From the
A pea seedling amine oxidase cDNA has been isolated and
sequenced. A single long open reading frame has amino acid sequences
corresponding to those determined from active site peptide (Janes, S.
M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley,
D. M., Mure, M., and Klinman, J. P.(1992) Biochemistry 31,
12147-12154) and N-terminal sequencing experiments. The latter
reveals the protein to have a 25-amino acid leader sequence with
characteristics of a secretion signal peptide, as expected for this
extracellular enzyme. Comparisons of the amino acid sequence of the
mature pea enzyme (649 amino acids) with that of the mature lentil
enzyme (569 amino acids; Rossi, A., Petruzzelli, R., and Finazzi-Agr,
A.(1992) FEBS Lett. 301, 253-257) reveal important and
unexpected differences particularly with regard to protein length.
Sequencing of part of the lentil gene identified several frameshift
differences within the coding region resulting in a mature lentil
protein of exactly the same length, 649 amino acids, as the pea enzyme.
Multiple alignments of 10 copper amine oxidase sequences reveal 33
completely conserved residues of which 10 are found within 41 aligned
residues at the C-terminal tails, the region missing from the original
lentil sequence. One of only four conserved histidines is found in this
region and may represent the third ligand to the copper. The pea enzyme
contains around 3-4% carbohydrate as judged by deglycosylation
experiments. We have also demonstrated by hybridization analysis that
copper amine oxidase genes are present in a range of mono- and
dicotyledonous plants.
Copper amine oxidases (EC 1.4.3.6) have been identified in a
wide range of microbial, plant, and animal systems (for reviews see
McIntire and Hartmann, 1993; Knowles and Dooley, 1994). In microbes
they allow utilization of unusual amine substrates as nitrogen and/or
carbon sources, whereas in plants and animals their functions are less
clear. The copper amine oxidases catalyze the oxidation of a wide
variety of biogenic amines, including mono-, di-, and polyamines to the
corresponding aldehyde with release of NH
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
yielding 4-aminobutyraldehyde and 5-aminovaleraldehyde,
respectively. This enzyme is relatively abundant comprising at least
0.1% total soluble protein in etiolated pea seedling epicotyls (McGowan
and Muir, 1971; McGuirl et al., 1994) but despite extensive
study its function(s) remain unclear (Smith, 1985). Plant copper amine
oxidases are generally found in the apoplast, loosely associated with
the cell wall (Angelini et al., 1986; Federico and Angelini
1986; Federico et al., 1985, 1988; Slocum and Furey, 1991).
Di- and polyamines are also present within the apoplast, and the
H
The
copper amine oxidases are homodimers of subunit size approximately
70-95 kDa depending on the source. Each subunit contains one
Cu(II) and a quinone cofactor (for review see Klinman and Mu, 1994)
which has been identified as 2,4,5-trihydroxyphenylalanine quinone
(TPQ
We are interested in
the biological functions of plant copper amine oxidases, and we report
here the molecular cloning and sequence analysis of the pea seedling
amine oxidase gene. Important discrepancies between the translated pea
and corresponding lentil sequence (Rossi et al., 1992) are
considered, and a corrected lentil sequence is presented. The
implications of this revision for interpretation of comparative amino
acid sequence alignments to highlight important residues are discussed.
We have also examined the level of glycosylation of pea seedling amine
oxidase. Finally, we demonstrate the presence of copper amine oxidase
gene homologs in a range of plant species.
A progressive sequencing strategy was used
with design of further primers to complete the sequence of both strands
of the cDNA insert. Sequence data were compiled and analyzed on a
MicroVax 3600 computer using Staden(1986) software. Amino acid sequence
alignments were performed using CLUSTALV and SOMAP (Higgins et
al., 1992; Parry-Smith and Attwood, 1991).
Lentil cDNA clones
were sequenced in a similar manner using primers selected from the pea
seedling sequencing project.
Although polyadenylation signals are less well defined for plant
than for mammalian genes, the 3`-untranslated region contains two
potential polyadenylation signals. The first (2152-2157; Fig. 1) exactly matches the AATAAA consensus; however, the second
AATGAA (2223-2228; Fig. 1) lies only 21 residues from the
poly(A) tail. It seems likely that this second signal represents a
near-upstream element associated with polyadenylation of the 3`-end of
the mRNA (Hunt, 1994)
One residue difference
occurs within coding region around amino acid 476 as discussed above,
but the extra 79 amino acids in the pea protein represent a major
difference between the C-terminal ends of the two polypeptide chains.
Such a gross difference in size seemed surprising given the
phylogenetic similarity of the two plants: both Leguminoseae, the
similar physiological and biochemical properties of the two enzymes,
and the good sequence identity within the rest of the coding regions.
To investigate these differences further we first resequenced the
pea seedling amine oxidase gene from a second clone, but this proved to
be identical to the original sequence determination. We then isolated
mRNA from lentil seedlings and prepared cDNA. Using pea gene sequencing
primers we PCR amplified the region of the lentil amine oxidase gene
corresponding to nucleotides 179-2149 (Fig. 1). The PCR fragment
was cloned into the pCR vector (Invitrogen) and was sequenced. Only a
limited number of differences was identified between this lentil
sequence and that of Rossi et al.(1992); nonetheless, the
differences are very important, as shown in Fig. 2. The most
significant of the differences were several single base insertions and
a deletion which had the effect of altering, several times, the reading
frame for the encoded protein. Three independent insertions lead to
frameshift alterations in the region encoding residues 474-478
(positions 1568-1582; Fig. 1) and result in a sequence with
strong identity to the pea gene including the presence of the
``missing'' residue. Most significant are insertions of a C
at the positions corresponding to nucleotides 1814 and 1859 in the pea
sequence (Fig. 1) and deletion of a G which occurs in the lentil
sequence between nucleotides 2033 and 2034 in the pea sequence (Fig. 1). These result in a sequence that matches the pea
seedling amine oxidase sequence by extending the lentil coding region
to encode a mature protein of 649 amino acids, identical in size to the
pea protein. Fig. 2shows a comparison of part of the revised
(this study) and the previous (Rossi et al., 1992) lentil
amine oxidase cDNA sequence, highlighting the observed differences and
the consequences for the protein coding region.
We analyzed the extent of
glycosylation in pea seedling amine oxidase and compared this with the
porcine plasma amine oxidase, a protein known to be extensively N-glycosylated. Fig. 3shows the results of treating
denatured protein samples with N-glycosidase, an enzyme
capable of removing all N-linked carbohydrate moieties.
Comparing the mobility with that of untreated controls revealed a
significant shift in molecular mass of the pig plasma amine oxidase
protein from 91.5 to 82.5 kDa, indicating that the protein contains
about 9.8% carbohydrate. By contrast, the pea seedling amine oxidase
showed a smaller change in migration rate from 72.5 to 69.5 kDa,
suggesting that the protein contains some 4% carbohydrate, a value
similar to that reported by Rossi et al.(1992) for the lentil
enzyme.
The large difference in
the level of glycosylation of the pea seedling amine oxidase compared
with the pig plasma amine oxidase probably accounts to some extent for
differential success in crystallization studies. The pea seedling amine
oxidase has been crystallized and preliminary data determined
(Vignevich et al., 1993). This contrasts with pig plasma amine
oxidase where crystallization studies have resulted only in very small
crystals of poor quality. No significant improvement in crystal quality
could be achieved even when native pig plasma amine oxidase was treated
with a combination of glycopeptidase F and endoglycosidase. Such
treatment produced a simpler but still heterogeneous population of
species as judged by isoelectric focusing,
Mechanistic studies suggest that copper
amine oxidases mediate C-H bond cleavage through a proton abstraction
step involving a group of pK
Several studies strongly suggest the
involvement of three equatorial histidine ligands in Cu(II)
coordination (Scott and Dooley, 1985; Barker et al., 1986;
McCraken et al., 1987). Based on sequence alignments, Mu et al.(1994) identified three conserved histidines, two within
a H-X-H motif (residues 442-444; Fig. 4) and one
at position 357 (Fig. 4). However, previous assessments of
conserved histidines have been affected by the incomplete lentil
sequence that prevented the identification of a fourth conserved
histidine at position 603 within the strongly conserved C-terminal
tail. It seems probable that the H-X-H motif provides two of
the copper ligands since Cai and Klinman (1994a) found that a H456D
mutant of the yeast enzyme (equivalent to His
It is important to note
that in the H456D yeast amine oxidase mutant the quinone cofactor was
not formed, leading to the suggestion that the three-dimensional
structure of the active site, including the histidine ligands and the
copper, is critical for the formation of the TPQ (Cai and Klinman,
1994a). In a further study Cai and Klinman (1994b) expressed the H.
polymorpha methylamine oxidase in Saccharomyces
cerevisiae, a yeast that appears to produce no quinoproteins
(Large, 1986), although this requires more definitive proof. The
identification of TPQ in this expressed enzyme is taken to suggest a
self-processing mechanism of hydroxylation and oxidation steps leading
to the formation of TPQ from tyrosine in a copper-peroxide-mediated
reaction. Tanizawa and colleagues have provided even stronger evidence
for copper-mediated conversion of the active site Tyr to TPQ. They have
expressed TPQ-free forms of both A. globiformis phenylethylamine oxidase and histaminase in E. coli by
growing in copper-free media and have subsequently shown formation of
TPQ by addition of Cu(II) to the purified apoprotein under aerobic
conditions (Tanizawa et al., 1994; Matsuzaki et al.,
1994; Choi et al., 1995). Given that several residues appear
to be totally conserved in all amine oxidases, as illustrated in Fig. 4, it seems likely that all copper amine oxidases will have
some conserved active site features and will catalyze cofactor
formation in a similar copper-dependent manner.
The recent reports
of preliminary crystallographic data for two copper amine oxidases from
pea seedling (Vignevich et al., 1993) and E. coli (Roh et al., 1994) and from studies under way at Leeds
suggest that structural information will shortly become available to
assist interpretation of the structural and mechanistic roles of the
various conserved residues.
From these reports, it remains unclear whether the copper
amine oxidases fulfill a specialist role in a limited number of species
or whether they are present in all plants. To begin answering this
question we analyzed Southern blots of EcoRI digested genomic
DNA from a range of plant species by heterologous hybridization with a
radiolabeled pea seedling amine oxidase cDNA probe. Pea DNA digested
with EcoRI, HindIII, or XbaI was similarly
hybridized. The results, shown in Fig. 5, reveal hybridization
signals in all samples. The plants represent dicotyledons from the
Leguminoseae (lentil, pea, and soybean), Solanaceae (tobacco, tomato,
and potato), and Chenopodiaceae (sugar beet) and monocotyledons from
the Gramineae (rice, wheat, and barley).
In pea
there is evidence for two copper amine oxidases: an epicotyl enzyme,
the subject of the present report, and an embryonic enzyme. These are
reported to have distinct biochemical properties, and the embryonic
enzyme has been implicated in regulating the induction of the epicotyl
enzyme (Srivastava et al., 1977). The indication of more than
one genomic gene by Southern analysis (Fig. 5, right
panel) is consistent with these enzymes being encoded by distinct
genes. In this regard we have recently isolated two distinct classes of
genomic clones from a pea genomic library. One class shows sequence
identity with the cotyledon cDNA clone reported here, so it is possible
the other class represents the amine oxidase expressed in embryonic
tissues.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Professor D. M. Dooley for providing some pea
seedling amine oxidase, Dr. D. Bucke for the pea seedling cDNA library,
Denise Ashworth for an efficient oligonucleotide synthesis service, and
Mike Beck for computer expertise. We also thank Dr. P. F. Knowles for
providing pig amine oxidase and for valuable discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
H
O
. The pea seedling enzyme preferentially
catalyzes oxidation of the diamine substrates putrescine (R = NH
(CH
)
) and
cadaverine (R =
NH
(CH
)
) at the primary amino group
according to ,
O
formed by their oxidation may be important
in lignosuberization and cross-linking of extracellular macromolecules
such as extensins, both during normal growth and in response to stress
and wounding (Angelini and Federico, 1985, 1989; Federico and Angelini,
1988; Angelini et al., 1990; Slocum and Furey, 1991). Recently
a correlation has been demonstrated between copper amine oxidase and
peroxidase levels in chickpea tissues undergoing wound healing
(Angelini et al., 1990, 1993; Scalet et al., 1991).
Differential induction levels of amine oxidase and peroxidase have also
been observed in susceptible and resistant cultivars of chickpea upon
infection by Ascochyta rabiei, suggesting that they play a
role in the defense response (Angelini et al., 1993).
(
)or TOPA quinone) in the bovine serum
enzyme (Janes et al., 1990). Subsequently TPQ has been
identified biochemically or its presence inferred from amino acid
sequence homology in other copper-containing amine oxidases from Hansenula polymorpha (Bruinenberg et al., 1989; Mu et al., 1992), Klebsiella aerogenes (Sugino et
al., 1992), Escherichia coli K12 (Cooper et al.,
1992; Azakami et al., 1994), lentil (Pedersen et al.,
1992; Rossi et al., 1992), pea (Janes et al., 1992), Arthrobacter strain P1 (Zhang et al., 1993), Arthrobacter globiformis (Tanizawa et al., 1994),
human kidney (first identified as amiloride-binding protein; Barbry et al., 1990), rat kidney (Lingueglia et al., 1993),
and A. globiformis histaminase (Choi et al., 1995).
Preliminary structural studies have been reported for the copper amine
oxidases from pea seedlings (Vignevich et al., 1993) and from E. coli (Roh et al., 1994).
Strains
E. coli strain LE 392 (supE44, supF58, hsdR514, galK2, galT22, metB1, trpR55, lacY1)
was used as host for cDNA library screening, and DH1 (supE44,
hsdR17, recA1, endA1, gyrA96, thi-1, relA1) was used for routine
plasmid growth.
Amino Acid Sequence Analysis
A 10-µg
sample of pea seedling amine oxidase, kindly provided by Professor D.
M. Dooley (Montana State University), was coupled to p-phenylene diisothiocyanate glass and was subjected to
N-terminal amino acid sequencing by Edman degradation. Released amino
acids were assigned following high performance liquid chromatography
reverse phase analysis against suitable standards. Amino acid
sequencing was performed by the Biotechnology and Biological Sciences
Research Council protein microsequencing facility in the Department of
Biochemistry and Molecular Biology, University of Leeds.
Routine Molecular Biology
Procedures
Routine DNA manipulations were performed
according to Sambrook et al. (1989). Restriction endonuclease,
ligase, and DNA polymerase reactions were performed in the buffers and
under the conditions recommended by their manufacturers. PCR fragments
were cloned into the pCR vector according to the protocols supplied
with the TA cloning system (Invitrogen).
cDNA Library
A cDNA library prepared from
mRNA isolated from 6-day-old etiolated pea seedlings with a Stratagene
cDNA synthesis kit was a gift from Dr. D. Bucke (Schering Agrochemicals
Ltd.). The cDNA was cloned into EcoRI/XhoI digested
Uni-ZAP (Stratagene) to give a library of 1.5 10
independent clones, which was amplified to a titer of 3.8
10
plaque-forming units/ml.
Poly(A)
Eight- to 10-day postgermination
lentil and pea etiolated seedlings were dissected and the epicotyls
frozen in liquid NRNA Isolation and
cDNA Synthesis for PCR
and stored at -70 °C. Frozen
tissue was powdered in liquid N
using a pestle and mortar,
and mRNA was isolated using a Pharmacia Microprep reagent kit according
to the manufacturer's instructions. First-strand cDNA was
prepared from poly(A)
RNA with a Pharmacia
first-strand cDNA synthesis kit using either a 5`-tailed oligo(dT)
primer (Pharmacia) or a sequence specific primer.
PCR Amplification of a Lentil Amine Oxidase
Probe
PCR primers were designed to amplify the lentil
seedling amine oxidase cDNA coding region (Rossi et al., 1992)
for use as a heterologous probe to screen the pea cDNA library. The PCR
mix contained 100 pg of lentil cDNA, 100 pmol (2 µM) of
each primer (5`-TTTACACCATTGCATACTCAACATC-3`) and
(5`-CCACACAGCCAAAGTATCGTCTCC-3`), and a 50 µM concentration of each dNTP in a 50-µl reaction. The reaction
mix was overlaid with 50 µl of mineral oil and subjected to a
``hot-start'' by adding 1 unit of SuperTaq (HT Biotechnology)
during the last minute of the initial 5-min denaturation (94 °C)
step. Amplification was achieved by 30 cycles of 94 °C, 1 min; 55
°C for 1 min; 72 °C for 3 min. The reaction products were
separated through a 1.5% NuSieve agarose gel (FMC), and a DNA band of
the expected size of 1.74 kilobase pairs was recovered.
Pea cDNA Library Screening
Phage
dilutions were plated on LE392 to give 15,000 plaques/9-cm-diameter
plate. Duplicate plaque lifts were taken onto Hybond N membranes (Amersham) according to the manufacturer's
instructions. A 30-ng aliquot of the lentil seedling amine oxidase
fragment in NuSieve agarose was radiolabeled with
[
-
P]dCTP by random hexamer labeling
(Feinberg and Vogelstein, 1984), and the probe was separated from
unincorporated label by spermine precipitation (Hoopes and McClure,
1981). The membranes were prehybridized at 56 °C for 4 h in 5
SSPE (1
SSPE, 150 mM NaCl, 10 mM NaH
PO
H
O, 1 mM Na
EDTA, pH 7.4), 6% polyethylene glycol, 0.5% skimmed
milk powder (Marvel), 1% SDS, 0.1% sodium pyrophosphate, and 250
µg/ml sonicated and denatured calf thymus DNA (Gurr and McPherson,
1992). Hybridization at 56 °C was performed overnight in the same
buffer after the addition of the radiolabeled probe. The membranes were
finally washed in 0.1% SSC (1
SSC, 150 mM NaCl, 15
mM trisodium citrate), 0.1% SDS at 65 °C. Positive plaques
were purified by successive rounds of screening at lower plaque
densities.
DNA Sequence Analysis
cDNA clones were
rescued as pBluescript SK(-) plasmids by in vivo excision from Uni-ZAP as detailed by Stratagene. DNA sequence
analysis was performed with Sequenase V2.0 kits (Amersham) with product
analysis on 6% wedge gels. Initial sequencing reactions were performed
using vector-specific T3 and T7 primers and degenerate primers. Forward
and reverse orientation primers (primers a and b, respectively) were
designed by back-translation of the amino acid sequence of the
TPQ-containing peptide (Janes et al., 1992). These primers
were designed prior to the definitive identification of Tyr as the
residue modified to TPQ (Mu et al., 1992) and so included
coding potential for both Tyr and Phe at the codon corresponding to the
TPQ site. Primer c was designed from the N-terminal amino acid sequence
of the mature pea seedling amine oxidase determined during the course
of the present study. The primer a sequence was 5`-GTI GGI AA(TC) T(TA)
(TC) GA(TC) AA(TC) GT-3`. The primer b sequence was 5`-ATI AC(AG)
TT(AG) TC(AG) (AT)A(AG) TT(AGCT) CC-3`. The primer c sequence was
5`-CA(TC) GTI CA(GA) CA(TC) CCI (TC)T-3`. Parentheses indicate
degenerate positions.
Southern Blot Analysis
Genomic DNAs were
prepared from lentil and pea according the method of Dellaporta et
al.(1983) with additional purification by cesium chloride density
gradient centrifugation (Sambrook et al., 1989). Genomic DNAs
from barley, rice, soybean, sugar beet, tomato, vine, wheat, and Arabidopsis were kindly provided by Claire Scollan, Yi Li, and
Ruth Turnbull (Centre for Plant Biochemistry and Biotechnology, Leeds).
Ten-µg aliquots of DNA were digested with restriction enzymes
according to the manufacturer's recommendations and were
separated through a 1% agarose gel (15 20 cm) by
electrophoresis at 1.5 V/cm for 16 h before transfer to Hybond
N
, followed by prehybridization and hybridization as
described for the cDNA library screen. The filter was successively
washed in 5
SSC, 0.1% SDS at 56 °C; then 1
SSC,
0.1% SDS at 65 °C; and finally in 0.1% SSC, 0.1% SDS at 65 °C
with autoradiographic exposure between each wash.
Northern Blot Analysis
Poly(A) mRNA was isolated from 6-day-old etiolated pea and lentil
epicotyl tissue using a Pharmacia Microprep reagent kit according to
the manufacturer's instructions. The mRNA was electrophoresed at
5 V/cm in a 1% agarose, 1
MOPS (Sambrook et al., 1989)
gel containing 0.66 M formaldehyde and ethidium bromide
together with 0.24-9.5-kilobase RNA markers (Life Technologies,
Inc.). The gel was capillary blotted onto a Hybond N
membrane and fixed according to the manufacturer's
instructions. The membrane was prehybridized in 5
Denhardt's, 6
SSC, 100 µg/ml sonicated and denatured
calf thymus DNA at 65 °C overnight and hybridized overnight under
the same conditions with a pea seedling amine oxidase cDNA random
hexamer-labeled probe prepared as described previously. The membrane
was washed to 0.1
SSC, 0.1
SDS at 65 °C before
autoradiography.
Deglycosylation Analysis
Protein samples
of approximately 1 mg were denatured by boiling for 5 min and were then
deglycosylated with N-glycosidase in 20 mM sodium
phosphate buffer, pH 7.2, at 37 °C for 16 h. Control reactions
containing no N-glycosidase were also performed. Proteins were
analyzed on 0.1% SDS, 10% polyacrylamide gel and stained with Coomassie
Brilliant Blue (Hames, 1981).
Isolation and Characterization of Pea Seedling
Amine Oxidase cDNA
Of 150,000 plaques of the cDNA library
screened, 102 gave positive hybridization signals after the primary
screen using a radiolabeled lentil amine oxidase gene probe (Rossi et al., 1992). Several clones were taken through two further
rounds of screening, and pBluescript clones were rescued from Uni-ZAP
by in vivo excision. One clone, pPSAO1, contained an insert of
2.3 kilobases which probably represents a full-length cDNA based on
three lines of experimental evidence. First, Northern analysis of
poly(A) RNA from 8-10-day-old pea and lentil
seedlings reveal a single transcript of approximately 2.3 kilobases
(data not shown). Second, rescreening of the cDNA library with a
740-base pair fragment from the 5`-proximal half of the pPSAO1 insert
(bases 490-1230) failed to identify clones with inserts longer than
that of pPSAO1. Finally, PCR screening of the cDNA library with a
vector primer and internal PSAO-specific primers failed to amplify any
sequences longer than those amplified from pPSAO1.
Sequence Analysis
The DNA sequence of the
cDNA insert in pPSAO1 is shown in Fig. 1and was determined for
both strands using a progressive primer design strategy. A long open
reading frame extends from position 74 to 2098 (Fig. 1), and the
translated amino acid sequence shows good overall homology with the
published lentil amine oxidase cDNA sequence (Rossi et al.,
1992). The identity of the translated pea seedling amine oxidase was
confirmed by comparison with two regions of peptide sequence data.
First, the amino acid sequence of the TPQ-containing peptide determined
by Janes et al.(1992) matches a region of coding sequence
between 1298 and 1331 (Fig. 1). X indicates an
unassigned residue which corresponds to Tyr, the residue
proposed to form the TPQ cofactor. In the lentil sequence this residue
is found at an identical position in the polypeptide chain (Rossi et al., 1992). Second, the N-terminal region of pea seedling
amine oxidase was determined to be X-T-P-L-H-V-Q-H-P-L-D. This
latter sequence matches the amino acids encoded by bases 149-181 (Fig. 1), and the unassigned residue (X) can be
identified as valine. This region of amino acid sequence does not
correspond to the start of the open reading frame but is preceded by a
putative signal sequence of 25 residues with characteristics expected
of a secretion signal (von Heijne, 1986). This finding is consistent
with evidence that amine oxidase is an extracellular enzyme in the
Leguminoseae (Federico and Angelini, 1986; Federico et al.,
1988; Slocum and Furey, 1991). It also agrees with the interpretation
of the lentil cDNA sequence (Rossi et al., 1992), although in
this case the start of the open reading frame was not present.
Figure 1:
Sequence of the pea
seedling copper amine oxidase cDNA clone pPSAO1. The translated
sequence of the copper amine oxidase is shown as single letter amino
acid codes and numbered +1 from the start of the mature coding
region. The signal sequence cleavage site is indicated by an arrowhead. Boxed regions correspond to amino acids
determined by peptide sequencing. Circled residues are those
totally conserved in all copper amine oxidases. Heavy circles indicate the tyrosine (Y) that is modified to TPQ, and the four
conserved histidines. Potential polyadenylation signals are underlined. The poly(A) tail that starts after position 2249
has been omitted.
The
ATG codon at nucleotides 74-76 (Fig. 1) most probably
represents the amine oxidase translation initiation site. It should be
noted that a second in-frame ATG (position 92-96; Fig. 1)
occurs six codons into the coding region and could represent the
translation start site. However, in eukaryotes the first ATG
encountered by the translation machinery is usually selected as the
initiation site. The sequence context of the first ATG
(CTCACGATGGCT) matches well the consensus translation start site
(TAAACAATGGCT) defined by Joshi(1987) for plant genes.
Comparison of the Pea Seedling and Lentil Amine
Oxidase Sequences
Multiple sequence alignment of amine
oxidase protein sequences by Zhang et al.(1993) revealed the
lentil sequence to be significantly shorter at the C-terminal end than
any other amine oxidase. Since the lentil diamine oxidase sequence was
the first of plant origin to be reported, this difference in length
suggested some functional significance. However, the sequence of the
pea seedling amine oxidase, presented here, contradicts this view. Over
the first 555 residues of the mature plant proteins there is very good
sequence conservation between the pea and lentil sequences with only 30
amino acid differences, many of which are conservative changes. The
region corresponding to residues 474-478 (-G-S-S-K-R-; Fig. 1) is not a good match, and there is an extra amino acid in
the pea seedling sequence in this region compared with the lentil
sequence (-E-V-Q-E-). More significantly, the predicted lentil mature
protein contains 569 amino acids with a calculated molecular mass of
64.3 kDa (Rossi et al., 1992), whereas the pea protein has 649
residues and a calculated molecular mass of 73.7 kDa. This represents a
difference in length of 80 amino acids.
Figure 2:
Lentil amine oxidase cDNA sequence. The
numbering corresponds to the amino acid numbers shown in Fig. 1. The
nucleotide sequence shown was determined during this work and
highlights differences with a previous sequence determination (Rossi et al., 1992). The translated sequences correspond to that
derived from the nucleotide sequence presented (upper, bold) and the previously reported sequence (lower;
Rossi et al., 1992). Nucleotide sequence differences are
indicated by double underscoring. Those base changes giving
rise to amino acid differences between the lentil and pea sequences are
indicated by closed circles. Open circles indicate
amino acid differences between the two lentil translation products. Open circles marked with downward pointing
arrowheads, which indicate the positions of base insertions, or
the upward arrowhead, which represents a deletion of the base
shown in lowercase letters, represent sites of frameshifts.
The consequence is to extend the original coding region as highlighted
by the boxed regions.
Deglycosylation
In the pea sequence there
are four potential N-glycosylation sites (N-X-S/T
where X is any residue) at amino acids 131, 334, 364, and 558.
Three of these sites are also found in the corrected lentil sequence at
131, 364, and 558; however, the site at 334 is a D-G-T in lentil. On
the basis of hydropathy profiles Rossi et al.(1992) proposed
that the site at 364 would be unlikely to be modified, leaving two
potential sites at 131 and 558.
Figure 3:
SDS-polyacrylamide gel electrophoretic
analysis of the levels of N-glycosylation of pea seedling and
porcine plasma amine oxidases (PSAO and PPAO,
respectively). Treatments are indicated above each lane.
Control samples were incubated either at room temperature or 37 °C
for 16 h in the absence of N-glycosidase; test samples were
incubated at 37 °C with the enzyme. Molecular masses were
calculated from the protein markers. In both the pea and pig treated lanes the major band moves to a lower molecular mass, although
the change is more pronounced for the pig
sample.
Rossi etal.(1992) suggested that the
difference in molecular mass between the translated lentil seedling
amine oxidase of 64.3 kDa and that determined by SDS-polyacrylamide gel
electrophoresis to be 70 kDa was probably due to glycosylation at two
sites. Although we cannot rule out varietal differences between the
lentils used in the present study and those used by Rossi et
al.(1992) such an explanation seems unlikely to account for the
significant difference in size of the amine oxidase proteins. Given the
phylogenetic similarity between lentil and pea and the similar
physicochemical properties and levels of glycosylation, we suggest that
the amine oxidases are of identical size.
(
)presumably due to the inability of N-glycosidase to gain access to all N-linked
carbohydrate sites in the native enzyme.
Comparative Amino Acid Sequence Analysis with Other
Copper Amine Oxidases
The pea seedling and corrected lentil
copper amine oxidase sequences allow refinement of amino acid sequence
alignments as shown in Fig. 4. Such comparisons highlight highly
conserved residues that are most likely of functional or structural
importance. An alignment of representative sequences of bacterial,
yeast, plant, and mammalian amine oxidases is shown in Fig. 4and
was prepared using the program CLUSTALV (Higgins et al., 1992)
with manual adjustments using SOMAP (Parry-Smith and Attwood, 1991).
There are 33 completely conserved residues mainly within two regions:
the central region (residues 286-459), approximately centered on
Tyr, and the C-terminal end. Thirteen of the conserved
residues are a Gly or Pro, which would be consistent with conserved
structural features such as turns. The present alignment provides the
first opportunity to appreciate fully the level of homology within the
C-terminal tail region. It reveals a very high level of sequence
conservation with 10 of the 33 conserved residues present within an
alignment window of only 41 residues (592-632; Fig. 4),
suggesting an important structural or functional role for this region.
Figure 4:
Amino
acid sequence alignment of copper amine oxidases. The totally conserved
residues are shown in inverse highlighting as are the three
positions corresponding to acidic positions (either aspartate or
glutamate). Positions at which a residue occurs in at least 60% of the
sequences are shown in boldface. PSAO, pea seedling
(present study); LSAO, lentil (present study; Rossi et
al., 1992); ECAO, E. coli (Azakami et
al., 1994); KPAO, Klebsiella (Sugino et
al., 1992); AGAO, A. globiformis (Tanizawa et al., 1994); ARAO, Arthrobacter strain P1
(Zhang et al., 1993); HPAO, H. polymorpha (Bruinenberg et al., 1989; Mu et al., 1992); BSAO, bovine serum; ABPHK, human kidney (Barbry et al., 1990); ABPRAT, rat kidney (Lingueglia et
al., 1993).
Residues previously reported to be conserved within amine oxidases
(Zhang et al., 1993; Mu et al., 1994) are also found
in pea seedling amine oxidase. These include the consensus N-Y-D/E
(residues 386-388) within which the Tyr has been shown to become
modified to TPQ in many amine oxidases (Janes et al., 1992; Mu et al., 1992). In pea seedling amine oxidase the peptide
sequence of the phenylhydrazine-labeled peptide was determined to be
V-G-N-blank-D-N-V-I-D-X-E (Janes et al., 1992). The
blank assignment within this sequence corresponds to Tyr in the pea sequence. In addition to Tyr
, two
further Tyr residues (286 and 523) are conserved. The residue
designated as unknown (X) is Trp
; however, it is
unclear why this is residue is not identified during amino acid
sequencing experiments.
around 5
(Farnum et al., 1986). The group involved is most likely a
carboxylate. Cai and Klinman (1994a) have investigated the role of
Glu
in the amine oxidase of the yeast H. polymorpha (equivalent to Asp
in pea). This residue is adjacent
to the TPQ, but study of the E406N variant enzyme ruled it out as the
active site base. Another acidic residue could function in this
capacity, and the alignment (Fig. 4) reveals several candidates.
Two glutamates (residues 348 and 608) and five aspartates (residues
180, 300, 318, 451, and 592) are conserved in addition to a further
three conserved acidic positions (Glu or Asp; residues 288, 388, and
609), although the latter group includes the Asp/Glu site at 388 which
is adjacent to the TPQ consensus and has been discounted as the base
(Cai and Klinman, 1994a).
in Fig. 4) affected the copper ligand field and the geometry of the
copper site, although copper was still bound at a reduced level. It
remains to be established whether His
or His
represents the third copper ligand.
Occurrence of Amine Oxidase Genes in Other
Plants
The copper-containing amine oxidases are very common
in the Leguminoseae and have been reported in certain other classes of
plant including Solanaceae (tobacco; Mizusaki et al., 1972;
Feth et al., 1986; Davies et al., 1989; Hyoscyamus niger (Hashimoto et al., 1990),
Cucurbitaceae (cucumber; Percival and Purves, 1974), Euphorbiaceae (Euphorbia characias, latex; Rinaldi et al., 1982),
Theaceae (tea; Tshushida and Takeo, 1985), and Compositae (Helianthus tuberosum, Jerusalem artichoke; Torrigiani et
al., 1989). There is evidence, particularly from the Leguminoseae,
of a role for copper amine oxidases in conjunction with peroxidases in
lignification and cross-linking of cell wall components during normal
growth, in wounding and a resistance response (Angelini and Federico,
1985, 1989; Federico and Angelini, 1988; Angelini et al.,
1990, 1993; Slocum and Furey, 1991; Scalet et al., 1991). The
correlation between levels of di- and polyamines and amine oxidase in
the apoplast supports a role as an HO
-producing
system. In other cases, such as tobacco (Feth et al., 1986;
Davies et al., 1989) and Hyoscyamus (Hashimoto et
al., 1990) copper amine oxidases have a role in alkaloid
biosynthesis, whereas in tea the enzyme is responsible for the
production of flavor components such as theanine (Tshushida and Takeo,
1985).
Figure 5:
Southern blot analysis of plant genomic
DNAs probed with the pea seedling cDNA probe.
The stronger band(s),
present in each track, is likely to represent a gene(s) that is quite
homologous to the pea cDNA probe and probably encodes a copper amine
oxidase. The presence of multiple bands indicates the likely occurrence
of other, less homologous genes. It is interesting to note the presence
of a very weak, but nonetheless detectable band in the porcine DNA, a
mammal known to have copper amine oxidases. This study provides
evidence for the widespread occurrence of copper amine oxidase genes in
the plant kingdom and could readily be extended to screen further
species. Although the hybridization results do not show whether genes
are expressed, it seems likely that most plants will have a functional
copper amine oxidase gene(s). For example, a copper-containing amine
oxidase has recently been shown to be expressed in maize (Suzuki and
Hagiwara, 1993), and we have recently cloned two copper amine oxidase
genes from Arabidopsis.(
)
(
)
/EMBL Data Bank with accession number(s) L39931 (Fig. 1).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.