©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Molecular Analysis of the Pea Seedling Copper Amine Oxidase (*)

Alex J. Tipping (§) , Michael J. McPherson (¶)

From the (1)Department of Biochemistry and Molecular Biology and the Centre for Plant Biochemistry and Biotechnology, University of Leeds, Leeds LS2 9JT, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 and HO. 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 ,

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 HO 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).

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()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).

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.


EXPERIMENTAL PROCEDURES

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)RNA Isolation and cDNA Synthesis for PCR

Eight- to 10-day postgermination lentil and pea etiolated seedlings were dissected and the epicotyls frozen in liquid N 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 NaHPO HO, 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.

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.

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).


RESULTS AND DISCUSSION

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.

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)

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.

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.


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.

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.


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.

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,()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.

Mechanistic studies suggest that copper amine oxidases mediate C-H bond cleavage through a proton abstraction step involving a group of pK 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).

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 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.

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.

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).

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).


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.()

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.()


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L39931 (Fig. 1).

§
Supported by a Biotechnology and Biological Sciences Research Council studentship.

Recipient of a Leverhulme Trust/Royal Society Senior Research Fellowship. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, U. K. Fax: 44-1132-333144; E-mail: GEN6MJM@BIOVAX.LEEDS.AC.UK.

The abbreviations used are: TPQ, 2,4,5-trihydroxyphenylalanine quinone; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid.

P. F. Knowles, personal communication.

S. G. Mand M. J. McPherson, in preparation.

A. J. Tipping and M. J. McPherson, unpublished data.


ACKNOWLEDGEMENTS

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.


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