Báo cáo khoa học: An important lysine residue in copper⁄quinone-containing amine oxidases

An important lysine residue in copper⁄quinone-containing amine oxidases Anna Mura1, Roberto Anedda2, Francesca Pintus1, Mariano Casu2, Alessandra Padiglia1, Giovanni Floris1 and Rosaria Medda1

1 Department of Applied Sciences in Biosystems, University of Cagliari, Italy 2 Department of Chemical Science, University of Cagliari, Italy

Keywords amine oxidase; copper; NMR; quinoprotein; xenon

Correspondence R. Medda, Department of Applied Sciences in Biosystems, University of Cagliari, Cittadella Universitaria, I-09042 Monserrato (CA), Italy Fax: +39 070 6754524 Tel: +39 070 6754517 E-mail: rmedda@unica.it

(Received 4 October 2006, revised 27 February 2007, accepted 15 March 2007)

doi:10.1111/j.1742-4658.2007.05793.x

The interaction of xenon with copper ⁄ 6-hydroxydopa (2,4,5-trihydroxy- phenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediter- ranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. 129Xe chemical shift provided evi- dence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the ana- lyzed plant amine oxidases showed that the kc value of the xenon-treated enzymes was reduced by 40%. Moreover, whereas the measured Km value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the Km value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the kc value of the xenon-treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramat- ically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity.

One,

referred to as a ‘reductive half-reaction’, involves the oxidation of amine to aldehyde and the formation of a reduced form of the TPQ cofactor:

Eox þ R (cid:2) CH2 (cid:2) NHþ

3 ! Ered þ R (cid:2) CHO

The other, known as ‘the oxidative half-reaction’, involves the reoxidation of the enzyme and contem- poraneous release of ammonia and hydrogen peroxide:

Copper ⁄ quinone-containing amine oxidases [(deami- nating) (copper-containing) amine:oxygen oxidoreduc- tase; EC 1.4.3.6] (Cu ⁄ TPQ AOs) found in bacteria, yeasts, fungi, plants and mammals catalyze the oxida- tive deamination of primary amines to the correspond- ing aldehydes while reducing molecular oxygen to hydrogen peroxide [1]. The ping-pong catalytic mech- anism of Cu ⁄ TPQ AOs can basically be divided into two half-reactions.

Abbreviations AO, amine oxidase; AGAO, Arthrobacter globiformis amine oxidase; BSAO, bovine serum amine oxidase; Cu-AO, copper amine oxidase; DABY, 1,4-diamino-2-butyne; ELAO, Euphorbia characias amine oxidase; HPAO, Hansenula polymorpha amine oxidase; LSAO, lentil seedling amine oxidase; PKAO, pig kidney amine oxidase; PSAO, pea seedling amine oxidase; TPQ, 6-hydroxydopa(2,4,5-trihydroxyphenethylamine) quinone (TOPA); TPQaq, CuII-aminoquinol; TPQsq, CuI–semiaminoquinolamine radical; XRD, X-ray diffraction.

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Ered þ O2 þ H2O ! Eox þ NHþ

4 þ H2O2

each subunit

are homodimers;

quinone. This observed semiquinone radical has been postulated to be covalently linked to a lysyl e-amino group of the protein [11], even though this hypothesis was ruled out by the same authors in a later paper [12]. Again in A. globiformis AO, two lysine residues, Lys184 and Lys354, situated close to the entrance of a suitable channel through which substrates and prod- ucts can access and exit the TPQ active site, have been found to be essential for the catalytic activity of the holoenzyme [12], although they do not seem to be involved in the formation of TPQ in the apoenzyme [5,12].

A nucleophilic residue has been shown with cer- tainty to be involved in the inhibition mechanism of AOs during the oxidation of 1,4-diamino-2-butyne (DABY) [13,14], 1,5-diamino-2-pentyne [15], the aro- matic monoamine tyramine [16], and other selective AO inhibitors [17]. Although the involvement of a lysine has been postulated [14,17], compelling evidence has not been presented.

AOs (molecular mass @70–90 kDa) contains an active site composed of a tightly bound Cu2+ and a quinone of 2,4,5-tri- hydroxyphenylalanine (TPQ or TOPA) [2]. Six AOs [3–8] (including a lysyl oxidase, from Pichia pastoris) have been crystallized previously, and characterized by single-crystal X-ray diffraction (XRD). The well- defined active site within these enzymes presents the following peculiar structural and functional features (Table 1): (a) TPQ is derived from the copper-cata- lyzed oxidation of a post-translationally modified tyrosine residue in the consensus sequence Asn-Tyr- Asp ⁄ Glu of the polypeptide chain [9]; (b) the copper ion is coordinated with the imidazole groups of three conserved histidine residues and with two water mole- cules (equatorial We and axial Wa) ) TPQ is close but not bound to the Cu2+, and appears to have high rotational mobility; (c) after the amine nucleophilic attack, the proton abstraction requires the presence of a base, which has been identified in a conserved aspar- tate residue; (d) a tyrosine residue seems to play an important role in the active site as a result of its hydrogen bond to O-4 of TPQ.

Finally, an important lysine has been suggested in the active site the crystal structure of pea AO at forming a hydrogen bond with the phe- (Lys296), nolic group of TPQ [4], although Duff et al. [18] later demonstrated that the published crystal showed TPQ in a nonproductive ‘on-copper conformation’. The role of Lys296 in the ‘off-copper conformation’ is therefore still unclear. The ‘on-copper and off-cop- per conformations’ refer to the orientation of TPQ and copper, as is clearly described by Dawkes & Phillips [19].

tyrosine

in biologically

Moreover, several amino acid residues have been shown to be critical in the proper positioning of TPQ during catalysis [10]. One amino acid implicated in the catalytic mechanism of some Cu ⁄ TPQ AOs is a lysine residue (see below for references), although its func- tion is somewhat elusive. For example, in the recom- binant AO from Arthrobacter globiformis, during TPQ formation from the oxidation of an intrinsic tyrosine in the amino acid sequence due to a post-translational event, the copper ion catalyzes the insertion of an oxygen atom into the ring to generate dihydroxyphenylalanine, which, upon oxidation and through the formation of the CuI ⁄ semiquinone radical to dihydroxyphenylalanine intermediate, gives

rise

Table 1. Conserved amino acid residues in Cu ⁄ TPQ-containing AOs.

Enzyme

BSAO

PSAO

ECAO

HPAO

AGAO

TPQ Hys

387 442 444 603 300 286

466 524 526 689 383 369

405 456 458 624 319 305

382 431 433 592 290 284

470 519 521 683 385 371

Asp Tyr

It is well known that the noble gas xenon specifically interacts with the hydrophobic interior of proteins, and an increasing number of papers in the recent lit- erature confirm that 129Xe NMR spectroscopy is a very good technique for the characterization of cavities and channels related compounds [20–27]. Moreover, it is generally believed that xenon atoms can induce structural changes in some of the cavities or channels that they are bound to, both in solution [28] and in the solid state [29]. Xenon has been used as a probe for dioxygen-binding cavities in copper AOs by recording XRD data under pressure of xenon gas [7,18], and in a recent paper [30] we demon- strated that, under 10 atm of xenon gas, an AO from lentil seedlings can generate the free radical intermedi- ate of TPQ (TPQsq) in the absence of substrates, a process that probably involves a lysine residue at the active site. In this article, we investigate the binding of xenon to highly purified AOs from various sources, and our results strongly support the hypothesis that a lysine residue is implicated in the catalytic mechanism of plant enzymes.

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Results

129Xe chemical shift and spin-lattice relaxation time in AO solution

the

much smaller than the T1 value of xenon in the buffer (ELAO T1 ¼ 4.3 ± 0.5 s; PKAO T1 ¼ 5.5 ± 0.8 s; buffer T1 ¼ (cid:3) 500 s). These features, which were also observed in LSAO and other protein solutions [30], are due to the fast exchange of xenon between both speci- fic and nonspecific sites of the proteins and the buffer, and they also confirm that there is an interaction between the dissolved xenon and the interior of the protein. However, such 129Xe NMR experiments can- not provide a more detailed characterization of the interaction between xenon and the protein, and the actual location of a possible involved cavity or cavities remains unknown and would require further studies; this, however, is beyond the purpose of this work.

Owing to the high enzyme concentrations (0.25– 0.35 mm) and the low ionic strength (1 mm) of the buffer used in the experiments in the presence of xenon, we were unable to obtain significant results with bovine serum AO (BSAO), on account of its tendency to form an irreversible inactive precipitate under such experimental conditions.

Xenon-induced spectroscopic features in plant enzymes

corresponding

0.35 mm,

the TPQ cofactor,

In a recent paper [30], 129Xe chemical shift and spin- lattice relaxation time studies in the presence of lentil seedling AO (LSAO) showed that the chemical shift of 129Xe changes as a function of protein concentration (10.4 p.p.m.Æmm)1), and that relaxation time (T1 ¼ 3.2 s) is significantly reduced as compared to T1 in the buffer ((cid:3) 500 s). These changes are commonly used as a tool to produce evidence of xenon–protein interactions [30]. In the present study, three AOs [pea seedling AO (PSAO), Euphorbia characias AO (ELAO) and pig kidney AO (PKAO)] were tested by 129Xe NMR spectroscopy. Figure 1 shows the 129Xe NMR spectra of the PKAO and ELAO AOs compared with the 129Xe NMR spectra of LSAO and xenon dissolved in buffer solution. The presence of a single resonance in the protein solution indicates that xenon undergoes fast exchange in all available environ- ments. Under 10 atm of xenon gas, the 129Xe NMR in AO samples is shifted downfield (ELAO signal 3.96 p.p.m. per to 11.3 p.p.m.Æmm)1, and PKAO 1.4 p.p.m. per 0.15 mm, cor- responding to 9.4 p.p.m.Æmm)1) as compared to the resonance of the same amount of xenon in the buffer, which is used as a reference and set to 0 p.p.m. More- over, the T1 value of all native enzymes is found to be

the Owing to the presence of oxidized form of AOs has a distinctive pink color and absorbs in the visible region: BSAO shows an electronic absorption band at 476 nm (e476 ¼ 3800 m)1Æcm)1) [31], PKAO at 490 nm (e490 ¼ 4000 m)1Æcm)1) [32], PSAO and LSAO at 498 nm (e498 ¼ 4100 m)1Æcm)1) [33,34], and ELAO at 490 nm (e490 ¼ 6000 m)1Æcm)1) [35].

Addition of a substrate to a solution containing AOs in the absence of air caused the visible absorption band to disappear immediately, indicating the rapid forma- tion of a reduced TPQ intermediate, the TPQaq, which can behave differently in plant AOs and mammalian AOs. Hence, a successive different behavior occurs. In plant AOs, TPQaq equilibrates rapidly with the TPQsq species by transferring one electron to copper, which is in turn reduced from the cupric to the cuprous state, and the solution immediately turns yellow as a result of the formation of new absorption bands centered at 464, 434 and 360 nm [36] (Fig. 2). In PKAO, the transfor- mation of TPQaq to TPQsq was observed only in the presence of CN– [37] (Fig. 2). On the other hand, BSAO, an enzyme which is not formed in the radical species during the normal catalytic cycle [38], stayed in the reduced aminoquinol form.

Fig. 1. 129Xe NMR spectra of AOs. 129Xe (10 atm) spectra in a solu- tion (sodium phosphate buffer 1 mM, pH 7.0, 20% D2O) containing 0.35 mM ELAO, 0.28 mM LSAO and 0.15 mM PKAO. Shifts refer to the 129Xe chemical shift in buffer. The 129Xe NMR spectrum of PSAO, not shown, is very similar to the LSAO spectrum.

As previously reported [30], when a solution con- taining LSAO (10 lm) was equilibrated with 10 atm of xenon gas without a substrate, after a marked lag per- iod ((cid:3) 6 h), bleaching of the 498 nm band started with

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A

A

0.2

0.3

0.15

0.2

0.1

0.1

e c n a b r o s b A

e c n a b r o s b A

0.05

0 300

500

600

0 300

500

600

400 Wavelength (nm)

400 Wavelength (nm)

B

B

0.15

0.2

0.1

0.1

e c n a b r o s b A

0.05

e c n a b r o s b A

0 300

500

600

0 300

400

600

400 Wavelength (nm)

500 Wavelength (nm)

(A) Native LSAO, 16 lM,

Fig. 3. Absorption spectra changes of ELAO and PKAO native enzyme under 10 atm of xenon gas. Conditions: (A) ELAO, 11 lM, and (B) PKAO, 19 lM, in 1 mM sodium phosphate buffer (pH 7.0). The spectra of the reduced forms (–––) were recorded after 48 h.

in Fig. 2. Absorption spectra of AOs. 1 mM sodium phosphate buffer (pH 7.0), under anaerobic condi- tions before (- - -) and after (–––) addition of 10 mM putrescine. (B) PKAO, 19 lM, in 1 mM sodium phosphate buffer (pH 7.0), before (- - -) and after (–––) addition of 10 mM cadaverine in anaerobic con- ditions and in the presence of 100 lM CN–.

Characteristics of xenon-treated AOs

that of

fea- contemporaneous formation of TPQsq spectral tures. Similar behavior was observed with AOs from pea seedlings and E. characias latex (Fig. 3). This spe- cies reached its maximum concentration after 48 h. After readmission of oxygen, the absorption spectrum of oxidized TPQ was recovered, and approximately 1 mol of ammonia and 1 mol of hydrogen peroxide per mole of the ELAO (or PSAO) active site were detected at the end of the experiment.

the Km for

The results obtained with mammalian proteins were different. For PKAO, where the semiquinolamine rad- ical appears in the presence of the substrate and CN– [37], bleaching of the 490 nm band started with a marked time lag ((cid:3) 6 h) after addition of 10 atm of xenon gas (Fig. 3). It is interesting to note that the radical species formed neither in the presence nor in the absence of CN–. As observed in plant enzymes, the absorption spectrum of oxidized TPQ was recovered after readmission of oxygen, and 1 mol of ammonia and 1 mol of hydrogen peroxide per mole of active site were detected.

the native

In BSAO, no changes in the spectral features were observed under 10 atm of xenon gas, indicating that the TPQ cofactor remained in its oxidized form.

After exhaustive dialysis, the xenon-treated LSAO was allowed to react with a substrate under anaer- obic conditions, and behavior similar to that of the native enzyme was observed. Nevertheless, the cata- lytic activity of xenon-treated LSAO towards putres- cine was shown to be about 40% of the native LSAO (Table 2), whereas the kc for benzylam- ine did not change (Table 2). Also, whereas the Km values for oxygen and benzylamine were similar with the native and xenon-treated LSAO, amine putrescine was considerably higher (Table 2). The kc ⁄ Km ratio, a more useful measure of substrate specificity, was shown to be dramatically reduced, and a comparison with those obtained for other AOs is shown in Table 2. Very similarly to LSAO, in activity was also seen in PSAO and in loss ELAO. The catalytic activity of xenon-treated PKAO towards cadaverine and benzylamine was shown to enzyme be about 20% of that of its (Table 2). Xenon-treated BSAO, which retains oxidized form, showed the same activity as the cor- responding native enzyme (Table 2).

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Table 2. Kinetic parameters of Cu ⁄ TPQ-containing AOs.

irreversible

the

Enzyme

kc s)1

Km (mM)

kc ⁄ Km

LSAO

646

grass pea [15], and for mammalian AOs from pig kid- ney [40] and from beef serum [17]; and (b) it has been postulated that inhibition of all enzymes involves an intermediate aminoallenic com- pound that forms covalently bound pyrrole in the reac- tion with a nucleophile at the active site.

2.2 44.2

1.95

Xenon-treated LSAO ELAO

190

155a 1b 62a 0.9b 38b 0.18b 13.3a 0.17b

0.45 7 0.43

700

Xenon-treated ELAO PSAO

140a

1.1 35.5 1.1

Xenon-treated PSAO BSAOc

10

0.16

PKAO

45

0.24 0.45 1.4 0.46 0.2 0.4 1.9 0.4 0.2 0.45 1.5 0.45 0.1 2.2 0.1 0.12 0.23 0.25

1.91 3.9 0.18

Xenon-treated PKAO

0.5b 53.2a 0.5b 1d 0.35b 4.5e 0.23b 0.9e 0.046b

The exact mechanism of inhibition was elusive, and it was only in grass pea AO that the involved nucleo- phile was identified as Glu113, a residue corresponding to a Lys113 in PSAO [14]. DABY was also shown to be a mechanism-based inactivator for native LSAO and ELAO, with a kinh of 0.1 min)1 and a half-max- inactivation of 4 · 10)5 m for ELAO (Fig. 4), imal and a kinh of 5 min)1 and a half-maximal inactivation of 4 · 10)4 m for LSAO. Moreover, all the xenon-trea- ted AOs were inactivated by the reaction with DABY, clearly indicating that the lysine residue involved in the reduction of TPQ under xenon pressure is not the nu- cleophilic residue involved in the DABY inhibition mechanism; that is, the reactive turnover product of DABY binds an amino acid residue without interfering with the TPQ function.

Discussion

a Using putrescine as substrate. b Using benzylamine as substrate. c In BSAO, there are no differences in the kinetic parameters before and after xenon treatment (not shown). d Using spermine as substrate. e Using cadaverine as substrate. SDs are not reported.

Oxidative deamination of a lysine residue

interesting reports have In the past decade, several been published on the catalytic mechanism of AOs, and a significant number of essential amino acid resi- dues have been identified by site-specific mutagenesis. In this article, we show that a lysine is an important residue and that it plays a key role in modulating the activity of plant AOs, as in the mammalian AO from pig kidney, and we tentatively assign this role to a lysine at the active site.

100

The oxidative deamination of a lysine residue was monitored through the formation of a-aminoadipic-d- semialdehyde-derivatized fluoresceinamine by HPLC (see Experimental procedures) [39]. As reported previ- ously [30], with xenon-treated native LSAO, 1 mol of allysine residue per mole of monomeric enzyme was detected. Identical results were obtained for PSAO, ELAO, and PKAO, whereas with BSAO, where no reduction occurred, no allysine residue was detected.

)

%

Reaction with the mechanism-based inhibitor

40

p p a k / 1

20

i

10

0

( y t i v i t c a l a u d s e R

0

0.06

0.09

0.03 1/[DABY] (µM -1)

0

10

20

30

Time (min)

The experimental findings clearly show that plant and mammalian AOs under 10 atm of xenon are reduced without the presence of an amine substrate. In the presence of xenon, plant enzymes form yellow TPQsq, whereas in pig enzyme the bleached species TPQaq is observed. A lysine residue at the active site may be implicated in this mechanism. An important method in studying the structure–function of an enzyme is to find specific inhibitors and follow their effects. Our interest in the present study is in the mechanism-based inhib- itor DABY, for the following two reasons: (a) the inhibitor has been found to be a suicide substrate for plant copper AO (Cu-AO) from pea seedlings [14] and

Fig. 4. Inactivation of ELAO by DABY. The enzyme (6 nM) was pre- incubated with the indicated concentrations of DABY at 25 (cid:2)C in 1 mM sodium phosphate buffer (pH 7.0). The concentrations of DABY were: d, 10 lM; s, 20 lM; ., 30 lM; w, 40 lM. Inset. Dou- ble reciprocal plot of apparent first-order rate constants of inactiva- tion (kapp) vs. DABY concentrations.

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mechanism. Duff et al. have recently reported a new crystal form of the P. pastoris lysyl oxidase that has a covalent crosslink between two lysine residues, Lys778 and Lys66 [44]. Whereas Lys778 can readily reach the TPQ cofactor in the active site of the enzyme without any other conformational changes, Lys66 is in a well- ordered region and cannot do so. The authors pro- posed that the lysyl oxidase oxidized Lys778 to the corresponding aldehyde allysine, which can react spon- taneously with Lys66, which is is nearby and appropri- ately oriented.

(Fig. 5).

All plant AOs used in the present study contain 38 lysines in each subunit (Protein Data Bank accession numbers: ELAO AF171698; PSAO L39931; LSAO X64201). Because, as reported previously [30], the elu- tion profiles resulting from HPLC analysis of the AO proteolytic digestion with trypsin and lysyl endopepti- dase are very complicated, it is extremely difficult to determine with certainty which lysine residue is conver- the identity in amino acid ted into allysine. As sequences of ELAO, LSAO and PSAO is about 92%, it would be safe to accept that both enzymes have an almost identical structure; that is, the three enzymes could have two identical subunits, each containing three structural domains (D2, D3, and D4). As observed in the crystal structure of PSAO, copper ion and TPQ are in close proximity (shorter distance (cid:3) 6 A˚ ) [4], but they are not coordinated. Moreover, a slight displacement of TPQ would be required to facili- tate the extremely fast electron transfer between TPQaq and TPQsq, and the TPQ side chain appears suffi- ciently flexible such a change. to accommodate Although TPQ has been found to be characterized by considerable conformational flexibility, it has also been pointed out that when an amine substrate attacks the TPQ at C-5, H+ abstraction of the active site base Asp300 would require it to rotate by 180(cid:2) [4]. This sig- nificant displacement would contrast with the previous observation that the TPQ cofactor could remain fixed during the catalytic cycle [41–43]. Currently, new forms of PSAO native protein crystal are available [18] in the so-called ‘off-copper conformation’. In this structure, the O-4 of TPQ is hydrogen bonded to the hydroxyl group of conserved tyrosinyl residue Tyr286, and the TPQ orientation is in the active form, with the aspartic active site base residue (Asp300) in an excellent posi- tion for abstraction of the Ca proton from the sub- strate, so that TPQ does not rotate during the catalytic

X-ray crystallography of PSAO has also demonstra- ted that a lysine residue, Lys296, is located in domain D4, between b-sheet C-5 and a helix H-8, close to the entrance to a channel found to be suitable for moving substrate and products to and from the copper ⁄ TPQ active site buried in the protein interior. This residue forms a hydrogen bond with the phenolic group of TPQ when in a nonproductive ‘on-copper conforma- tion’ [4], but its role in the ‘off-copper conformation’ is still unknown. This amino acid is conserved in In LSAO (Lys296) and ELAO (Lys302) BSAO (Protein Data Bank accession number S69583), the residue corresponding to Lys296 in LSAO is Thr381 (Fig. 5), but an arginine is present at position 382. Although the amino acid sequence of PKAO is unknown, there may be a threonine residue, as in human kidney AO (Thr369), considering its great homology with known reported sequences [45] (Fig. 5). In this case, a lysine (Lys370) flanks the threonine resi- due that could react with TPQ. This is evidence for the importance of a lysine residue in the active site for the formation of the radical species in plant enzymes and the aminoquinol in PKAO under xenon pressure. As the arginine residue in BSAO possesses a highly basic guanidine group, it could be unreactive with TPQ under xenon pressure.

Fig. 5. Partial amino acid sequence alignment of some AOs. The active site base aspartate residue is in yellow; the lysine residue at the active site, probably involved in the formation of the radical in plant AOs, is in green; the nucleophilic residue probably involved in the mechanism- based inhibition by DABY is in red. The Gene Bank accession numbers of each sequence are: PSAO, AB026253; LSAO, X64201; ELAO, AF171698; and BSAO, S69583. HKAO (human kidney AO) is from Novotny et al. [45].

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is

Another

interesting result

[14], implicated in the

as reported by Fre´ bort et al. the Lys113 in PSAO could be formation of pyrrole. This residue could correspond to Asp113 in LSAO, Asp117 in ELAO, and Asp179 in BSAO (Fig. 5).

three Cu-AOs

In a recent paper,

amine oxidase

three xenon derivatives,

that xenon-treated LSAO shows lower activity and a higher Km value for diamine putrescine as substrate, but not for aromatic monoamine benzylamine. However, xenon treatment of PKAO was accompanied by loss of activity for both cadaverine and benzylamine. These results are most compatible with two different mechanisms being involved in the interaction between enzyme and sub- strate. It is possible, only in the plant enzyme, that the e-amino group of Lys296 may interact with the posit- ive charge of the amino group of putrescine, as shown in Fig. 6. This residue could have an important role in conferring substrate specificity, with consequences for catalytic efficiency when lysine is transformed into allysine.

residue

[Arthrobacter globiformis (AGAO), PSAO and P. pastoris lysyl oxidase] were investigated by Duff et al. [18] by XRD under high xenon pressure, with the aim of finding a potential dioxygen-binding cavity close to the active site of Cu-AO that is related to enzyme function. In all the xenon proved to be bound at a variety of cavities and with a range of occupancies. The xenon sites closest to the Cu ⁄ TPQ center in each structure are: Xe–Cu (cid:4) 7.5 A˚ and Xe–TPQ (cid:4) 9.5 A˚ . From this study, the authors concluded that the results do not give enough evidence of a xenon-binding site in a region of the molecule close to the active site to justify the suggestion of a potential transient dioxygen-binding site.

Thanks to DABY, a mechanism-based inhibitor, we can confirm that an amino acid residue is impli- cated in the mechanism-based inhibition that is dif- ferent from the residue implicated in TPQ reduction under xenon pressure. A nucleophile is implicated in the DABY inhibition mechanism, and

interactions with unpaired electrons

In addressing the usefulness of 129Xe NMR spectros- copy in the characterization of biological compounds in solution, it must be pointed out that these systems are generally characterized by complex structures and often by the presence of more than one specific site for ligands and ⁄ or substrates. The nearest neighbor resi- dues of the bound xenon atoms in the cavities are pre- dominantly nonpolar side chains, but they include polar side chains and backbone peptide groups. This, together with the fact that the observed 129Xe chemical shift is dynamically averaged among different binding sites and at the same time interacts with the protein surface, makes it difficult to separate the individual contributions so as to show whether a particular xenon-binding site is responsible for the different com- ponents observed in the studied AOs in solution. Hyperfine in radical species and ⁄ or paramagnetic metal ions could be a further source of information, as long as they can be distinguished from other structural or dynamic fac- tors affecting NMR parameters.

Fig. 6. Active site of plant AOs. The model of the active site shows the possible interaction with two substrates: benzylamine, which represents a substrate with an apolar chain, and putrescine, with a positively charged amino group. The positively charged e-amino group of lysine exerts a repulsive force towards substrates charac- terized by the presence of a positively charged amino group, such as putrescine, leading to a lower catalytic efficiency when lysine is transformed into allysine. Neither lysine nor allysine can interact with the apolar chain of benzylamine, leading to this amino acid residue being responsible for the different substrate specificities.

These 129Xe NMR outputs cannot provide local interaction involved. information on the host–guest Experimental evidence of the fast diffusion of xenon within AOs clearly opposes the static and average pic- tures given by single-crystal XRD structures, which seem to show that xenon atoms are localized at specific sites. Moreover, it is worth noting that, as the single- crystal XRD results utterly ignore the fundamental dynamic features involved in the functionality of the in solution, hypotheses on biological biomolecules activities based on crystal structures should be consi- dered critically.

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Concluding remarks

using cadaverine as substrate) [32], pea seedlings (PSAO; kc ¼ 140 s)1 using putrescine as substrate) [33], lentil seed- lings (LSAO; kc ¼ 155 s)1 using putrescine as substrate) latex (ELAO; kc ¼ 23 s)1 using [34] and E. characias putrescine as substrate) [35] were prepared according to the described procedures. The activities of

The TPQsq radical represents the highly reactive spe- cies with the oxygen molecule in the catalytic cycle of plant AOs. Thus, the radical species observed under 10 atm of xenon without a substrate in plant AOs only, and the fact that lysine was identified at the act- ive site, could reveal key aspects of the structure–func- tion relationship among various AOs. Moreover, plant enzymes show a high affinity for putrescine and a lower activity for benzylamine. In contrast, xenon-trea- ted plant AOs show a high loss in catalytic activity towards putrescine, but not towards benzylamine. The transformation of a lysine residue, probably Lys296, into allysine, four residues from the active site base identified in a conserved aspartate residue (Asp300), could have an important role in the recognition of sub- strates with a positively charged amino group.

the tested enzymes were measured according to the procedures reported in the related refer- ences. Oxygen uptake was determined with a Clark-type electrode coupled to an OXYG1 Hansatech oxygraph (Hansatech Instruments Ltd, King’s Lynn, UK). The tem- perature of the reaction chamber was kept at 37 (cid:2)C by using a circulating water bath. The solution (1 mL) con- taining the enzyme in a 1 mm sodium phosphate buffer (pH 7.0) was maintained for 20 min at a constant level of oxygen, as previously reported [46,47], and the reaction was started by addition of the related substrate. The Km values for AOs using different substrate concentrations at a satur- ating concentration of oxygen (219 lm), or varying concen- trations of oxygen at a saturating concentration of substrate, were calculated from initial velocity data fitted to the Michaelis–Menten equation by nonlinear regression and by double reciprocal plots by Michaelis–Menten analysis in a 1 mm sodium phosphate buffer (pH 7.0). Benzylamine oxidase activity was measured in a 1 mm sodium phosphate buffer (pH 7.0), by monitoring the increase in absorbance of UV light at 250 nm using an e250 ¼ 12.8 mm)1Æcm)1 for benzaldehyde [36]. Catalytic center activity (kc) is defined as mole of substrate consumed per mole of active sites · s)1.

Spectroscopic methods

In conclusion, although the data reported in the pre- sent article may well be valid generally, the exact loca- tion and nature of the observed interactions between xenon and the enzymes studied remain somewhat hypothetical and are not of any functional significance. Nevertheless, from our results, we conclude that xenon is capable of forcing a conformational change in AOs, such that most of them react with one of their own lysine residues. As reported for other amino acid resi- dues, changes in active site architecture and charge dis- tribution seem to be critical during catalysis in AOs. Thus, further comparative investigation of the active site in AOs from plants, mammals and bacteria is nee- ded to understand whether these enzymes, which differ in structure and action mechanism, follow a similar metabolic pathway.

Experimental procedures

Materials

UV ⁄ visible experiments Absorption spectra of AOs in a 1 mm sodium phosphate buffer (pH 7.0) were recorded at 25 (cid:2)C with an Ultrospec 2100 spectrophotometer (Biochrom Ltd, Cambridge, UK). Anaerobic experiments were performed with a Thunberg- type spectrophotometer cuvette (Soffieria Vetro, Sassari, Italy). Solutions were subjected to several cycles of evacu- ation followed by flushing with argon.

129Xe NMR experiments

All reagents were of the highest purity degree available. 1,4-Diaminobutane dihydrochloride (putrescine), 1,5-dia- minopentane dihydrochloride (cadaverine), benzylamine and N,N¢-bis(3-aminopropyl)-1,4-butane hydrochloride diamine tetrahydrochloride (spermine) were purchased from Sigma Aldrich (St Louis, MO). Xenon chemical shift meas- urements were made using 92% enriched 129Xe (Chemical Research 2000; Rome, Italy). DABY was synthesized as previously reported [13].

Experiments were carried out as previously reported [20]. Briefly, samples of native AOs in a 1 mm sodium phos- phate buffer (pH 7.0), with 20% D2O, were degassed using three freeze–pump–thaw cycles, pressurized with 10 atm of xenon gas into Wilmad high-pressure NMR tubes (outside diameter 5 mm and internal diameter 7.1 mm; outside diameter 5 mm and internal diameter 2.2 mm; Buena, NJ) and allowed to equilibrate for 48 h. 129Xe NMR spectra were recorded on a Varian VXR-300 spectrometer (Varian, Palo Alto, CA), and 129Xe NMR spin lattice relaxation times (T1) of native AOs were measured using the inversion

Enzymes AOs from bovine plasma (BSAO; kc ¼ 0.35 s)1 using ben- zylamine as substrate) [31], pig kidney (PKAO; kc ¼ 4.5 s)1

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Guss J (2003) The crystal structure of Pichia pastoris lysyl oxidase. Biochemistry 42, 15148–15157. recovery method with an acquisition time of 1 s and a recycling delay of 3T1. 8 Lunelli M, Di Paolo ML, Biadene M, Calderone V,

Assays of products

Battistutta R, Scarpa M, Rigo A & Zanotti G (2005) Crystal structure of amine oxidase from bovine serum. J Mol Biol 346, 991–1004.

9 Mu D, Janes SM, Smith AJ, Brown DE, Dooley DM & Klinman JP (1992) Tyrosine codon corresponding to topa quinone at the active site of copper amine oxi- dases. J Biol Chem 267, 7979–7982. 10 Mure M (2004) Tyrosine-derived quinone cofactors. Acc Chem Res 37, 131–139. fluoresceinamine) Ammonia production was determined from the amount of NADH consumed in the presence of glutamate dehydroge- nase, and hydrogen peroxide formation was detected with the peroxidase ⁄ 4-hydroxy-3-methoxyphenylacetic acid method [36]. a-Aminoadipic-d-semialdehyde (allysine) residue was derivatized to a decarboxylated fluoresceinamine (a-amino- adipic-d-semialdehyde-derivatized and determined by HPLC as previously reported [30,39].

Acknowledgements

11 Matsuzaki R, Suzuki S, Yamaguchi K, Fukui T & Tanizawa K (1995) Spectroscopic studies on the mechanism of the topa quinone generation in bacterial monoamine oxidase. Biochemistry 34, 4524–4530.

12 Matsuzaki R & Tanizawa K (1998) Exploring a channel to the active site of copper ⁄ topaquinone-containing phe- nylethylamine oxidase by chemical modification and site-specific mutagenesis. Biochemistry 37, 13947–13957.

This study was supported partly by MURST 60%, by investimenti della ricerca di FIRB (Fondo per gli base), and by Fondazione Banco di Sardegna (Sassari, Italy) funds.

13 Pecˇ P & Fre´ bort I (1992) 1,4-Diamino-2-butyne as the mechanism-based pea diamine oxidase inhibitor. Eur J Biochem 209, 661–665.

References

1 Floris G & Finazzi Agro` A (2004) Amine oxidases. In

Encyclopedia Biological Chemistry (Lennarz WJ & Lane MD, eds), Vol. 1, pp. 85–89. Academic Press Inc., New York, NY. 14 Fre´ bort I, Sˇ ebela M, Svendsen I, Hirota S, Endo M, Yamauchi O, Bellelli A, Lemr K & Pecˇ P (2000) Molecular mode of interaction of plant amine oxidase with the mechanism-based inhibitor 2-butyne-1,4-diam- ine. Eur J Biochem 267, 1423–1433. 2 Janes SM, Mu D, Wemmer D, Smith AJ, Kaur S,

Maltby D, Burlingame AL & Klinman JP (1990) A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 248, 981–987. 15 Lamplot Z, Sˇ ebela M, Malonˇ M, Lenobel R, Lemr K, Havlisˇ J, Pecˇ P, Qiao C & Sayre LM (2004) 1,5-Dia- mino-2-pentyne is both a substrate and inactivator of plant copper amine oxidases. Eur J Biochem 271, 4696–4708. 3 Parson MR, Convery MA, Wilmot CM, Yadav KDS,

Blakeley V, Corner AS, Phillips SEV, McPherson MJ & Knowles PF (1995) Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A˚ resolu- tion. Structure 3, 1171–1184. 16 Padiglia A, Floris G, Longu S, Schinina` ME, Pedersen JZ, Finazzi Agro` A, De Angelis F & Medda R (2004) Inhibition of lentil copper ⁄ TPQ amine oxidase by the mechanism-based inhibitor produced from tyramine. Biol Chem 385, 323–329. 4 Kumar V, Dooley DM, Freeman HC, Mithchell Guss J, 17 Shepard EM, Smith J, Elmore BO, Kuchar JA, Sayre

Harvey I, McGuirl MA, Wilce MCJ & Zubak VM (1996) Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A˚ resolution. Structure 4, 943–955. LM & Dooley DM (2002) Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases. Eur J Biochem 269, 3645–3658. 18 Duff AP, Trambaiolo DM, Cohen AE, Ellis PJ, Juda

GA, Shepard EM, Langley DB, Dooley DM, Freeman HC & Mitchell Guss J (2004) Using xenon as a probe for dioxygen-binding sites in copper amine oxidases. J Mol Biol 344, 599–607. 5 Wilce MCJ, Dooley DM, Freeman HC, Mitchell Guss J, Matsunami H, McIntire WS, Ruggiero HC, Tanizawa K & Yamaguchi H (1997) Crystal structures of the copper- containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone. Biochemistry 36, 16116–16133. 6 Li R, Klinman JP, Scott Mathews F (1998). Copper

19 Dawkes HC & Phillips SEV (2001) Copper amine oxi- dase: cunning cofactor and controversial copper. Curr Opin Struct Biol 11, 666–673.

amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 A˚ resolution reveals the act- ive conformation. Structure 6, 293–307.

FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS

2593

20 Tilton RF & Kuntz ID Jr (1982) Nuclear magnetic reso- nance studies of xenon-129 with myoglobin and haemo- globin. Biochemistry 21, 6850–6857. 7 Duff AP, Cohen AE, Ellis PJ, Kuchar JA, Langley DB, Shepard EM, Dooley DM, Freeman HC & Mitchell

A. Mura et al.

Lysine residue and copper–quinoproteins

seedling amine oxidase for crystallization studies. Plant Physiol 106, 1205–1211.

21 Rubin SM, Spence MM, Goodson BM, Wemmer DE & Pines A (2000) Evidence of non-specific surface interac- tions between laser-polarized xenon and myoglobin in solution. Proc Natl Acad Sci USA 97, 3472–3475. 34 Floris G, Giartosio A & Rinaldi A (1983) Diamine oxi- dase from Lens esculenta seedlings: purification and properties. Phytochemistry 22, 1871–1874. 35 Padiglia A, Medda R, Lorrai A, Murgia B, Pedersen

22 Locci E, Dehouck Y, Casu M, Saba G, Lai A, Luhmer M, Reisse J & Batik K (2001) Probing proteins in solu- tion by 129Xe NMR spectroscopy. J Magn Reson 150, 167–174. 23 Rubin SM, Spence MM, Pines A & Wemmer DE JZ, Finazzi Agro` A & Floris G (1998) Characterization of Euphorbia characias latex amine oxidase. Plant Phy- siol 117, 1363–1371.

(2001) Characterization of the effects of non-specific xenon–protein interactions on 129Xe chemical shifts in aqueous solution: further development of xenon as a biomolecular probe. J Magn Reson 152, 79–86. 36 Medda R, Padiglia A, Bellelli A, Sarti P, Santanche` S, Finazzi Agro` A & Floris G (1998) Intermediates in the catalytic cycle of lentil seedling copper-containing amine oxidase. Biochem J 332, 431–437. 24 Locci E, Casu M, Saba G, Lai A, Reisse J & Batik K 37 Dooley DM, McGuirl MA, Peisach J & McCracken J

(2002) The potential of 129Xe NMR relaxation measure- ments for the study of heme proteins. Chem Phys Chem 3, 812–814. (1987) The generation of an organic free radical in sub- strate-reduced pig kidney diamine oxidase-cyanide. FEBS Lett 214, 274–278. 38 Su Q & Klinman JP (1998) Probing the mechanism of

25 Corda M, Era B, Fais A & Casu M (2004) Structural investigation of pig metmyoglobin by 129Xe NMR spectroscopy. Biochim Biophys Acta 1674, 182–192. 26 Dubois L, Da Silva P, Landon C, Huber G, Ponchet proton coupled electron transfer to dioxygen: the oxida- tive half-reaction of bovine serum amine oxidase. Bio- chemistry 37, 12513–12525. 39 Akagawa M, Sasaki T & Suyama K (2002) Oxidative

M, Novelle F, Berthault P & Desvaux H (2004) Probing the hydrophobic cavity of lipid transfer protein from Nicotiana tabacum through xenon-based NMR spectro- scopy. J Am Chem Soc 126, 15738–15746. deamination of lysine residue in plasma protein of dia- betic rats. Novel mechanism via the Maillard reaction. Eur J Biochem 269, 5451–5458. 40 He Z, Nadkarni DV, Sayre LM & Greenaway FT

(1995) Mechanism-based inactivation of porcine kidney diamine oxidase by 1,4-diamino-2-butene. Biochim Bio- phys Acta 1253, 117–127. 27 Desvaux H, Dubois L, Huber G, Quillin ML, Berthault P & Matthews BW (2005) Dynamics of xenon binding inside the hydrophobic cavity of pseudo-wild-type Bac- teriophage T4 lysozyme explored through xenon-based NMR spectroscopy. J Am Chem Soc 127, 11676–11683. 28 Moglich A, Koch B, Gronwald W, Hengstenberg W,

41 Plastino J, Green EL, Sanders-Loehr J & Klinman JP (1999) An unexpected role for an active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha. Biochemistry 38, 8204–8216. 42 Cai D, Dove J, Nakamura N, Sanders-Loehr J & Brunner E & Kalbitzer HR (2004) Solution structure of the active-centre mutant I14A of the histidine-contain- ing phosphocarrier protein from Staphylococcus carno- sus. Eur J Biochem 271, 4815–4824. 29 Soldatov DV, Moudrakovsky IL, Grachev EV &

Klinman JP (1997) Mechanism-based inactivation of a yeast methylamine oxidase mutant: implications for the functional role of the consensus sequence surrounding topaquinone. Biochemistry 36, 11472–11478. Ripmeester JA (2006) Micropores in crystalline dipep- tides as seen from the crystal structure, He pycnometry, and 129Xe NMR spectroscopy. J Am Chem Soc 128, 6737–6744. 30 Medda R, Mura A, Longu S, Anedda R, Padiglia A,

43 Schwartz B, Green EL, Sanders-Loehr J & Klinman JP (1998) Relationship between conserved consensus site residues and the productive conformation for the TPQ cofactor in a copper-containing amine oxidase from yeast. Biochemistry 37, 16591–16600. Casu M & Floris G (2006) An unexpected formation of the spectroscopic CuI-semiquinone radical by xenon- induced self-catalysis of a copper quinoprotein. Biochi- mie 88, 827–835.

44 Duff AP, Cohen AE, Ellis PJ, Hilmer K, Langley DB, Dooley DM, Freeman HC & Mitchell Guss J (2006) The 1.23 A˚ structure of Pichia pastoris lysyl oxidase reveals a lysine–lysine cross-link. Acta Crystallogr D62, 1073–1084.

45 Novotny WF, Chassande O, Baker M, Lazdunski M & Barbry P (1994) Diamine oxidase is the amiloride-bind- ing protein and is inhibited by amiloride analogues. J Biol Chem 269, 9921–9925. 31 Turini P, Sabatini S, Befani O, Chimenti F, Casanova C, Riccio PL & Mondovı` B (1982) Purification of bovine plasma amine oxidase. Anal Biochem 125, 294–298. 32 Padiglia A, Medda R, Lorrai A, Paci M, Pedersen JZ, Boffi A, Bellelli A, Finazzi Agro` A & Floris G (2001) Irreversible inhibition of pig kidney copper-containing amine oxidase by sodium and lithium ions. Eur J Bio- chem 268, 4686–4697.

FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS

2594

46 Mills SA & Klinman JP (2000) Evidence against reduc- tion of Cu2+ to Cu+ during dioxygen activation in a 33 McGuirl MA, McCahon CD, McKeown KA & Dooley DM (1994) Purification and characterization of pea

A. Mura et al.

Lysine residue and copper–quinoproteins

FEBS Journal 274 (2007) 2585–2595 ª 2007 The Authors Journal compilation ª 2007 FEBS

2595

copper amine oxidase from yeast. J Am Chem Soc 122, 9897–9904. Role of copper in bacterial amine oxidase: spectroscopic and crystallographic studies of metal-substituted enzymes. J Am Chem Soc 125, 1041–1055. 47 Kishishita S, Okajima T, Kim M, Yamaguchi H, Hirota S, Suzuki S, Kuroda S, Tanizawa K & Mure M (2003)