Plant Science 155 (2000) 11–20

Purification and cloning of the two domain glyoxalase I fromwheat bran

Katja S. Johansen a,*, Ib Svendsen b, Søren K. Rasmussen a

a Plant Biology and Biogeochemistry Department, Risø National Laboratory, PBK-301, DK-4000 Roskilde, Denmarkb Chemistry Department, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, 2500 Valby, Denmark

Received 19 August 1999; received in revised form 25 October 1999; accepted 22 November 1999

Abstract

Investigation of proteins extracted from wheat bran lead to the isolation of a 37 kDa polypeptide extracted from apolyacrylamide gel. Extensive internal peptide sequence information of this protein identified it as a glyoxalase I. Glyoxalase Iactivity in crude wheat bran extract was measured to 1 U/mg protein (1U=1 mmol S-lactoyl glutathione formed/min).Degenerate primers were designed and used for PCR-RACE-based cloning of the corresponding composite cDNA sequence(AJ243528). The wheat bran glyoxalase I amino acid sequence is very similar to the translated sequence of a RNA transcriptinduced by desiccation of the resurrection grass Sporobulus stapfianus, suggesting a role for glyoxalase in de- or rehydration ofplant tissue. The 37 kDa wheat enzyme belongs to a group of monomeric glyoxalases and is composed of two similar halves eachrepresenting the full-length human glyoxalase I enzyme. A survey of glyoxalase I sequences, including one (not previouslyreported) from Drosophila melanogaster, is presented and alignments of these sequences show that amino acid residues involvedin co-ordinating zinc or interaction with the substrate are conserved. The alignments indicate a non-linear evolution of glyoxalaseI enzymes. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Glyoxalase I; Triticum aesti6um ; Sequence alignment; Gene duplication; Drosophila melanogaster ; Amino acid sequence

www.elsevier.com/locate/plantsci

1. Introduction

The glyoxalase system consists of (1) glyoxalaseI (S-D-lactoylglutathione methylglyoxal lyase (iso-merising), EC 4.4.1.5) which is capable of formingS-(2-hydroxyacyl)glutathione from most 2-oxoaldehydes in the presence of glutathione, andof (2) glyoxalase II (S-2-hydroxyacylglutathionehydrolase, EC 3.1.2.6) which hydrolyse thethioester formed in the first reaction to the free2-hydroxyacids and glutathione. Two-oxoalde-

hydes are electrophilic and cytotoxic compoundsand the coupled reaction may therefore havearisen to detoxify these compounds. Research onthe ubiquitous glyoxalase system in mammals wasinitiated more than 80 years ago, and at the timethis system was believed to be of great importanceand a major part in the pathway of triosecatabolism. Now it is evident that the glyoxalasesystem is responsible for catalysing only a smallpercentage of the trioses generated in the citricacid cycle. It has been suggested that the glu-tathione thiolester S-D-lactoylglutathione, pro-duced by the glyoxalase I reaction, may havespecific cellular functions in cell proliferation, dif-ferentiation and other processes. The glyoxalasesystem is reviewed and discussed by Thornalley[1,2].

Glyoxalase I is believed to be a zinc metallo-protein, with the best characterised example being

Abbre6iations: EBI, European bioinformation institute; EMBL,European molecular biology laboratory; kDa, kilodalton; NAA,naphthalene-acetic acid; NCBI, National center for biotechnologyinformation; PVPP, polyvinylpolypyrrolidone; SDS-PAGE, sodiumdodecyl-sulphate polyacylamide gel electrophoresis; TFA, trifl-uoroacetic acid.

* Corresponding author. Tel.: +45-467-741-23; fax: +45-467-741-22.

E-mail address: katja.johansen@risoe.dk (K.S. Johansen)

0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 5 0 - 2

K.S. Johansen et al. / Plant Science 155 (2000) 11–2012

the human enzyme. The crystal structure of hu-man glyoxalase I has recently been solved to aresolution of 2.2 A, [3]. The enzyme consists of twomonomers, each built up from two equal domains,and it has been suggested that the active dimericprotein has evolved from monomeric proteinsthrough 3D domain swapping [4]. Supporting thistheory, the Pseudomonas putida glyoxalase I hasnow been shown to be active both as a monomerand as a dimer, albeit with a much lower Kcat/Km

value for the monomer [5]. The dimeric form canbe slowly converted to and from the monomerform as glutathione is removed from or added tothe solution.

The residues involved in binding of the zinc ionwere identified in the crystal structure of humanglyoxalase I, but no structural homology to aglutathione binding domain like the one seen inglutathione-S-transferases could be found in thehuman sequence [3]. Instead three residues in-volved in polar interactions with glutathione wereidentified.

Interestingly, a non-zinc glyoxalase I from Es-cherichia coli, coded for by the gloA gene, hasbeen characterised [6]. Zinc has no effect on theactivity of the enzyme whereas nickel greatly en-hances the activity and was shown to be bound ina 1:1 ratio with the dimer.

Glyoxalase-I activity has been studied in severalhigher plant species, and in some cases the enzymehas been further characterised. In tomato (Lycop-ersicon esculentum) [7] an 848 bp cDNA clone wasidentified by differential screening for salt-inducedgenes, and the glyoxalase activity confirmed byexpression in yeast. Using a similar approach inthe resurrection grass Sporobolus staphianus [8] a1.2 kb cDNA clone was found in desiccatingplants. In addition a cDNA clone encoding a186-residue long glyoxalase I has been isolatedfrom epicotyls of Cicer arietinum grown underosmotic stress conditions [9]. Recently, the glyox-alase I cDNA from Brassica juncea was clonedand shown to confer resistance towards stresswhen expressed in E. coli and tobacco [10]. It hasbeen suggested that the increased expression ofglyoxalase I is linked to a high demand for ATPgeneration and enhanced glycolysis in stressedplants [7,9,10].

Glyoxalase I protein has also been purified fromB. juncea seedlings (27 kDa protein) [11], fromGlycine max cell suspensions (in which the enzyme

is a dimer of 26 and 29 kDa polypeptides) [12],and from Aloe 6era (44 kDa protein) [13], amongothers.

Blue light was shown to promote cell prolifera-tion and glyoxalase I activity in Amaranthus panic-ulatus cells [14]. Incubation of dark-grown callusin the presence of the calcium ionophore A23187overcame the requirement for light, suggesting arole for calcium in glyoxalase I activation and cellproliferation. Likewise auxin (NAA) induces gly-oxalase I activity and cell division in tobaccoprotoplasts [15] and soybean suspension cultures[12].

Here we present for the first time the cDNAsequence and extensive protein sequence data for awheat bran glyoxalase I. The duplicate nature ofthe sequence and the relationship to other knownglyoxalase I sequences is discussed.

2. Materials and methods

2.1. Plant material

Commercially available wheat bran (RingstedDampmølle A/S) was used as starting material.

2.2. Protein extraction

Wheat bran was extracted for 30 min at 4°C in1:5 w/v water containing 5 mM b-mercap-toethanol and 10% (g/g wheat bran) PVPP. Theextract was passed through a mesh and then cen-trifuged for 20 min at 8000×g to precipitate thestarch. Ammonium sulphate was added to 30%saturation and the resulting supernatant saturatedto 60% ammonium sulphate. The pellet was solu-bilised and dialysed against 50 mM sodium acetate(pH 4.6) overnight. The sample (20 ml, 80 mgprotein) was loaded on a 6 ml Resource S column(Pharmacia-LKB) equilibrated with the samebuffer and eluted with a linear gradient of acetatebuffer containing 1.0 M sodium chloride.

Proteins in the fractions eluting at 250–400 mMsodium chloride (6.5 ml, 16 mg protein) wereseparated on a NuPage (NOVEX) 4–12% SDS-PAGE gel and the major band at 37 kDa cut out.The gel was run with Mes buffer at 200 mV (assuggested by the manufacture) and with M12(NOVEX) as the molecular weight standards.

K.S. Johansen et al. / Plant Science 155 (2000) 11–20 13

2.3. Amino acid sequencing

Cleavage of the protein in the gel was doneaccording to Kellner [16]. EndoLysC protease wasused in the ratio 1:4 (with the target protein) in 1%ammonium bicarbonate at room temperatureovernight. A control gel piece not containingprotein was treated in the same way. The resultingpeptides were extracted with 70% TFA and sepa-rated by HPLC on a Vydac C18 using an acetoni-trile gradient in 0.1% TFA from 5–60% over 1 h.The eluate was monitored at 216 nm, the relevantpeaks collected by hand and dried in a Savantrotor vaporator. Before sequencing, the sampleswere redissolved in 30% acetic acid. Amino acidsequencing was performed on a model 470A se-quenator connected to a model 120A phenylthio-hydantion analyser (both Applied Biosystems)according to the manufacturers instructions.

2.4. cDNA cloning and sequencing

Total RNA was extracted from wheat seedlings[17] and mRNA isolated with the help of Dynal-beads mRNA DIRECT kit (Dynal, Norway) ac-cording to the manufactures protocols. Firststrand DNA synthesis was performed using ran-dom hexamers. PCR was then performed usingdegenerate primers (cGSP3 5%-GGC CTA CAACTA CGG NGT NGA CTA CG, cGSP2 5%-CGA CAT CAT NGC GAT NGT GTA CTT G).Specific primers (GSP01, GSP02, GSP03, GSP04,GSP05, GSP08) were designed and employed in5%and 3% race using the 5%RACE System kit fromGIBCO BRL (Life Technology).

PCR products were extracted from a 1%agarose gel and blunt end cloned in the Sma I siteof pUC 18 (Pharmacia).

Automated DNA sequencing was done with anApplied Biosystems 377 Prism. The ABI BigDyeterminator cycle sequencing ready reaction kit wasused to make the sequencing reactions on a PerkinElmer thermocycler. Sequence traces were proof-read using Sequencher 3.0 (GeneCode). ThecDNA sequences were translated by the MacVec-tor 6.5 (Oxford Molecular) software.

2.5. Database search and sequence analysis

The BLAST service at NCBI [18] was exploitedfor the identification of the peptide sequences.

Glyoxalase I sequences were found using either theEntrez search engine or the sequence retrievalsystem at EMBL. The FASTA3 program at EBIidentified more sequences with similarity to theknown glyoxalases. The sequences were importedinto the MacVector 6.5 (Oxford Molecular) pro-gram and aligned using the default parameter forClustalW formatted alignments. The N-terminalpeptide sequence was tested with SignalP v1.1software at Center for Biological Sequence Analy-sis (CBS) (www.cbs.dtu.dk) for the presence ofsecretory signal peptide motifs.

2.6. Glyoxalase assay

The glyoxalase activity was measured in crudewheat bran extract, and the assay was done ac-cording to Racker [19]. The assay mixture con-tained 100 mM sodium phosphate buffer pH 7.5,3.5 mM methylglyoxal, 1.7 mM reduced glu-tathione, and 16.0 mM magnesium sulphate in afinal volume of 1 ml. The mixture was transferredto quartz cuvettes and incubated at 25°C. Then0.02 ml wheat bran extract (or boiled extract ascontrol) was added, and the formation of estermonitored by measuring the increase in ab-sorbance at 240 nm in a Shimadzu spectrophoto-meter for 20 min.

3. Results and discussion

3.1. The sequence of wheat bran glyoxalase I

Following ammonium sulphate fractionation ofthe crude wheat bran extract, ion exchange chro-matography was carried out. The protein contain-ing fractions were analysed by SDS-PAGE and amajor protein band visible after staining withcoomassie blue, corresponding to a molecularweight of 37 kDa, was cut out (Fig. 1). Theprotein was cleaved in the gel by EndoLysCprotease and successfully subjected to amino acidsequencing. From the extensive internal aminoacid sequence data obtained the 37 kDa peptidesequence was identified as a glyoxalase I due to itshigh degree of similarity to known glyoxalase Isequences.

Primers were designed based on the peptidesequence and were utilised to isolate the corre-sponding cDNA sequence (in several fragments)

K.S. Johansen et al. / Plant Science 155 (2000) 11–2014

Fig. 1. Coomassie stained 4–12% NuPage gel of wheat branproteins. Lane 1, 60 mg protein of pooled fractions from ionexchange chromatography of wheat bran proteins; lane 2,M12 molecular weight markers. The arrow indicates theposition of the major protein band that was cut out of the geland subjected to peptide sequencing.

via coupled reverse transcription PCR. Fig. 2shows the alignment of the translated wheat se-quence, and the peptide sequences obtained fromthe 37 kDa protein, together with the monocotsequences from S. staphianus and rice (Oryza sa-ti6a). There are a few positions at which thetranslated cDNA sequence and the peptide datado not agree. When these residues are comparedwith the equivalent residues found in homologousproteins (Fig. 4) it seems plausible that the cDNAsequence represents a gene other than that encod-ing the 37 kDa protein. Residue 89 is Ser in thewheat translation, Citrus paradise, Brassica oler-acea and Arabidopsis thaliana 2 but Lys in thewheat 37 kDa peptide, S. staphianus, O. sati6a andA. thaliana 1. Because the protein was isolatedfrom bran and the cDNA sequence from seedlingsit is very likely that the cDNA represents anothergene.

The 40 N-terminal amino acids of the translatedwheat bran sequence were tested and found not tocontain any signal peptide motif. The wheat se-quence presented here does not include a startMet.

Fig. 2. Alignment of the wheat, rice and S. stapfianus sequences with the amino acid sequences from the purified wheat glyoxalaseI peptide. Identical residues are in bold on a dark background; similar residues are on a grey background. Areas of similarity areboxed and the consensus sequence is written as the last line. The abbreviations correspond to the accession numbers as listed inTable 1.

K.S. Johansen et al. / Plant Science 155 (2000) 11–20 15

Table 1EMBL/Genbank entriesa identified as glyoxalase I sequences

TypedSpeciescAccessionb

AC002130 Arabidopsis thaliana LongAC002131 LongArabidopsis thaliana

ShortBrassica junceaY13239Brassica oleraceaZ74950 Long

ShortAJ224520 Cicer arietinumShortCitrus paradisiZ97064

Drosophila melanogasterAC005452 ShortECU57363 ShortEscherichia coli

ShortGlycine maxAJ010423Haemophilus influenzaeU57364 Short

ShortL07837 HumanShortLycopersicon esculentumZ48183

Neisseria meningitidisY14298 ShortLongAB017042 Oryza sati6aShortPseudomonas putidaL33880

Saccharomyces cere6isiaeX99240 LongShortSTU57364 Salmonella typhimuriumLongSchizosaccharomyces pompeQ09751

Sporobolous staphianusSSY10782 LongSynecocus spPcc6803D63999 Short

LongTriticum aesti6umAJ243528e

ShortVPU06949 Vibrio parahaemolyticus

a The entries identified as glyoaxalase I sequences are listedalphabetically.

b Accession numbers.c Scientific name.d Indication of the length of the sequences.e This study.

lated group of enzymes, the glutathione S-trans-ferases (GST), are known to be induced by auxin,and in fact the GST from G. max (AF048978)[20]has a very high degree of identity to the soybeanX68819 sequence (alignment not shown). There-fore, the sequence X68819 is not included in thealignments shown here.

The Drosophila glyoxalase I sequence was foundby alignment of the human glyoxalase peptide tothe translated sequence of nucleotide 46873-48936of chromosome 2R (2R, region 43B2–43C2, is theaccession number listed in Table 1). This sequencehas, to our knowledge, not previously been iden-tified as an open reading frame and is thereforethe first example from insects, and only the secondof animal origin glyoxalase I to be reported. TheDrosophila peptide shown in the alignments pre-sented here has been generated manually by re-moving four introns from the nucleotide sequence.The introns were identified by alignment to thehuman polypeptide and it was not possible toidentify the N-terminal Drosophila sequence.

The alignment in Fig. 3 further suggests that theV. parahaemolyticus sequence is truncated by atleast six amino acids in the N-terminus. It starts atthe fully conserved Met (Met 36 in the human andMet 7 in the E. coli sequence) and is thus lackingthe essential His-or Gln-34 that is involved withthe binding of zinc.

3.3. Gene duplication

The sequences from S. stapfianus, wheat, B.oleracea, A. thaliana, C. paradisi and O. sati6aencodes 280–290 amino acid proteins (Fig. 4)from almost twice as long transcript as the se-quences shown in Fig. 3. The two halves, residues(according to Fig. 4) 1–150 and 151–295, respec-tively, each represents a subunit of the humanglyoxalase I enzyme. This is also the case for theSchizosaccharomyces pombe and Saccharomycescere6isiae yeast enzymes (not shown). The geneduplication has most likely led to a functionalglyoxalase I monomer.

The alignment in Fig. 4 shows large areas ofidentity between all long plant glyoxalases. Theyeast sequences are also of the long type but arenot included in the figure because each of the twohalves of these glyoxalase I sequences are, whencut in two halves, more similar to the relativelyshort plant and animal enzyme sequences (see also

3.2. Sequence similarity

Several glyoxalase I sequences from both pro-and eukaryotic organisms can be found in EMBL/Genbank (Table 1). Glyoxalases can be grouped intwo according to their size and Figs. 3 and 4 showalignments of the long (280–295 amino acids, Fig.3) and short (up to 190 amino acids, Fig. 4)glyoxalase sequences, respectively. Most of thepublished sequences encode proteins of the shorttype and, these enzymes may all function ashomodimers.

It should be noted that EMBL accessionX68819, which has been denoted soybean glyox-alase I cDNA, shares very little identity with therest of the glyoxalase I sequences. Most impor-tantly the well-conserved metal binding residues(see Section 3.4) are not evident in this sequence.The soybean glyoxalase expression was initiallyfound to be induced by auxin [12], and Kalia et al.[15] has illustrated the similarity of this sequenceto other auxin-regulated gene products. The re-

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Fig. 5). The similarity is obvious because thesesequences (together with the P. putida sequence)have some areas, residue 82–96 and 105–124, thatare not present in the rest of the microbial or thelong plant sequences. In that respect, the wheathalf-sequences has more in common with the mi-crobial (short) sequences and interestingly Deswaland Sopory [21] has noticed that B. juncea and themicrobial glyoxalases are sharing an alkaline pHoptimum.

Fig. 5 shows the tree-diagram relating all glyox-alases. Wheat and the other long glyoxalase Isequences are clustered in two branches: one formonomer I and a second for monomer II. Theyeast sequences are grouped together and are dis-tant from the rest of the long sequences and closer

to the short sequences. The two animal sequencesare closely related, and are unexpectedly placedcloser to B. juncea than B. juncea is to B. oleraceain the dendrogram.

Unequal crossovers can explain the duplicatenature of the glyoxalase I, but the rather strangephylogenetic relationship is intriguing.

3.4. Residues co-ordinating zinc

Fig. 3 shows that the residues predicted fromthe crystal structure of the human enzyme to beco-ordinating zinc ions are conserved among allglyoxalase I sequences. The four residues involvedare Gln-34, Glu-100 from one domain and His-128 and Glu-176 from the other domain (numbers

Fig. 3. Sequence alignment of relatively short glyoxalase I sequences. Identical residues are in bold on a dark background; similarresidues are on a grey background. Areas of similarity are boxed and the consensus sequence is written as the last line. Theabbreviations correspond to the accession numbers as listed in Table 1.

K.S. Johansen et al. / Plant Science 155 (2000) 11–20 17

Fig. 4. Sequence alignment of the relatively long glyoxalase I sequences. Identical residues are in bold on a dark background;similar residues are on a grey background. Areas of similarity are boxed and the consensus sequence is written as the last line.The abbreviations correspond to the accession numbers as listed in Table 1. The entry AC002130 is named A. Thaliana 1 and theentry AC002131 A. thaliana 2. AC002130 has a long N-terminal extension, and the first 62 amino acids has been deleted in A.thaliana 1b in order to optimise the alignment.

according to the human sequence [3]). Glu-100,His-128 and Glu-176 are present in all the glyox-alase sequences, but in many of the sequences theGln-34 is changed to a His via a single base pairchange. Both residues are known to be able toco-ordinate zinc and most likely this change do notdisrupt the zinc binding site. All microbial se-quences and the long plant sequences have His asthe first zinc co-ordinating residue instead of Glnwhich is seen in the rest of the short glyoxalase I.This is in agreement with His in B. juncea glyoxalaseI being involved in the catalysis [21]. As discussedby Cameron et al. [3] the Glu-100 and Glu-176

might be involved in both co-ordination of zinc anddirectly in the catalytic reaction as active base.

In the long glyoxalase I sequences the corre-sponding zinc binding residues are His-29, Glu-80His-98 and Glu-147 (numbers according to thealignment in Fig. 4). In the second half of theenzyme another putative zinc binding site can befound and the residues involved are Gln-160, Glu-211 and Gln-229 but the last residue seems to besubstituted with a valine (Val-281), a substitutionlikely to disrupt the binding of zinc [3]. Based onthe alignment in Fig. 4 it is possible that the Glu-291could be involved instead of Val-281 because this

K.S. Johansen et al. / Plant Science 155 (2000) 11–2018

residue is fully conserved among the long plantglyoxalase I sequences.

3.5. Residues interacting with glutathione

Three residues in the human glyoxalase I crystalstructure were found to be making polar interac-tions with the g-glutamyl residue of glutathione.Of these, only two are conserved among all thesequences (Arg-38 in the short corresponds toArg-33 in the long and Asn-104 in the shortcorresponds to Asn-84 in the long glyoxalase Isequences, respectively) whereas the last (Arg-124)is only found in some of the short sequences andnot at all in the long glyoxalase sequences. Twoaromatic rings (Phe-68 and 167) were found to bein the plane of the peptide bond of glutathione,but only the equivalent of the last of these (Phe-167) is conserved. Phe-68 is present in some of theshort but a conservative substitution has rendereda Tyr at that position in the E. coli, V. para-

haemolyticus, S. typhimurium, L. influenzae (Tyr-63) and all of the long sequences (Tyr-194). Thesignificance of these observations in relation to thereaction mechanism is uncertain.

3.6. Different spatial distribution of introns in thegene sequences

The human and the Drosophila gene sequenceseach contains four introns, but not at the samepositions, relative to the protein sequence, al-though at the second and the fourth intron theposition is only shifted one amino acid residuefrom each other. The glyoxalase I gene from A.thaliana contains a total of seven introns with thefirst located at the very beginning of the A.thaliana 1b sequence. Neither of those seven splicesites coincides with the afore-mentioned intronsfrom the animal genes. The introns have mostlikely been introduced after duplication of theancestral glyoxalase I sequence.

3.7. The function of glyoxalase I in wheat bran

Glyoxalase I activity was measured in crudeextract of wheat bran to 1 U/mg protein. Thisactivity is comparable to the levels found in otherplant extracts, 0.2–0.4 U/mg in N. tabacum proto-plasts [15], 0.5 U/mg in un-stressed to 1.8 U/mg insalt stressed B. juncea seedlings [10]. Wheat bran isnot an active tissue and is not likely to have a highdemand for ATP. It has been speculated thatincreased glycolysis to generate ATP leads to in-creased levels of methylglyoxal and induction ofglyoxalase I activity in stressed plants [7,9,10].Induction of transcription of the glyoxalase I geneduring desiccation of resurrection grass [22], andthe presence of the protein in dry wheat branmight therefore suggest new roles for glyoxalase I,either in protecting cells during server water defi-ciency or during re-hydration when the tissue be-comes metabolically active.

A number of enzymes having the potential to beactive upon hydration are found in resting cerealgrains. Among these are b-amylase in barley [23]and wheat [24], which is secreted to the endospermin order to utilise the carbon reserves and storedenergy in the starch grains. It seems more likelythat glyoxalase I is functioning in the living cells ofthe grain where it destroys methylglyoxal, a toxicby-product from the citric acid cycle.

Fig. 5. Dendrogram of the relationship between all knownglyoxalase I sequences. The long sequences have been dividedin two equal halves after Arg-146 (Fig. 4). The N-terminalhalf is monomer I and the C-terminal is monomer II, thesequence accessions are listed in Table 1.

K.S. Johansen et al. / Plant Science 155 (2000) 11–20 19

4. Conclusion

A wheat bran glyoxalase I peptide and cDNAsequence have been identified, adding to the grow-ing number of glyoxalase I sequences being pub-lished from all species and plants in particular.Two distinct types of glyoxalases exist and plantenzymes of both types are found. One type isfunctioning as a homo-dimer the other type hasthrough a gene-duplication evolved as a functionalmonomer of almost the size of the dimer glyox-alase I enzyme. The three known sequences frommonocots are all belonging to the long monomerictype. The long plant glyoxalase I enzymes haveclearly not evolved directly from the ancestralshort plant sequence and the evolutionary rela-tionship between all glyoxalase I sequences isintriguing.

Acknowledgements

The Danish Research Academy and the firstframework (1996–2001) under the Cereal Networkin Denmark in part, supported this work. Theauthors would like to thank Dr Tomas H. Robertsfor proofreading the manuscript.

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