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: rmed
Trang 1amine oxidases
Anna Mura1, Roberto Anedda2, Francesca Pintus1, Mariano Casu2, Alessandra Padiglia1,
Giovanni Floris1and Rosaria Medda1
1 Department of Applied Sciences in Biosystems, University of Cagliari, Italy
2 Department of Chemical Science, University of Cagliari, Italy
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 reduccorrespond-ing molecular oxygen to
hydrogen peroxide [1] The ping-pong catalytic
mech-anism of Cu⁄ TPQ AOs can basically be divided into
two half-reactions
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:
Eoxþ R CH2 NHþ3 ! Eredþ R CHO
The other, known as ‘the oxidative half-reaction’, involves the reoxidation of the enzyme and contem-poraneous release of ammonia and hydrogen peroxide:
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 kcvalue 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
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, Cu II -aminoquinol; TPQsq, Cu I –semiaminoquinolamine radical; XRD, X-ray diffraction.
Trang 2Eredþ O2þ H2O! Eoxþ NHþ4 þ H2O2
AOs are homodimers; each subunit (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
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 tyrosine ring to generate
dihydroxyphenylalanine, which, upon oxidation and
through the formation of the CuI⁄ semiquinone radical
intermediate, gives rise to dihydroxyphenylalanine
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
Finally, an important lysine has been suggested in the crystal structure of pea AO at the active site (Lys296), forming a hydrogen bond with the phe-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]
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 in biologically 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
Table 1 Conserved amino acid residues in Cu ⁄ TPQ-containing
AOs.
Enzyme
Trang 3129Xe chemical shift and spin-lattice relaxation
time in AO solution
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 the relaxation time
(T1¼ 3.2 s) is significantly reduced as compared to T1
in the buffer ( 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
signal in AO samples is shifted downfield (ELAO
3.96 p.p.m per 0.35 mm, corresponding 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 T1value of all native enzymes is found to be
much smaller than the T1value of xenon in the buffer (ELAO T1¼ 4.3 ± 0.5 s; PKAO T1 ¼ 5.5 ± 0.8 s; buffer T1¼ 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 nonspecispeci-fic 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
Owing to the presence of the TPQ cofactor, the oxidized form of AOs has a distinctive pink color and absorbs in the visible region: BSAO shows an electronic absorption band at 476 nm (e476¼ 3800 m)1Æcm)1) [31], PKAO at 490 nm (e490¼ 4000 m)1Æcm)1) [32], PSAO and LSAO at 498 nm (e498¼ 4100 m)1Æcm)1) [33,34], and ELAO at 490 nm (e490¼ 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
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 ( 6 h), bleaching of the 498 nm band started with
Fig 1 129 Xe NMR spectra of AOs 129 Xe (10 atm) spectra in a
solu-tion (sodium phosphate buffer 1 m M , pH 7.0, 20% D2O) containing
0.35 m M ELAO, 0.28 m M LSAO and 0.15 m M PKAO Shifts refer to
the 129 Xe chemical shift in buffer The 129 Xe NMR spectrum of
PSAO, not shown, is very similar to the LSAO spectrum.
Trang 4contemporaneous formation of TPQsq spectral
fea-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 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 ( 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
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
Characteristics of xenon-treated AOs 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 that 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 native and xenon-treated LSAO, the Km for the 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, loss in activity was also seen in PSAO and in ELAO The catalytic activity of xenon-treated PKAO towards cadaverine and benzylamine was shown to
be about 20% of that of the native enzyme (Table 2) Xenon-treated BSAO, which retains its oxidized form, showed the same activity as the cor-responding native enzyme (Table 2)
600 500
400 300
0.3
0.2
0.1
0
Wavelength (nm)
A
600 500
400 300
0.15
0.1
0.05
0
Wavelength (nm)
B
Fig 3 Absorption spectra changes of ELAO and PKAO native enzyme under 10 atm of xenon gas Conditions: (A) ELAO,
11 l M , and (B) PKAO, 19 l M , in 1 m M sodium phosphate buffer (pH 7.0) The spectra of the reduced forms (–––) were recorded after 48 h.
600 500
400 300
0.2
0.15
0.1
0.05
0
Wavelength (nm)
A
600 500
400 300
0.2
0.1
0
Wavelength (nm)
B
Fig 2 Absorption spectra of AOs (A) Native LSAO, 16 l M , in
1 m M sodium phosphate buffer (pH 7.0), under anaerobic
condi-tions before (- - -) and after (–––) addition of 10 m M putrescine (B)
PKAO, 19 l M , in 1 m M sodium phosphate buffer (pH 7.0), before
(- - -) and after (–––) addition of 10 m M cadaverine in anaerobic
con-ditions and in the presence of 100 l M CN –
Trang 5Oxidative deamination of a lysine residue
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
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
grass pea [15], and for mammalian AOs from pig kid-ney [40] and from beef serum [17]; and (b) it has been postulated that the irreversible 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
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-imal inactivation of 4· 10)5m for ELAO (Fig 4), and a kinh of 5 min)1 and a half-maximal inactivation
of 4· 10)4mfor LSAO Moreover, all the xenon-trea-ted AOs were inactivaxenon-trea-ted 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
In the past decade, several interesting reports have 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
Table 2 Kinetic parameters of Cu ⁄ TPQ-containing AOs.
Enzyme
k c
s)1
K m
(m M ) k c ⁄ K m
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.
30 20
10 0
100
10
Time (min)
40 20 0 1/[DABY] (µ M -1 )
Fig 4 Inactivation of ELAO by DABY The enzyme (6 n M ) was pre-incubated with the indicated concentrations of DABY at 25 C in
1 m M sodium phosphate buffer (pH 7.0) The concentrations of DABY were: d, 10 l M ; s, 20 l M ; , 30 l M ; w, 40 l M Inset Dou-ble reciprocal plot of apparent first-order rate constants of inactiva-tion (kapp) vs DABY concentrations.
Trang 6All 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-ted into allysine As the identity in amino acid
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
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 to accommodate such a change
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 [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 abstracposi-tion of the Ca proton from the
sub-strate, so that TPQ does not rotate during the catalytic
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
X-ray crystallography of PSAO has also demonstra-ted that a lysine residue, Lys296, is locademonstra-ted 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 conformaconforma-tion’
is still unknown This amino acid is conserved in LSAO (Lys296) and ELAO (Lys302) (Fig 5) In 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].
Trang 7Another interesting result is that xenon-treated
LSAO shows lower activity and a higher Kmvalue 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
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 residue is
implicated in the DABY inhibition mechanism, and
as reported by Fre´bort et al [14], the Lys113 in PSAO could be implicated in the formation of pyrrole This residue could correspond to Asp113 in LSAO, Asp117 in ELAO, and Asp179 in BSAO (Fig 5)
In a recent paper, three Cu-AOs [Arthrobacter globiformis amine oxidase (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 three xenon derivatives, 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 7.5 A˚ and Xe–TPQ 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
In addressing the usefulness of129Xe 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 observed129Xe 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 interactions with unpaired electrons 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
These 129Xe NMR outputs cannot provide local information on the host–guest interaction involved Experimental evidence of the fast diffusion of xenon within AOs clearly opposes the static and average pic-tures given by single-crystal XRD strucpic-tures, 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 biomolecules in solution, hypotheses on biological activities based on crystal structures should be consi-dered critically
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.
Trang 8Concluding remarks
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 relastructure–func-tionship 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
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 interacloca-tions 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
All reagents were of the highest purity degree available
1,4-Diaminobutane dihydrochloride (putrescine),
diamine tetrahydrochloride (spermine) were purchased from
Sigma Aldrich (St Louis, MO) Xenon chemical shift
Research 2000; Rome, Italy) DABY was synthesized as
previously reported [13]
Enzymes
using cadaverine as substrate) [32], pea seedlings (PSAO;
putrescine as substrate) [35] were prepared according to the described procedures
The activities of 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
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
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
Spectroscopic methods
UV⁄ visible experiments
Absorption spectra of AOs in a 1 mm sodium phosphate
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
Experiments were carried out as previously reported [20] Briefly, samples of native AOs in a 1 mm sodium
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)
were recorded on a Varian VXR-300 spectrometer (Varian,
Trang 9recovery method with an acquisition time of 1 s and a
Assays of products
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
[36] a-Aminoadipic-d-semialdehyde (allysine) residue was
derivatized to a decarboxylated fluoresceinamine
determined by HPLC as previously reported [30,39]
Acknowledgements
This study was supported partly by MURST 60%, by
FIRB (Fondo per gli investimenti della ricerca di
base), and by Fondazione Banco di Sardegna (Sassari,
Italy) funds
References
1 Floris G & Finazzi Agro` A (2004) Amine oxidases In
MD, eds), Vol 1, pp 85–89 Academic Press Inc.,
New York, NY
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
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
4 Kumar V, Dooley DM, Freeman HC, Mithchell Guss J,
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
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
amine oxidase from Hansenula polymorpha: the crystal
structure determined at 2.4 A˚ resolution reveals the
act-ive conformation Structure 6, 293–307
7 Duff AP, Cohen AE, Ellis PJ, Kuchar JA, Langley DB,
Shepard EM, Dooley DM, Freeman HC & Mitchell
Guss J (2003) The crystal structure of Pichia pastoris lysyl oxidase Biochemistry 42, 15148–15157
8 Lunelli M, Di Paolo ML, Biadene M, Calderone V, 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
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
phe-nylethylamine oxidase by chemical modification and site-specific mutagenesis Biochemistry 37, 13947–13957
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
14 Fre´bort I, Sˇebela 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
15 Lamplot Z, Sˇebela 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
16 Padiglia A, Floris G, Longu S, Schinina` ME, Pedersen
JZ, Finazzi Agro` A, De Angelis F & Medda R (2004)
mechanism-based inhibitor produced from tyramine Biol Chem 385, 323–329
17 Shepard EM, Smith J, Elmore BO, Kuchar JA, Sayre
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
19 Dawkes HC & Phillips SEV (2001) Copper amine oxi-dase: cunning cofactor and controversial copper Curr Opin Struct Biol 11, 666–673
20 Tilton RF & Kuntz ID Jr (1982) Nuclear magnetic reso-nance studies of xenon-129 with myoglobin and haemo-globin Biochemistry 21, 6850–6857
Trang 1021 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
22 Locci E, Dehouck Y, Casu M, Saba G, Lai A, Luhmer
M, Reisse J & Batik K (2001) Probing proteins in
167–174
23 Rubin SM, Spence MM, Pines A & Wemmer DE
(2001) Characterization of the effects of non-specific
aqueous solution: further development of xenon as a
biomolecular probe J Magn Reson 152, 79–86
24 Locci E, Casu M, Saba G, Lai A, Reisse J & Batik K
measure-ments for the study of heme proteins Chem Phys Chem
3, 812–814
25 Corda M, Era B, Fais A & Casu M (2004) Structural
spectroscopy Biochim Biophys Acta 1674, 182–192
26 Dubois L, Da Silva P, Landon C, Huber G, Ponchet
M, Novelle F, Berthault P & Desvaux H (2004) Probing
the hydrophobic cavity of lipid transfer protein from
spectro-scopy J Am Chem Soc 126, 15738–15746
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
NMR spectroscopy J Am Chem Soc 127, 11676–11683
28 Moglich A, Koch B, Gronwald W, Hengstenberg W,
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 &
Ripmeester JA (2006) Micropores in crystalline
dipep-tides as seen from the crystal structure, He pycnometry,
6737–6744
30 Medda R, Mura A, Longu S, Anedda R, Padiglia A,
Casu M & Floris G (2006) An unexpected formation of
xenon-induced self-catalysis of a copper quinoprotein
Biochi-mie 88, 827–835
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
33 McGuirl MA, McCahon CD, McKeown KA & Dooley
DM (1994) Purification and characterization of pea
seedling amine oxidase for crystallization studies Plant Physiol 106, 1205–1211
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
JZ, Finazzi Agro` A & Floris G (1998) Characterization
of Euphorbia characias latex amine oxidase Plant Phy-siol 117, 1363–1371
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
37 Dooley DM, McGuirl MA, Peisach J & McCracken J (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 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 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
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 & 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
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
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
46 Mills SA & Klinman JP (2000) Evidence against