH, Smith AJ, Brown DE,Dooley DM, Mure M, Klinman JP. 1992. Identification oftopaquinone and its consensus sequence in copper amineoxidases. Biochemistry 31, 12147-54.
Laemmli UK. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 277, 680-5.
Luhova L, Slavik L, Frebort I, Halata M, Pec P. 1995. Aminooxidase from Trigonella foenum-graecum seedlings.Phytochemistry 38, 23-5.
Luhova L, Slavfk L, Frebort I, Sebela M, Zajoncova L, Pec P.1996. Comparison of kinetic properties between plant andfungal amine oxidase. Journal of Enzyme Inhibition 10,251-62.
Macholan L. 1965. Darstellung der 2-hydroxyderivative descadaverins und putrescins. Collection of CzechoslovakChemical Communications 30, 2074-9.
Macholan L. 1972. 3-Hydroxycadaverine, a new substrate fordiamine oxidase, Enzymologia 42, 303-15.
Macholan L. 1974. Selective and reversible inhibition of diamineoxidase by 1,5-diamino-3-pentanone. Collection ofCzechoslovak Chemical Communications 39, 653-61.
Macholan L, Hubalek F, Subova H. 1975. Oxidation of
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
l,4-diamino-2-butene to pyrrol, a sensitive test of diamineoxidase activity Collection of Czechoslovak ChemicalCommunications 40, 1247-58.
Matsuzaki R, Fukui T, Sato H, Ozaki Y, Tanizawa K. 1994.Generation of the topa quinone cofactor in bacterial monoam-ine oxidase by cupric ion-dependent autooxidation of aspecific tyrosyl residue. FEBS Letters 351, 360^4.
Matsuzaki R, Suzuki S, Yamaguchi K, Fukui T. Tanizawa K.1995. Spectroscopic studies on the mechanism of the topaquinone generation in bacterial monoamine oxidase.Biochemistry 34, 4524-30.
Maycock AL, Suva RH, Abeles RH. 1975. Novel inactivatorsof plasma amine oxidase. Journal of American ChemicalSociety 97, 5613-4.
Mclntire WS, Hart maun C. 1992. Copper-containing amineoxidases. In: Davidson VL, ed. Principles and applications ofquinoproteins. New York: Marcel Dekker, 97-171.
Medda R, Padiglia A, Floris G. 1995. Plant copper-amineoxidases. Phytochemistry 39, 1-9.
Morpurgo L, Agostinelli E, Mondovi B, Avigliano L, Silvestri R,Stefancich G, Artico M. 1992. Bovine serum amine oxidase:half-site reactivity with phenylhydrazine, semicarbazide andaromatic hydrazides. Biochemistry 31, 2615-21.
Narayan AS, Siegel RC, Martin G. 1972. On the inhibition oflysyl oxidase by /3-aminopropionitrile. Biochemical andBiophysical Research Communication 46, 745-51.
Padiglia A, Cogoni A, Floris G. 1991. Characterization of amineoxidases from Pisum, Lens, Lathyrus, and deer.Phytochemistry 30, 3895-7.
Parsons MR, Convery MA, Wilmot CM, Yadav KDS, BlakeleyV, Corner AS, Phillips SEV, McPherson MJ, Knowles PF.1995. Crystal structure of a quinoenzyme: copper amineoxidase of Escherichia coli at 2 A resolution. Structure3, 1171-84.
Paz M, FlOckiger R, Boak A, Kagan HM, Gallop PM. 1991.Specific detection of quinoproteins by redox-cycling staining.Journal of Biological Chemistry 266, 689-92.
Pe£ P, Chudy J, Macholan L. 1991. Determination of theactivity of diamine oxidase from pea with Z-1,4-diamino-2-butene as a substrate. Biologia (Bratislava) 46, 665-72.
Fenugreek amine oxidase 1907
Pet P. Frebort I. 1992. Some amines as inhibitors of peadiamine oxidase. Journal of Enzyme Inhibition 5, 323-9.
Raimondi L, Banchelli G, Bertocci B, Lodovici M, Ignesti G,Pirisino R, Buffoni F, Bertini V, De Munno A. 1985. Reactionof pig plasma benzylamine oxidase with beta-aminopropionitrile. Agents and Actions 16, 95-8.
Rossi A, Petruzelli R, Finazzi-Agr6 A. 1992. cDNA-derivedamino-acid sequence of lentil seedlings' amine oxidase. FEBSLetters 301, 253-7.
Sebela M, Studnickova M, Wimmerova M. 1996. Differentialpulse polarograpbic study of the redox centres in pea amineoxidase. Bioelectrochemistry and Bioenergetics 41, 173-9.
Skyvova M, Macholan L. 1970. Inhibition of pea diamineoxidase by aliphatic amino ketones and their reaction withpyridoxal 5-phosphate. Collection of Czechoslovak ChemicalCommunications 35, 2345-54.
Steinebach V, Benen JAE, Bader R, Postma PW, De Vries S,Duinc JA. 1996. Cloning of the maoA gene that encodesaromatic amine oxidase of Escherichia coli W3350 andcharacterization of the overexpressed enzyme. EuropeanJournal of Biochemistry 237, 584-91.
Steinebach V, Groen BW, Wijmenga SS, Niessen WMA,Jongejan JA, Duine JA. 1995. Identification of topa quinoneas illustrated for pig kidney diamine oxidase and Escherichiacoli amine oxidase. Analytical Biochemistry 230, 159-66.
Tang S-S, Trackman PC, Kagan HM. 1983. Reaction of aorticlysyl oxidase with /J-aminopropionitrile. Journal of BiologicalChemistry 258, 4331-8.
Tipping AJ, McPherson MJ. 1995. Cloning and molecularanalysis of the pea seedling copper amine oxidase. Journal ofBiological Chemistry 270, 16939^46.
Wimmerova M, Glatz Z, Janiczek O, Macholan L. 1993.Improved chromatographic purification of pea seedlingsdiamine oxidase. Preparative Biochemistry 23, 303-19.
Yoshida A, Takenaka Y, Tamaki H, Suzuki H, LJeno T, KmnagaiH, Frebort I, Adachi O. 1997. Vanillin production fromvanillylamine by microbial amine oxidase. Proceedings ofKansai Branch Meeting of Japan Society for Bioscience,Biotechnology and Agrochemistry, Kyoto, Japan.
Downloaded from https://academic.oup.com/jxb/article-abstract/48/11/1897/560185by gueston 07 April 2018
![Fenugreek-Solorio Et Al. [FINAL]](https://static.documents.pub/doc/80x56/55cf9748550346d03390bee8/fenugreek-solorio-et-al-final.jpg)
