van Berkel, Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands Fax: +31 317484801 Tel: +3
Trang 1Mamoudou H Dicko1,2,3, Harry Gruppen2, Riet Hilhorst1,*, Alphons G J Voragen2
and Willem J H van Berkel1
1 Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, The Netherlands
2 Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, The Netherlands
3 Laboratoire de Biochimie, CRSBAN, UFR-SVT, Universite´ de Ouagadougou, Burkina Faso
Keywords
glycoform; hemeprotein; isoenzyme;
peroxidase; sorghum
Correspondence
M H Dicko, Laboratoire de Biochimie,
CRSBAN, UFR-SVT, Universite de
Ouagadougou, 03 BP 7021,
Ouagadougou 03, Burkina Faso
Fax: +226 50337373
Tel: +226 70272643
E-mail: mdicko@univ-ouaga.bf
W J H van Berkel, Laboratory of
Biochemistry, Department of
Agrotechnology and Food Sciences,
Wageningen University, PO Box 8128,
6700 ET Wageningen, The Netherlands
Fax: +31 317484801
Tel: +31 317482861
E-mail: willem.vanberkel@wur.nl
*Present address
PamGene, PO Box 1345, 5200
BJ’s-Hertogenbosch, The Netherlands
Database
Sequence data for sorghum peroxidase
described here has been submitted to the
UnitProt knowledgebase under the
accession number P84516
(Received 2 February 2006, revised 18
March 2006, accepted 22 March 2006)
doi:10.1111/j.1742-4658.2006.05243.x
The major cationic peroxidase in sorghum grain (SPC4) , which is ubiqui-tously present in all sorghum varieties was purified to apparent homogen-eity, and found to be a highly basic protein (pI 11) MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34227 and
35629 Da and it contains a type-b heme Chemical deglycosylation allowed
to estimate sugar contents of 3.0% and 6.7% (w⁄ w) in glycoform I and II, respectively, and a mass of the apoprotein of 33 246 Da High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains The N-terminal sequence of SPC4 is not blocked by pyroglutamate MS analysis showed that six pep-tides, including the N-terminal sequence of SPC4 matched with the predic-ted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids The N-terminal frag-ment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor SPC4 is activated by calcium ions Ca2+ binding increased the protein conformational stability by rais-ing the meltrais-ing temperature (Tm) from 67 to 82C SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives In spite of the conserved active sites, SPC4 differs from BP1 in being active with aro-matic compounds above pH 5
Abbreviations
ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid); BP1, barley peroxidase isoenzyme-1; HPAEC, high performance anion exchange chromatography; HRP C, horseradish peroxidase isoenzyme C; GlcNAc, N-acetyl-glucosamine; SPC4, major sorghum cationic peroxidase; TFMS, trifluoromethanesulfonic acid.
Trang 2Plant secretory peroxidases (donor: hydrogen peroxide
oxidoreductase, EC 1.11.1.7) are class III peroxidases
that contain a FeIII–protoporphyrin-IX as the
pros-thetic group linked to a proximal His residue They
catalyze the conversion of a large number of
sub-strates, notably phenolic compounds for biosynthetic
and catabolic functions In general, they use hydrogen
peroxide as electron acceptor [1] Multigene families of
peroxidases exist, and in the genomes of rice (Oryza
sativa) and thale cress (Arabidopsis thaliania) up to 138
and 73 of peroxidase genes, respectively, were
discov-ered [2,3] Moreover, the ongoing project of sorghum
genome sequencing has allowed us to currently
iden-tify 160 stretches of sorghum peroxidase genes (http://
peroxidase.isb-sib.ch/index.php) The physiological
functions of peroxidases are associated with defense
mechanisms, auxin metabolism and the biosynthesis of
cell-wall polymers such as lignin and suberin [1,4,5]
Most peroxidases are glycoproteins occurring in
dif-ferent glycoforms, which may contain difdif-ferent glycan
chains [4] For instance, barley peroxidase (BP1)
con-sists of two forms; one glycosylated at Asn300 (BP1a)
and the other (BP1b) nonglycosylated [6,7] The major
glycan chain in BP1a represents 70% of the total
carbo-hydrate content and has as structure Mana1–6(Xylb1–
2)Manb1–4GlcNAcb1–4(Fuca1–3)GlcNAc [6] Next to
iron, Ca2+is an important metal cofactor of heme
per-oxidases Class III peroxidases are known to contain
two distinct Ca2+-binding sites, one localized on the
proximal side and the other on the distal side of the
heme Ca2+ both modulates the enzyme activity and
stability [8]
Cereal peroxidases hitherto characterized are from
barley [6], wheat [9], rice [10], and maize [11] All these
enzymes are monomers with molecular masses ranging
from 35 to 40 kDa The crystal structure of BP1, with
two helical domains and four disulfide bridges
(C18-C99, C51-C56, C106-C301 and C186-C213) is highly
similar to the structure of the archetypical horseradish
peroxidase (HRP C) Although BP1 shares structural
similarities and catalytic properties with HRP C, its
behavior is atypical, as it is unable to form compound
I at pH values greater than 5 [7]
Relatively little is known about the structure and
properties of sorghum peroxidase [Sorghum bicolor (L)
Moench] Sorghum is the fifth most important cereal
crop in the world after wheat, rice, maize, and barley
Properties of a crude sorghum peroxidase preparation
such as pI (9–10) and molecular mass (43 kDa) have
been reported [12] However, until now no sorghum
grain peroxidase has been purified to homogeneity and
characterized When screening for peroxidase activity
in the seeds of 50 sorghum varieties originating from
different parts of the world, the cationic peroxidase was ubiquitously present in all varieties [13,14] It was also the most abundant isoenzyme in both ungerminated and germinated sorghum grains [14] In other cereals, the cationic isoenzymes are also the most abundant enzymes and account for more than 80% of total activity [6,15]
In recent years, it has been shown that cationic per-oxidases are more active with phenolic compounds than anionic peroxidases and laccases [16] Thus, cationic peroxidases may be of interest for biocatalytic applica-tions such as the production of useful polymers, the treatment of waste water streams polluted with toxic aromatic compounds, and various other clinical and biotechnological applications [17] Cationic peroxidases may also find interest in food biotechnology by modifi-cation of functional properties of food proteins and carbohydrates [18,19] The other reason to characterize the peroxidase from sorghum is the fact that during food preparation, the peroxidase present could have a large effect on the properties of the prepared foods (beer, porridge, couscous, etc.) [14,18,19] The resulting oxidation products have effects on human health Therefore, knowledge of biochemical properties of the major peroxidase can help on sorghum processing
In this study, we have purified and characterized the cationic peroxidase isoenzyme from sorghum grain
Results and discussion
Purification of major peroxidase from sorghum seed
At least four sorghum peroxidase cationic isoenzymes, denoted SPC1, SPC2, SPC3 and SPC4, according to their order of elution, could be distinguished and separ-ated by the Mono-S cation exchange chromatographic step (Fig 1A) SPC4 was by far the most abundant iso-enzyme Zymography (Fig 2A) showed that this enzyme has an experimental pI value > 9 Three inde-pendent repetitions of all purification steps were per-formed to confirm the profile and abundance of isoenzymes within sorghum grain The purification by three chromatographic steps resulted in a final enrich-ment of SPC4 by 105-fold, with an activity yield of 28% (Table 1) The purity of SPC4 was assessed by the single protein band obtained by SDS⁄ PAGE (Fig 2B) and the high RZ value (4.0) The purification of SPC4 is sum-marized in Table 1 The final specific activity of SPC4 for the H2O2-dependent oxidation of ABTS was
1071 UÆmg)1 The purified enzyme was soluble in aque-ous acetone, methanol and ethanol up to proportions of 40% (v⁄ v) of organic solvent The enzyme eluted from a Superdex G 75 column in one symmetrical peak with an
Trang 3apparent mass of 32 kDa (Fig 1B) Together with the
molecular masses obtained by SDS⁄ PAGE (38 kDa,
Fig 2B) and MALDI-TOF-MS (34283–35631 Da,
Fig 3A), this shows that SPC4 is a monomer
Carbohydrate composition
MALDI-TOF-MS analysis revealed that SPC4 consists
of two species with masses of 34 283 and 35 631 Da,
respectively (Fig 3A) Chemical deglycosylation of the
enzyme yielded a single protein peak with a mass of
33 449 Da (Fig 3B) This indicates that the
hetero-geneity of the enzyme is exclusively related to its glycan
composition and that SPC4 has two glycoforms For
convenience, the species with a mass of 34 283 Da is further referred to as glycoform I and the species with a mass of 35 631 Da as glycoform II The chemical deglycosylation was not complete because it leaves one unit of GlcNAc (203 Da) remaining on the polypeptide chain at each attachment site [20] Thus, the molecular mass of fully deglycosylated SPC4 is at most 33 246 Da The sugar contents estimated by MALDI-TOF-MS are 3.0% and 6.7% in glycoform I and II, respectively Carbohydrate analysis of SPC4 by HPAEC showed
an average carbohydrate content of approximately 5.4% (Table 2) From the overall sugar content (HPAEC) and the estimated sugar contents of the indi-vidual glycoforms (MALDI-TOF-MS), the proportions
of glycoforms I and II can be calculated to be 35 and 65%, respectively HPAEC analysis showed that the main sugar constituents of the glycan chains are fucose, mannose, xylose, and N-acetylglucosamine (Table 2) MALDI-TOF-MS analysis of HRP C as positive control showed masses of the native and
deglycosylat-ed form of 43 663 Da and 35 505 Da, respectively (Fig 3C,D) Since HRP C has eight glycan chains [21],
at least 8 GlcNAc residues will remain after chemical deglycosylation Thus, the fully deglycosylated HRP C
Fig 1 Purification of cationic isoforms of sorghum peroxidase (A)
Mono-S cation exchange chromatography: peroxidase activity (o),
absorbance at 280 nm (—), absorbance at 403 nm (- - -), and 0–1 M
NaCl gradient (—) (B) Elution profile of Mono S purified SPC4 on
Superdex 75 PG.
Fig 2 Zymogram and SDS ⁄ PAGE of major
cationic sorghum peroxidase (A)
Zymogra-phy: lane 1, crude extract and lane 2,
purif-ied SPC4 (B) SDS ⁄ PAGE of purification
steps of SPC4: lane M, marker proteins;
lane 1, crude extract; lane 2, acetone
precip-itate; lane 3, preparative Superdex 75
frac-tion; lane 4, unbound Resource-Q fracfrac-tion;
lane 5, Mono-S fraction; lane 6, analytical
Superdex 75 fraction.
Table 1 Purification of the major sorghum peroxidase.
Step
Total activity (U)
Total protein (mg)
Specific activity (UÆmg)1)
Yield (%)
Trang 4would have a mass of 33 881 Da (35 505–203· 8 Da),
which is in good agreement with data obtained by
electrospray ionization mass spectrometry [22], and
also with the calculated mass based on the primary
structure (Table 2) The mass of the sugar moiety in
HRP C is therefore 9782 Da, corresponding to 22.4%
(w⁄ w) HPAEC analysis of the HRP C sugar
composi-tion revealed a carbohydrate content of 22.1% (w⁄ w)
The comparison of sugar composition between SPC4
and HRP C is illustrated in Table 2 The sugar content
of SPC4 is much lower than that observed with HRP
C as well as from other cationic peroxidases except for BP1, which also has a low sugar content (Table 3)
Spectral properties The UV-visible spectrum of native SPC4 (Fig 4A) is interpreted in terms of the spin and coordination state
Fig 3 MALDI-TOF-MS analysis of native and deglycosylated forms of SPC4 and HRP C (A) Native SPC4, (B) deglycosylated SPC4, (C) native HRP C, and (D) deglycosylated HRP C.
Table 2 Molecular mass and sugar composition of SPC4 and HRP.
Mass of intact protein (Da)
Mass of carbohydrate moiety (Da)
Proportion of carbohydrate (%, w ⁄ w)
Number of residues (mol ⁄ mol) determined by HPAEC
HRP d
(present study)
a MS, mass spectrometry analysis of the two glycoforms I and II; b HPAEC, high performance anion exchange chromatography analysis of both glycoforms;cSPC4, sorghum cationic peroxidase (the average molecular mass and sugar composition of the two glycoforms was con-sidered) d Horseradish peroxidase according to the present study e Horseradish peroxidase according to theoretical prediction [21].
Trang 5of the resting enzyme The absorption spectrum of
native SPC4 showed characteristics typical of high-spin
iron(III) heme proteins, with a maximum in the Soret
region at 403 nm and a b-band at 497 nm [23]
(Fig 4A) There is also a charge-transfer band
(por-phyrin to iron) [1] in the spectrum between 630 and
640 nm Moreover, with the spectrum of the extracted
heme, a Q0vband (vibrational transition of the iron p
electrons) [1] at 532 nm and a porphyrin to iron charge
transfer band at 637 nm were clearly observed The
Q0vband at 532 nm was not visible in the native
per-oxidase because it is obscured by b-band and charge
transfer bands [1] These spectral properties are
charac-teristic for an iron(III)-containing protoporphyrin-IX
The molar absorption coefficient of SPC4 at 403 nm
was determined to be approximately 104 mm)1Æcm)1
Figure 4(B) shows the mass spectral analysis of the
extracted heme cofactor of SPC4 The mass of 616 Da
corresponds to the mass of
iron(III)–protoporphyrin-IX, confirming that SPC4 contains a type-b heme The
peak with a mass of 563 Da is ascribed to the partial
loss of iron by the protoporphyrin-IX The
MALDI-TOF-MS spectrum (Fig 4B) also shows an intense
peak with a mass of 650, which is assigned to a
heme-H2O2 adduct Thus, SPC4 is a type-b heme-con-taining peroxidase, which shares similar molecular properties with cereal peroxidases [1,6,15]
Far UV-circular dichroism spectroscopy indicated that SPC4 contains 42 ± 6% a-helix, 35 ± 7% b-sheet and 24 ± 7% b-turns (not shown) These val-ues should be taken with caution as in peroxidase structures predicted from CD spectra the a-helix con-tent can be underestimated Nevertheless, this secon-dary structure content is similar to that of other plant peroxidases [24]
Amino acid composition and N-terminal sequence analysis
The amino acid composition of SPC4 together with those of other cationic peroxidases is given in Table 3 The average amino acid calculated mass of cationic peroxidases is 106.7 Da (Table 3), allowing estimation
of 311 amino acid residues in SPC4 From this amino acid composition, a theoretical pI value of 11 was cal-culated, assuming that all eight cysteines are involved
in disulfide bridges [1,7,25,26] The low ratio (Asx + Glx)⁄ (Arg + Lys) of SPC4 and its pI value
Table 3 Amino acid composition of SPC4 and other cationic plant peroxidases.
a Results of SPC4 are presented in number of amino acid ⁄ protein and in mole percentage (mol ⁄ mol) in brackets b Rice [10], c wheat [9], d bar-ley [6], e Cenchrus ciliaris [53], f horseradish [25], g peanut [54], h Scutellaria baicalensis [55], i turnip [56] j Calculated molecular weights using software to compute pI ⁄ MW ⁄ titration curve, available at http://expasy.ch/tools/#primary ⁄ k
, sugar composition not given. lUniProtKB ⁄ TrEMBL accession number.
Trang 6indicate that in comparison to other cationic
peroxid-ases, SCP4 is highly basic (Table 3)
Like BP1 [6], the N-terminal sequence of SPC4 is
not blocked by pyroglutamate, in contrast to most
other peroxidases [25] The first 20 amino acid residues
are shown in Fig 5 A TBLASTN search at the
Gram-ene website (http://www.gramGram-ene.org) indicated that
the SPC4 gene is localized in the sorghum chromosome
1 At the Institute for Genomic Research (http://
www.tigr.org), the best match with 100% identity was
found with gene indice TC102191 (213 amino acids)
MALDI-TOF-MS analysis (Fig 6) showed that six
peptides, including the N-terminal sequence of SPC4
matched with the predicted tryptic peptides of
TC102191, indicating that TC102191 codes for the
N-terminal part of the sequence of SPC4 SPC4 has a
signal peptide of 31 amino acids (Fig 5) Since the
expected full length of SPC4 is about 311 amino acid
residues, the C-terminal sequence of about 129 amino
acids is unknown Among the currently 160 stretches
of sorghum peroxidase genes that are identified (http://
peroxidase.isb-sib.ch/index.php), SPC4 corresponds to SbPrx50 With the currently ongoing sorghum genome project (http://fungen.botany.uga.edu), the full seq-uence of this gene will be available soon
The sequence of the N-terminal part of SPC4 was analyzed by searching for domain database (RPS-BLAST at NCBI: http://www.ncbi.nlm.nih.gov/blast), protein families database (Pfam9Sanger Institute: http://www.sanger.ac.uk/software/Pfam) and for
speci-fic protein motifs, domains and families (InterProScan
at EBI: http://www.ebi.ac.uk/InterProScan) The RPS-BLAST and Pfam searches indicated with expect val-ues of 4e-59 and 1.1e-50, respectively, that SPC4 belongs to the Class III of plant secretory peroxidases like HRP C Furthermore, the InterProScan software, which integrates several tools for the analysis of domain and family of proteins, clearly showed that SPC4 contains all the fundamental motifs characteris-tic of Class III plant peroxidases The TBLASTN search against the nonredundant database at NCBI (http://www.ncbi.nlm.nih.gov/blast) indicated that SPC4 is most closely related to cereal peroxidases (Fig 5) Because the N-terminal sequences of the mature peroxidases from rice, wheat and maize are as yet unknown, the alignment of sequences in Fig 5 is made by including the signal peptides of peroxidases (precursors) The N-terminal fragment of SPC4 has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%), and maize (58%), indicative for a common ancestor [27]
SPC4 consists of two domains and has an N-ter-minal extension of one and eight residues, compared
to BP1 [6] and HRP C [25], respectively The key cata-lytic residues (Arg46, Phe49, His50, Asn78, Pro150 and His180) and cysteines involved in intramolecular disulfide bridges (Cys19-Cys100; Cys52-Cys57; Cys107) are all conserved (Fig 5) The structural motif
-P-X-P-is found at sequence positions 150–152 Th-P-X-P-is region -P-X-P-is involved in the substrate binding of plant peroxidases [26] In particular, Pro150, which is completely con-served in the plant peroxidase superfamily (class III),
is crucially involved in substrate binding and oxidation [7,26] Another important residue of SPC4 concerns Thr68, which is equivalent to Thr67 of BP1 This resi-due is conserved in most cereal peroxidases (Fig 5), but not in HRP C Structural studies have shown that the distal heme pocket of BP1 is significantly different
to that of other plant peroxidases In BP1, at pH above 5, the distal His makes a hydrogen bond with Thr67 and not with the distal Asn70 as in HRP C As
a result, the orientation of the distal His residue is altered and located too far from the heme iron atom
to be able to catalyze the formation of compound I In
Fig 4 Heme analysis of SPC4 (A) Spectral properties of SPC4.
The absorption spectrum of purified SPC4 was recorded in 50 m M
sodium acetate pH 5 The inset shows the spectrum of the
extract-ed heme (B) MALDI-TOF-MS analysis of SPC4 heme.
Trang 7addition, Phe48 (equivalent to Ph49 in SPC4) moves
toward the heme iron, and in doing so, the accessibility
of the heme iron is diminished [7] Given the high
sequence identity with BP1 it may be conceivable that
a similar situation applies in SPC4
The only putative glycosylation site present in the
sequenced N-terminal fragment of SPC4 is Asn78
However, Asn78 is an active site residue of class III
peroxidases that is not glycosylated [6] Thus, as found for most peroxidases, the glycosylation sites of SPC4 are localized in the C-terminus part of the enzyme
Catalytic properties SPC4 was stable between pH 3 and pH 7 for 2 h at
25C The enzyme showed optimal activity with
Fig 5 Multiple sequence alignment of
major cationic sorghum peroxidase (P84516)
with other cereal peroxidases The
N-term-inal fragment of SPC4 is aligned with barley
BP1 (Q40069), rice Prx23 (Q94D M 0), wheat
WSP1 (Q8LK23), and maize peroxidase
(O04710) The codes under brackets are
UniProtKB⁄ TrEMBL entries The highly
con-served catalytic residues among all class III
peroxidases are marked with asterisks The
N-terminal sequence of SPC4 obtained by
Edman sequencing is underlined The signal
peptides of SPC4 and BP1 are shown in the
boxes.
Fig 6 MALDI-TOF-MS peptide mass fingerprint of SPC4.
Trang 8ABTS, ferulic acid and N-acetyl-l-tyrosine at pH 3.8,
5.5 and 6.5, respectively (Fig 7) These different pH
optima are in line with reported properties of other
peroxidases [28,29] For instance the pH optima found
for the activity of lettuce (Lactuca sativa) peroxidase
were 4.5, 6.0, 5.5–6.0, and 6.0–6.5 for the substrates
tetramethylbenzidine, guaiacol, caffeic acid, and
chlo-rogenic acid, respectively [28] It is known that there is
no correlation between the pH optima of peroxidase
activity and their pI values because both anionic
(pI 3.5) and cationic (pI 8.8) horseradish peroxidases
display for instance the same optimum pH for the
oxi-dation of p-coumaric acid [29] The substrates oxidized
at low pH (ABTS and ferulic acid) have higher
cata-lytic efficiencies (Table 4) than those oxidized at higher
pH values (N-acetyl-l-tyrosine) maybe because of the
higher oxidation potential of the reaction intermediates
compound I and II at low pH [29] The difference in
the optimum pH of peroxidase activity between
sub-strates may also reflect the pH-dependence of their
ionization potentials A pH-dependence of peroxidase
activity as a function of substrate could be explained
by several reasons A change in pH would affect the
extent to which each functional groups of the amino
acid involved in substrate binding, or catalytic residues
ionizes, and thus the conformation of the peroxidase
molecule A change in the structural conformation will
obviously affect the shape of the active site, and thus
either increase or decrease the enzyme’s affinity for
substrate molecules [1] This hypothesis is further
sup-ported by the fact that different amino acids can be
involved for plant peroxidases binding to
physiologi-cally relevant substrates [29] The pH-dependence of the contribution from electrostatic repulsion or attrac-tion during substrate binding and release can also be considered The better and maybe faster binding of electron donors have been suggested to justify the dif-ference in the oxidation of phenolic substrates by plant peroxidases [29] Furthermore, some substrates are oxidized in a single-electron reaction (ABTS) and oth-ers in a two-electron reaction (phenolic compounds), and some products undergo nonenzymatic polymeriza-tion reacpolymeriza-tions after peroxidase oxidapolymeriza-tion of substrates from which they derived [1] Such kinetic differences might alter the overall pH-activity profile
Nevertheless, SPC4 remarkably differs from BP1 [23] in being active with aromatic compounds above
pH 5 This activity, which is also apparent from the zymography analysis (Fig 2A), is intriguing in view of the structural relationship mentioned above
Stafford and Brown [30] reported an oxidative dimerization of ferulic acid by sorghum grain extracts Furthermore, using a crude extract from sorghum variety NK300, a high peroxidase activity
on ferulic acid and no activities on tyrosine and other phenolics were observed [31] Here we found that purified SPC4 has a high preference for hydroxycinnamates, including ferulic acid and p-cou-maric acid, which are among the most abundant phenolic compounds in sorghum [32] Kinetic studies performed at pH 5.5 showed that the catalytic effi-ciency of SPC4 with phenolic compounds decreased
in the following order: ferulic acid > p-coumaric acid > N-acetyl tyrosine methyl ester > N-acetyl tyrosine > tyrosine > catechol > Gly-Tyr-Gly (Table 4)
Fig 7 Dependence of SPC4 activity on pH The enzyme (10 n M )
was incubated with 10 m M ABTS (d), 125 l M ferulic acid (s), or
250 l M N-acetyl tyrosine (m) in the presence of 5 m M H2O2, in
dif-ferent 50 m M McIlvaine buffers (pH 2.5–8), at 20 C Enzymes
activities were monitored as described in the Experimental
proced-ures Vertical bars indicate the standard error of each experiment.
Table 4 Substrate specificity a of sorghum peroxidase.
Substrate
Substrate
k max
(nm)
Substrate molar absorption coefficient (m M )1Æcm)1)
Product
k max
(nm)
Apparent
V max ⁄ K m
( M )1Æs)1)
Indole-3-acetic acid
N-acetyl tyrosine methyl ester
a The substrates are ranked by order of preference The reaction was followed by bsubstrate disappearance or cproduct formation according to d [18], e [29], f [34], g [51], and h [52].
Trang 9The relatively high reactivity with hydroxycinnamic
acid derivatives suggests that the enzyme may be
involved in the formation of diferulate linkages in
the plant cell wall On the other hand, the rather
low catalytic efficiency of SPC4 with tyrosine and
tyrosine-containing peptides suggests that the enzyme
is less involved in protein cross-linking through
di-tyrosine formation
SPC4 also displayed auxin (3-indole acetic acid)
activity This activity, which takes place in the absence
of added hydrogen peroxide, is mechanistically
differ-ent for cationic and anionic peroxidases [33] and not a
property of all plant peroxidase isoforms [34] The
physiological significance of auxin metabolism by plant
peroxidases is still an area of debate Some peroxidases
regulate the level of auxin either by direct degradation
or by oxidizing endogenous flavonoids, which are
inhibitors of auxin transport [35] The activity of SPC4
on auxin might be related to the presence of His48
(His40 in HRP C) in the distal domain near the heme,
which is believed to play a role in auxin recognition
based on sequence similarity with auxin binding
pro-teins [33]
The activity of SPC4 was stimulated in the presence
of CaCl2 The maximum increase of activity of the
purified enzyme was two-fold with an apparent
semi-maximal activation at 0.7 mm CaCl2 A similar, but
somewhat stronger activation, was observed for BP1
for which the calcium binding sites are not fully
occu-pied [23] The Ca2+ activation of SPC4 is of interest
because not all peroxidases are activated by Ca2+[15]
HRP C for instance contains two structural calcium
ions (proximal and distal) that are also of functional
significance [26] Binding of Ca2+decreased the
intrin-sic tryptophan fluorescence intensity of SPC4 From
the binding curve, a dissociation constant for the
SPC4–Ca2+ ion complex, Kd¼ 2.4 ± 0.3 mm, was
determined The affinity of SPC4 for Ca2+ was
some-what higher than that of BP1 (Kd¼ 4 mm) [15] The
calcium status of BP1 is anomalous, with the distal
calcium-binding site substituted by sodium [7] Based
on the sequence alignments, the distal binding site in
SPC4 is formed by Asp51, Asp58, Ser60 (side chains)
and Asp51, Val54, Gly56 (main chain carbonyls) The
entire sequence of SPC4 is needed to establish the
proximal calcium binding site The binding of Ca2+
has been proposed to change the electronic properties
of the heme iron or the topology of the heme vicinity
and might improve substrate binding [7,8,15,23] With
SPC4, such structural perturbations must be small
because circular dichroism analysis revealed that Ca2+
binding does not change the secondary structure of the
enzyme (not shown)
Thermal stability
In the absence of added CaCl2, SPC4 readily lost activity when incubated at temperatures above 55C (Fig 8A) However, in the presence of excess Ca2+ ions, the enzyme kept its full activity at up to 65C for 90-min incubation (Fig 8B) Arrhenius plots (Fig 8C) of the thermoinactivation data revealed straight lines and showed that Ca2+ binding only slightly increases the activation energy of heat inactiva-tion of SPC4 from 157 ± 12–170 ± 14 kJÆmol)1 The increased stability of SPC4 in the presence of Ca2+ ions was confirmed by fluorescence experiments Upon
Fig 8 Thermoinactivation of SPC4 The enzyme (270 n M ) was incubated at different temperatures in 50 m M sodium acetate pH 5, either in the absence (A) or presence (B) of 5 m M CaCl 2 : 55 C (d),
60C (s), 65 C (m), 70 C (n), 75 C (n), 80 C (h), 85 C (X);
90 C (r), 95 C (e) (C) Arrhenius plot for heat inactivation of SPC4 in the absence (r) or presence (e) of calcium Vertical bars indicate the standard error of each experiment.
Trang 10heating, both in the absence and presence of Ca2+
ions, a strong increase in protein tryptophan
fluores-cence was observed (Fig 9A,B) Independent of the
presence of Ca2+ions, and treating the data according
to van Mierlo et al [36], SPC4 followed a simple
two-state mechanism of heat-induced unfolding This is in
agreement with other plant peroxidases [37]
Thermal unfolding of SPC4 induced not only an
increase of fluorescence intensity but also a
bathochro-mic shift of the fluorescence maximum from 338 to
348 nm (not shown) Tmvalues of 67C and 82 C for
the free and calcium-bound form, respectively, were
found In the absence of Ca2+, the melting
tempera-ture of SPC4 was between that of HRP C (Tm¼
60C) and the African palm tree peroxidase (Tm¼
74C) [37] In the presence of Ca2+, the Tmof SPC4
is near that of soybean peroxidase, which is one of the
most stable plant peroxidases with a Tm of 90C in
the presence of calcium [38]
In conclusion, the major isoenzyme in sorghum
grain (SPC4) was shown to be a cationic peroxidase
having two glycoforms with unusual basic character
and a high heat stability in the presence of calcium
The enzyme has relatively low carbohydrate content It shares similar molecular properties with other cereal peroxidases such as barley peroxidase 1 but has dis-tinct catalytic properties in being active on aromatic compounds above pH 5 Therefore, the enzyme may develop as an alternative peroxidase for biochemical and clinical assays, and biocatalysis
Experimental procedures
Chemicals
Horseradish peroxidase [HRP, EC 1.11.1.7] (grade II, lot N16H9522), p-coumaric acid, ferulic acid, l-tyrosine, tri-fluoromethanesulfonic acid, and indole-3-acetic acid were from Sigma-Aldrich (Zwijndrecht, the Netherlands) N-ace-tyl tyrosine, N-aceN-ace-tyl tyrosine methyl ester and Gly-Tyr-Gly were from Bachem, Bubendorf, Switzerland Hydrogen per-oxide was from Merck (Darmstadt, Germany) Modified trypsin (EC 3.4.21.4) sequencing grade was from Roche Diagnostics GmbH (Mannheim, Germany) Electrophoresis gels (IEF, pH 3–9) were purchased from Amersham
Biorad (Richmond, CA, USA) Immobilon-P transfer mem-brane was from Millipore Corporation (Bedford, MA, USA) Maltodextrin MD05 standards were obtained from Spreda (Burghof, Switzerland) Low molecular weight standard proteins were from Amersham Pharmacia Biotech (Uppsala, Sweden) All other chemicals were of analytical grade
Enzyme purification
The grains of sorghum variety [Sorghum bicolor (L) Moench var Cauga 108–15] grown in 1998 were used [13] Peroxidase isoenzymes were extracted from flour as des-cribed previously [13,14] Protein precipitation was
extract, followed by centrifugation (10 000 g, 30 min) The
resuspended in the extraction buffer and dialyzed overnight
Subsequent chromatography steps were performed at
monit-ored at wavelengths of 280 and 403 nm Reinheitszahl (RZ)
chro-matograms [4] The supernatant (150 mL) obtained after acetone precipitation and subsequent dialysis was loaded
Amersham Pharmacia Biotech, Uppsala, Sweden) equili-brated with starting buffer Proteins were eluted at a flow
A
B
Fig 9 Thermal unfolding of SPC4 as followed by intrinsic
trypto-phan fluorescence The enzyme (2.22 l M ) was heated in 10 m M
sodium acetate pH 5, either in the absence (A) or presence (B) of
5 m M CaCl 2 at a rate of 0.5 CÆmin)1 The excitation wavelength
was 295 nm The emission at 342 nm was monitored at 0.5-min
intervals Solid lines are the best fit of the two states unfolding
equation (Eqn 1) [36].