 |
INTRODUCTION |
Polyamine oxidase
(PAO),1 a flavin adenine
dinucleotide (FAD)-containing enzyme, catalyzes the oxidation of
polyamines at the secondary amino group, giving different products
according to the organism considered. In particular, vertebrate PAOs
(EC 1.5.3.11) participate in the interconversion metabolism of
polyamines, converting N1-acetyl derivatives of
spermine (N1-acetylSpm) and spermidine
(N1-acetylSpd) into Spd and putrescine,
respectively, plus 3-aminopropanal and H2O2,
(1-3). PAOs with similar characteristics occur in methylotrophic yeasts (4, 5) and amoebae (6). On the contrary, plant (1), bacterial
(7), and protozoan (8) PAOs oxidize spermidine and spermine to
4-aminobutanol or N-(3-aminopropyl)-4-aminobutanol, respectively, plus 1,3-diaminopropane and H2O2.
As these compounds cannot be converted directly to other polyamines,
this class of PAOs generally is considered to be involved in the
terminal catabolism of polyamines. Since PAOs play a crucial role in
polyamine catabolism, these enzymes are important drug targets, and in
fact, it has been shown that a number of polyamine analogues have an
antitumor effect in different cell lines (9-12).
As compared with large and detailed investigations on plant PAOs (1,
13-22), only little attention has been devoted to the animal
counterpart (2, 23-28). Recently, Wang et al. (29) and
Vujcic et al. (30) have reported the cloning and
characterization of novel mammalian PAO enzymes capable of oxidizing
preferentially Spm and for this reason named spermine oxidase (SMO)
(30). In particular, this enzyme was expressed in an in
vitro transcription/translation system (29) and into transiently
transfected human kidney cells (30). Based on these studies, it was
postulated that in addition to the traditional interconversion pathway
in which Spm is first acetylated by spermidine/spermine
N1-acetyltransferase and then oxidized by PAO,
mammalian cells contain an enzyme capable of directly oxidizing Spm to
Spd (30).
Data base searching analysis using PAOh1 cDNA sequence
recovered a mouse cDNA clone (Image Clone 264769) corresponding to the murine counterpart, which was supplied by the United Kingdom Medical Research Council Human Genome Mapping Resource Centre (Cambridge, United Kingdom) consortium and herein defined as mSMO. To
enhance the knowledge of enzymology of mammalian SMO and shed light on
the structure/function relationship of this enzyme, the mSMO cDNA
was further subcloned and expressed in secreted and secreted-tagged
forms into Escherichia coli BL21 DE3 cells. This paper
describes the expression and the main biochemical features of mouse
spermine oxidase. Notwithstanding the low amino acid sequence homology
shown by the animal and plant PAOs (45% sequence homology between MPAO
and mSMO), molecular modeling of mSMO based on MPAO three-dimensional
structure (18) suggests that the general features of MPAO active site
are conserved in mSMO.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
N,N1-bis(2,3-butadienyl)-1-4-butane-diamin
(MDL72527) was a generous gift from Dr. M. De Agasio (Consiglio
Nazionale delle Ricerche, Area della Ricerca di Roma, Montelibretti,
Italy). Spm, Spd, and pargilyne were purchased from Sigma.
N1-acetylSpm and
N1-acetylSpd originally purchased from Sigma are
no longer commercially available.
DNA and Genetic Methodology--
The methods described by
Sambrook et al. (32) were used for the extraction and
manipulation of plasmid DNA and general DNA in vitro
methods. Nucleotide sequencing was obtained for both strands using the
automated fluorescent dye terminator technique (PerkinElmer ABI model
373A). Analyses of data base sequences used on-line facilities of the
NCBI (www.ncbi.nlm.nih.gov). Multiple sequence alignment was
performed using the program ClustalW (available at
www.ebi.ac.uk/clustalw) (33).
Construction of mSMO Expression Plasmids--
A BLAST sequence
similarity search using the human cDNA PAOh1 (GenBankTM
accession number AY033889) resulted in the Image Clone 2647695 (GenBankTM accession number BC004831), which was supplied
by the United Kingdom Medical Research Council Human Genome Mapping
Resource Centre. To test the fidelity of this sequence, the murine
cDNA was resequenced, and by PCR amplification, two full-length
cDNAs were generated possessing modified 5' and 3' ends. In
particular, the two following synthetic oligonucleotides were used to
introduce the XhoI restriction site and produce a stop codon
and, alternatively, a longer open reading frame in-frame with the
downstream His-tagged sequence at the 3' end of mSMO cDNA:
PAO1-REV, 5'-AAATATCTCGAGGGAACACATTTGGCAGTGAGG-3', and PAO2-REV,
5'-TTTATACTCGAGGGGCCCCTGCTGGAAGAGGTC-3', respectively. The
oligonucleotide PAO3-FOR, 5'-CCATGCAAAGTTGTGAATCCAG-3', was used
coupled with the above described primers. Amplified PCR products were
restricted by XhoI and ligated with the restricted
MscI/XhoI pET25b vector (Novagen) to obtain two
genetic constructs. The first of these named pmSMO has a bacterial
periplasmic leader sequence at the 5' end. The second one named
pmSMO-HT has a bacterial periplasmic leader sequence and a His tag
sequence at 3' end. These two recombinant cDNA constructs were
utilized to transform E. coli Bl21 DE3-competent cells.
Expression of mSMO in E. coli Cells--
E. coli BL21
DE3 cells transformed with pmSMO and pmSMO-HT plasmids were grown at
30 °C in LB medium containing 50 µg/ml ampicillin to
A660 = 0.6 and then induced with
isopropyl-
-D-thiogalactopyranoside (1 mM
final concentration) followed by further cultivation for 5 h at
28 °C.
Periplasmic Fraction Purification--
The E. coli
BL21 DE3 cells were harvested by centrifugation at 4 °C for 10 min
at 10,000 × g, washed with 0.4 culture volumes of 30 mM Tris-HCl, pH 8.0, 20% sucrose, and 1 mM
EDTA, and incubated 5-10 min at room temperature. The suspension was
centrifuged at 10,000 × g for 10 min at 4 °C. The
pellet was resuspended in 0.05 culture volumes of ice-cold 5 mM MgSO4 with vigorous shaking for 10 min on
ice. The cell was then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant corresponding to the periplasmic fraction was finally collected.
Rapid Affinity Purification with pET His Tag Systems--
The
supernatant from E. coli BL21 DE3 cells transformed with
the plasmid pmSMO-HT was applied to a column (3 ml) with
Ni2+ cations immobilized on the His-Bind resin (Novagen)
equilibrated with binding buffer (5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl, pH 7.9). The column
was washed with 60 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl, pH 7.9, followed by elution with 750 mM imidazole, 0.5 M NaCl, and 20 mM
Tris-HCl, pH 7.9.
Assay of mSMO and Determination of the Kinetic
Constants--
Enzyme activity was measured spectrophotometrically by
following the formation of a pink adduct (
515 = 2.6 × 104 M
1 cm
1) as a
result of the oxidation and following condensation of aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid catalyzed by horseradish peroxidase (14). The measurements were performed in 0.2 M
sodium phosphate (NaPi) buffer, pH 8.0, with different substrates at various concentration. Enzyme activities were expressed in
international units (IU, the enzyme amount catalyzing the
oxidation of 1 µmol of substrate × min
1) on 1 liter of culture broth. Protein content was estimated by the method of
Bradford (34) with bovine albumin as a standard. SDS-PAGE was performed
according to the method of Laemmli (35). In the enzyme assays, the mSMO
concentration was 2.0 × 10
8 M.
Km and kcat values were
determined with Spm as a substrate at pH 8.0. In the mSMO inhibition
assays, the monoamine oxidase inhibitor pargyline and the PAO inhibitor
MDL72527 were used at the final concentration of 1.0 mM
with Spd concentration of 0.5 mM. The concentrations chosen
for each inhibitor were based on studies published previously (2).
Identification of mSMO Reaction Products--
The enzymatic
reaction was carried out in a final volume of 1 ml containing 0.1 M NaPi, pH 8.0, 0.2 mM Spm, and 0.1 unit of purified mSMO. Incubation was performed at 37 °C for 10 min and stopped by the addition of 200 µl, 20% (w/v) HClO4.
Precipitated proteins were removed by centrifugation, and the
supernatants were dansylated and analyzed by silica gel TLC according
to Flores and Galston (36) or oxidized by maize MPAO enzyme. The
amounts of O2 consumed and H2O2
formed from Spm by the oxidation with mSMO and from oxidized Spm by the
oxidation with MPAO were measured as described in Federico et
al. (37).
Molecular Modeling of mSMO--
The molecular model of mSMO was
built using the crystal structure of MPAO as a template (Protein Data
Bank code 1B37) (18). In detail, a multiple sequence alignment among
mSMO, MPAO, and other PAOs with known amino acid sequence was obtained
using the program ClustalW (33). The alignment was then manually
refined on the basis of mSMO secondary structure prediction obtained
using the Predict Protein server (38) available online
(dodo.cpmc.columbia.edu/pp/predictprotein.html), which checks for
the occurrence of insertions and deletions on surface loops. Based on
this alignment, the three-dimensional structure of mSMO was then built
using Modeler (Release 6), a program that models protein
three-dimensional structure by satisfaction of spatial
restraints (39).
 |
RESULTS |
Expression of mSMO in E. coli Cells--
The mSMO cDNA clone
was obtained as described under "Experimental Procedures." The two
recombinant cDNA constructs pmSMO (secreted form) and pmSMO-HT
(secreted-tagged form) were used to transform E. coli BL21
DE3 cells. After induction with
isopropyl-
-D-thiogalactopyranoside under the control of
the T7 promoter, the catalytically active proteins were both expressed
at a level of about 6 units/liter of culture broth. The enzyme activity
for both recombinant forms was measured spectrophotometrically as
described under "Experimental Procedures."
Protein Purification of mSMO--
mSMO was isolated from
E. coli BL21 DE3 cells by overexpression of pmSMO-HT and
purified by using His-Bind chromatography kit (Novagen). The SDS-PAGE
electrophoretic analysis was performed on pmSMO-HT transformed E. coli extract, and mSMO was purified. The last one results in a
single band of ~68 kDa as shown in Fig. 1.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 1.
Purified recombinant mSMO-HisTAG
protein analysis. SDS-PAGE analysis of the recombinant mSMO-HisTAG
protein (1 µg of the purified enzyme) after Coomassie Brilliant Blue
staining. MW, molecular weight marker (low range,
Sigma).
|
|
Kinetic Properties of mSMO--
The substrate specificity of mSMO
for several kinds of polyamines and acetylpolyamines has been
investigated under standard conditions at pH 8.0. The purified enzyme
could oxidize Spm rapidly and Spd extremely slowly
(N1-acetylSpd and
N1-acetylSpm). The oxidation rate for
N1-acetylSpm was approximately 1:1,000 of
that for Spm, whereas the rates for Spd and
N1-acetylSpd were >3,000-fold lower than that
for Spm, indicating that Spm is the preferential substrate for mSMO. No
activity was detected using putrescine and
N1-acetylcadaverine as substrates.
The TLC analysis of reaction products (data not shown) demonstrated
that Spm was oxidized by mSMO to Spd. Stoichiometric analysis of Spm
oxidation by mSMO and Spm oxidation by MPAO yielded a molar ratio of
substrate to O2 and H2O2 of 1:1:1.
These results confirmed that mSMO converts Spm into Spd plus
H2O2 and 3-aminopropanal (Fig.
2). The purified mSMO exhibited a pH
optimum of 8.0 in NaPi buffer; thus, the kinetic properties of the
secreted-tagged recombinant enzyme were determined using Spm as
substrate at pH 8.0. The values of Km and
kcat resulted to be 90 µM and 4.5 s
1, respectively. The absorption spectrum of the native
enzyme showed three peaks typical of oxidized flavoproteins (15) with
maxima at 278, 365 and 450 nm. The addition of equimolar amounts of
substrate (Spm) in anaerobic conditions induced the reduction of the
enzyme as indicated by the decrease of the absorbance bands in the
visible range at 365 and 450 nm, whereas reoxygenation of the enzyme
restored the initial spectrum, confirming the involvement of the flavin cofactor in the catalytic cycle (Fig. 3).
The determination of the flavin content in the protein results in a
flavin/mSMO stoichiometry of 1:1. To confirm that the enzyme
activity was attributable to a PAO and not to monoamine oxidases,
their specific inhibitors, MDL72527 and pargyline, respectively,
were included in the reactions. Only MDL72527 inhibited mSMO. The
inhibition was complete in our experimental conditions. Substrate
specificity and the pH optimum were also determined for the secreted
recombinant form, measuring the enzyme activity of the periplasmic
fraction, thus confirming the data obtained for the purified form.

View larger version (5K):
[in this window]
[in a new window]
|
Fig. 2.
Amine oxidation reaction catalyzed by
mSMO. Scheme of the cleavage on the Spm substrate operated by
mSMO.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Absorbance spectra of mSMO: effect of adding
Spm to oxidized enzyme and oxygenation of the reduced form.
Absorbance spectra were recorded in anaerobiosis with 1 ml of 10 µM mSMO in 0.1 M NaPi, pH 7.5, using a
Thumberg-type cuvette. a, native enzyme in anaerobic
condition; b, native enzyme in anaerobic condition + 20 µM Spm; c, enzyme after reoxygenation.
|
|
Structural Analysis and Active Site Modeling of mSMO--
The
primary structure of mSMO was deduced from the cDNA sequence. Its
open reading frame predicts a 555-amino acid protein with a calculated
Mr of 61,8523. Amino acid sequence alignment between mSMO and PAOh1 (29) has revealed that they share a 95.1% sequence identity, whereas the sequence comparison with the plant PAOs
shows a lower identity ranging from 23.4 to 26.5%. Multiple amino acid
sequence alignments of these two proteins together with other known
members of the plant PAO family are shown in Fig.
4.

View larger version (94K):
[in this window]
[in a new window]
|
Fig. 4.
Amino acid sequence comparison of animal
(mSMO and PAOh1) and plant (MPAO1, BPAO1, BPAO2, and APAO) polyamine
oxidase proteins. Multi-alignment was done using the
program ClustalW sequence alignment. Identical residues are indicated
by gray boxes. Signal peptides are underlined.
Residues in the maize MPAO enzyme organizing the catalytic U-tunnel are
in boldface letters: the ones putatively involved
in the catalytic activity are labeled by an asterisk, the
ones composing the tunnel entrance (carboxylate ring and aromatic
portion) are labeled by a ¤. Numeration is shown at the
right side. Percentage of identity refers to mouse SMO
(Image Clone 264769). BPAO1 and BPAO2, barley
PAOs; APAO, Arabidopsis thaliana PAO.
|
|
Given the fairly low sequence identity between mSMO and MPAO (26.5%),
homology modeling techniques, which heavily rely on the availability of
a correct sequence alignment, must be applied carefully to allow the
construction of a reliable three-dimensional model of the entire mouse
protein structure. For this reason, a multiple sequence alignment among
mSMO, MPAO, and other PAOs with known amino acid sequence was obtained
using the program ClustalW (Fig. 4) (33). In addition, the alignment
was manually refined on the basis of mSMO secondary structure
prediction, which was obtained using the program Predict Protein (38),
to avoid the unlikely occurrence of insertions and deletions within
secondary structure elements. This procedure yielded the final
alignment between mSMO and MPAO, the only enzyme belonging to this
class with known three-dimensional structure (18). Based on this
alignment, the three-dimensional model of mSMO was built with the
program Modeler (39).
As shown in Table I, an analysis of mSMO
FAD binding pocket reveals a high degree of conservation of the
residues involved in the stabilization of this prosthetic group among
PAO and human monoamine oxidase enzymes (31). This result is not
surprising, although it can be used as an internal check for the
correctness of the sequence alignment used for mSMO model
construction.
View this table:
[in this window]
[in a new window]
|
Table I
Common FAD binding amino acids in MPAO, hMAO A, hMAO B, and mSMO
Function and position of the 15 amino acids of MPAO associated with FAD
binding and their corresponding ones in hMAO A, hMAO B, and mSMO. hMAO
A and B, human MAO A and B; mSMO, mouse SMo. Identical amino acids are
represented by an equal sign.
|
|
Fig. 5 shows a comparison of the active
site structure of MPAO and mSMO. In this case, the degree of
conservation is lower, but several residues are conserved or
conservatively substituted. In particular, the overall "tunnel"
shape of the active site observed in MPAO (18) is maintained in mSMO,
although the central part of the tunnel is wider because of the
substitutions T298W and T439W (numbering refers to MPAO). The
substitution of Glu-62 involved in substrate binding in MPAO by a His
residue in mSMO is the major difference observed between MPAO and mSMO
active sites. This substitution is particularly interesting in that it
can provide an explanation for the different pH profile of the activity
observed in mSMO (see "Discussion"). Finally, it is interesting to
note both in mSMO and in PAOh1 the conservation of Lys-300, a
residue that has been hypothesized to play a structural role in the
correct positioning of the FAD cofactor by forming an indirect hydrogen bond with FAD through a bridging water molecule (40).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
Stereoview of the residues building up the
catalytic tunnel of MPAO (A) and mSMO
(B). The isoallosazinic ring of FAD is
shown in green, whereas the substrate spermine is shown in
red. Enzyme substrate complexes have been modeled using as a
template the structure of MPAO in complex with the Spm analogue
MDL72527 (Protein Data Bank code 1BQ5) (18). Residues are numbered
according to MPAO amino acid sequence. The figure has been made with
Grasp (41).
|
|
 |
DISCUSSION |
Plant or animal recombinant PAOs have been never reported to be
expressed in E. coli cells to our knowledge. This is the
first report of a vertebrate polyamine oxidase overexpressed in
secreted and secreted-tagged forms in such a heterologous system. SMO
recombinant enzymes are targeted to the periplasmic membrane
compartment, and the catalytically active proteins are expressed at a
level of ~6 units/liter of culture broth. The data obtained for the secreted recombinant mSMO perfectly match the ones obtained for the
secreted-tagged enzyme form.
The analysis of the expressed mSMO protein gave an improved
understanding of the biochemical features of this enzyme, since the
purified recombinant enzyme was able to oxidize only Spm and failed to
act upon Spd and the N1-acetyl derivatives. The
enzyme specificity reported herein is in agreement with the one
described in transfected human cells by Vujcic et al.
(30).
The precise nature of the reaction products and the cleavage position
on the Spm substrate are those typical of an animal PAO enzyme. In line
with this finding, the specific inhibition of mSMO enzyme activity was
obtained with MDL72527 but not with pargyline.
The absorption spectrum of the native enzyme showed the typical
three-banded spectrum for flavoproteins. Furthermore, the addition of
the Spm substrate in anaerobic conditions resulted in a dramatic
absorbance decrease, while reoxygenation of the enzyme restored the
initial spectrum, indicating the involvement of a FAD group in the
catalytic cycle.
An analysis of the molecular model of mSMO as compared with MPAO
three-dimensional structure suggests that the catalytic tunnel of the
former enzyme is wider. It is tempting to speculate that the preference
for Spm over Spd as a substrate observed for mSMO is linked to the
different shape of the catalytic tunnel in which the short Spd
substrate would be bound in a "floppy" fashion, thus rendering less
efficient the enzyme catalysis, which has been hypothesized to rely on
an "in register" binding of the substrates.
The substitution of Glu-62 by a His residue in mSMO can provide an
explanation for the peculiar pH dependence of the activity. In fact, it
can be reasonably assumed that His-62 is partially protonated at pH
values lower than 7.0. Thus, the binding of the cationic polyamine
substrates would be unfavorable and enzyme activity would be very low
as observed experimentally. At pH values higher than 7.0, His-62 would
deprotonate, thus facilitating substrate binding and leading to an
increase in enzyme activity, which is maximal at pH values of
~8.0.
In conclusion, in mammalian cells, polyamine catabolism seems to be
mediated by the activity of two enzymes, PAO and the novel SMO,
described in this work (30). The precise significance of both polyamine
oxidase activities in the metabolism of polyamines remains to be
established, particularly regarding the role of each enzyme in
regulating polyamine concentrations in mammalian tissues in relation
with growth processes and malignant transformation.