1
tefan Jane
ek2,3
1 Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84238 Bratislava and 2 Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84251 Bratislava, Slovakia
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Abstract |
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Keywords: evolution/polygalacturonase/rhamnogalacturonase/xylogalacturonan hydrolase
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Introduction |
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Pectin, as a heteropolysaccharide, is a major constituent of the middle lamella of primary cell walls of dicotyledonous plants, composed of alternating homogalacturonan-smooth and rhamnogalacturonan-hairy regions (Williamson et al., 1998; Van der Vlugt-Bergmans et al., 2000
). The smooth regions are polymers of
-1,4-linked D-galacturonic acid units, partially esterified, which are split by PGs, EPGs, EPGDs, pectin lyases, pectate lyases and de-esterified by pectin methylesterases. The hairy regions consist of three different subunits, as identified in apples (Schols and Voragen, 1996
): (i) subunit I is xylogalacturonan, i.e. a galacturonan backbone heavily substituted with xylose (degraded by XGH); (ii) subunit II is a short section of rhamnogalacturonan backbone with many arabinan, galactan or arabinogalactan side-chains (degraded by arabinases and galactanases); and (iii) subunit III is rhamnogalacturonan consisting of alternating rhamnose and galacturonic acid residues (degraded by RGs).
More than 100 amino acid sequences of the family 28 glycoside hydrolases are available from GenBank (Benson et al., 2000) and SWISS-PROT (Bairoch and Apweiler, 2000
) sequence databases. Three three-dimensional structures have already been determined, those for the RG A from Aspergillus aculeatus (Petersen et al., 1997
), PG A from Erwinia carotovora ssp. carotovora (Pickersgill et al., 1998
) and PG II from Aspergillus niger (Van Santen et al., 1999
). All adopt the so-called parallel ß-helix structural domain first observed in pectate lyase C (Yoder et al., 1993
). This domain is a characteristic fold for a larger protein family of right-handed parallel ß-helix proteins and may consist of 712 coils forming either three or four parallel ß-sheets (Yoder and Jurnak, 1995
; Jenkins et al., 1998
).
Comparison of the three-dimensional structure of PG with that of RG enabled the similarities and differences in their active sites to be found (Pickersgill et al., 1998), which should be applicable also for the other members of family 28. The similarities in the presumed active sites as well as the overall structural similarity confirm the original classification of PGs and RGs into one sequence-based family (Henrissat, 1991
) despite their very low sequence identity (about 15%).
Several comparisons of amino acid sequences of bacterial, fungal and plant polygalacturonases were performed, but in most cases either a limited number of various enzymes were used for the comparison or attention was focused only on their isolated, best conserved sequence segments (Scott-Craig et al., 1990; Bussink et al., 1991
; Ruttkowski et al., 1991
; Tebbutt et al., 1994
; Kester et al., 1996
; Petersen et al., 1996
; Tenberge et al., 1996
; Huang and Allen, 1997
; Hadfield et al., 1998
; Stratilová et al., 1998
; Gognies et al., 1999
; Wubben et al., 1999
; Torki et al., 2000
). Thus, the regions comprising the residues 178_NTD, 201_DD, 222_GHG and 256_RIK (unless otherwise specified, all amino acid numbering throughout the text corresponds to the open reading frame of A.niger PG II) (Bussink et al., 1990
) have been found to be strictly conserved in all PGs, EPGs and EPGDs, with the Asp180, Asp201, Asp202, His223, Arg256 and Lys258 being probably involved in their active site (Rexová-Benková and Mra
ková, 1978
; Pickersgill et al., 1998
; Van Santen et al., 1999
; Armand et al., 2000
). However, in RGs these regions are 193_glD, 215_De, 237_sgG and 269_miK (A.aculeatus RG mature enzyme numbering) (Kofod et al., 1994
); the His223 of polygalacturonase is replaced by Gly238 of RG.
Moreover, Stratilová et al. (Stratilová et al., 1996) have described the potential role for a tyrosine residue in the function of a PG by chemical modification and spectrophotometric titration. The eventual position of this tyrosine was proposed by comparison of 36 different PG sequences which revealed a strictly conserved tyrosine residue equivalent to Tyr291 of A.niger PG II (Stratilová et al., 1998
).
Despite all these partial findings a serious deep analysis of all available amino acid sequences of the glycoside hydrolase family 28 is still lacking. Moreover, the family 28 is a quickly growing family of enzymes (more than 100 members). The need for this study may be supported also by running genome projects that yield numerous sequences of putative proteins having similarity to those of the family 28. Therefore, the aim of the present study was to: (i) compare as many as possible amino acid sequences of the members of this family of glycoside hydrolases; (ii) describe their evolutionary relationships in detail; and (iii) reveal their similarities and differences that would allow one to discriminate between them.
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Materials and methods |
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Results and discussion |
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In this study 115 amino acid sequences of PGs, EPGs and EPGDs were compared. One sequence represents the insects (animals), the one from the phytophagous mustard beetle Phaedon cochleariae (Girard and Jouanin, 1999), whereas all the others belong to bacteria, fungi and plants (Table I
) that form the three main groups. No polygalacturonase from archaeal origin is known. The group of plant enzymes covers 19 members of a gene family encoding PGs and EPGs in Arabidopsis thaliana (Torki et al., 2000
).
The amino acid sequence alignment of all 115 PG, EPG and EPGD sequences (Figure 1; the colour version of Figure 1
can be found at the URL: http://nic.savba.sk/~umikstef/PGs) confirmed that there are four strictly conserved sequence segments with one invariantly conserved residue, Tyr291, as recognized previously (e.g. Stratilová et al., 1998
). Remarkably there was only one further amino acid residue, Gly228, strictly conserved in all these enzymes (Pavenicová, 2000). This glycine is positioned close to the C-terminus of the third conserved region (222_GHG). Structurally, it is located in the seventh turn between the ß-sheets PB1 and PB2a of A.niger PG II (Van Santen et al., 1999
); however, as yet no function has been assigned to it.
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The second segment, 201_DD, is exclusively conserved in all polygalacturonases with specific amino acid residues neighbouring at both sides of this dipeptide. All bacterial and plant enzymes together with most of fungal EPGs have the glycine at the N-terminal side of 201_DD, while all fungal PGs (including the PG from insect) contain a glutamine in that position. Concerning the C-terminal side of the second segment, there is a cysteine residue conserved in all plant and fungal polygalacturonases, the insect PG as well as the two bacterial enzymes (Agrtu.pg and Yeren.epgd).
In the third segment, 222_GHG, there is an almost invariantly conserved (94.8%) Gly222 followed by two totally conserved His and Gly. The former glycine was replaced in one of the two fungal PGs from Colletotrichum lindemuthianum and all four known fungal EPGs by serine as well as in the enzyme from T.maritima. The fourth conserved segment, 256_RIK, contains a highly conserved (87.0%) Ile257 in addition to the strictly conserved Lys and Arg which is replaced by His in the insect PG. The isoleucine was not conserved in two bacterial PGs (Agrtu.pg and Thtma.pg; IleLeu), all bacterial EPGDs (by Ala, Gly and Leu), four fungal PGs (Botfu2.pg, Ophno.pg, Penex.pg and Pengr2.pg; Ile
Val) and five plant PGs (tomato abscission zone; Ile
Val).
With regard to the Tyr291 found previously to be conserved in polygalacturonases (Stratilová et al., 1998), the alignment of all 115 PGs, EPGs and EPGDs known at present confirmed that this tyrosine belongs to the invariantly conserved residues of these enzymes (Figure 1
).
Bacterial PGs and EPGDs
The group of bacterial PGs contains the sequences with a very low degree of mutual similarity. The seven PGs involved in the present study (Table I) exhibit only 4.7 and 11.2% identity and similarity, respectively, except for the two PGs from E.carotovora that share 96.0% sequence identity (Hinton et al., 1990
; Saarilahti et al., 1990
). Pair-wise similarity varies in the range between 11 and 50%, but in most cases does not reach 20%. The bacterial PG sequences have only 10 invariant residues (Asn197, Ile218, Gly248, Lys255, Gly282, Gly315, Val330 and the dipeptide 287_GV; Erwca2.pg numbering) in addition to the four well recognized conserved regions and presumably functional invariant tyrosine (Figure 1
). It is worth mentioning that in Ralstonia solanacearum EPGD, this tyrosine was identified only with help of the hydrophobic cluster analysis method (data not shown) due to an inserted oligopeptide segment (Huang and Allen, 1997
) absent in all other polygalacturonases (Figure 1
).
On the other hand, the overall sequence similarity among bacterial EPGDs is higher (23.7% identity and 37.3% similarity), the pair-wise similarity ranging from 25 to 60%. These sequences contain not only isolated invariant residues but also several identical segments that have been found to be characteristic of bacterial EPGDs only (Figure 1). The most important segments are 202_MTL, 255_NIRI, 378_FGNS, 399_NF, 417_AW, 467_GGGA and 584_PW (Erwch.epgd numbering).
Fungal PGs and EPGs and the insect PG
Forty-three fungal PGs form a substantial part of the entire set of polygalaturonases studied in this work (Table I). Their sequences are 8.9% identical and 17.4% similar with the average pair-wise similarity of about 60% ranging from lower than 20% to higher than 90%. In the amino acid sequence alignment (Figure 1
) several aromatic residues can be found as characteristic of these fungal PGs: Phe32, Phe74, Phe80, Trp85, Trp114, Trp115, Phe128, Phe129, Phe182, Phe214, Tyr272, Tyr283, Tyr326, Trp337 and Trp339 (Aspni2.pg numbering). Not all of them are conserved strictly (except of Trp115 and Phe182), but in most cases there are conservative (aromatic
aromatic) substitutions. Of these Phe80, Trp85 and Tyr272 (or their correspondences) are present also in the fungal EPGs, and the equivalents of Trp115 (though replaced by tyrosines) may be found in the whole set of sequences shown in Figure 1
. There is also one longer segment specific for fungal PGs (205_AinSG) positioned from the C-terminal side close to the active-site dipeptide 201_DD. Among the other conserved residues the two prolines, Pro148 (conserved also in the fungal EPGs) and Pro300 (not strictly conserved), could be of interest.
Fungal EPGs exhibit quite high degrees of identity (36.7%) and similarity (51.0%) with several longer conserved stretches, characteristic dipeptides and isolated invariant residues. In the N-terminal part, there is a segment starting with 60_DD and ending with 75_GG (Aspergillus tubigensis EPG numbering). The following regions are also typical for the EPGs from fungi: 121_SFKxxFQN, 166_LRPiL, 225_WDTYR, 248_SFKPN, 319_GGGG. In addition, Phe132, Phe133, Tyr143, Trp306, Tyr331 and Phe374 (although not invariantly conserved in all cases) as well as the two dipeptides, 355_TL and 364_LT (located in the C-terminal part), should be of importance for fungal EPGs.
With regard to the one representative of animal polygalacturonases, the insect PG from P.cochleariae, its sequence goes well with the fungal PGs and contains almost all sequence features characteristic of this group of PGs (Figure 1). There are 20 single residues or short segments conserved in fungal PGs that are identical with those from the insect PG.
Plant PGs and EPGs
In the group of plant polygalacturonases analysed in this study, the exact enzyme specificity has not been determined strictly due to the fact that many of them were not biochemically characterized in detail or were taken as putative proteins from sequencing the whole genome. It is not possible to say clearly in all cases whether the enzyme is a PG or EPG. There are (Figure 1) again several well conserved aromatic amino acid residues, such as Trp99 (LycesA.pg numbering), Trp157, Phe203, Trp331, Phe343 and Tyr382, as well as the other residues Ser245, Gly250, Gly269 (strictly conserved also in all bacterial PGs and EPGDs and almost in all fungal EPGs), Pro354 and Asp358 (substituted in three cases by Asn).
In agreement with the proposed classification system (Hadfield and Bennett, 1998; Torki et al., 2000
) the present set of 56 plant polygalacturonases can be divided into five clades: A, B, C, D, E plus the gymnosperm PG from Cryptomeria japonica (cedar) with a sequence without resemblance to the rest (except for the conserved sequence regions covering the active-site residues discussed above). This division is based on the evolutionary tree (Figure 2
) of all polygalacturonases (Table I
) reflecting the alignment shown in Figure 1
. The tree will be discussed later.
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With regard to sequence identity and similarity in the frame of the individual clades, the values are shown in Table II. However, in general, the conserved sequence regions containing the active-site residues are conserved in plant polygalacturonases as longer segments (Figure 1
).
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As pointed out by the published crystal structures of polygalacturonases (Pickersgill et al., 1998; Van Santen et al., 1999
) these enzymes contain some disulfide bridges stabilizing their molecules. However, the conservation of cysteines reflects taxonomy, i.e. the corresponding disulfides could be conserved only in the frames of the respective bacterial, fungal and plant groups as described above. There is only one cysteine residue conserved throughout all the polygalacturonases (Figure 1
) in the position of Cys45 (Aspni2.pg).
Bacterial PG from E.carotovora has two SS bridges, Cys41Cys62 and Cys115Cys125 (Pickersgill et al., 1998), but there is no conservation of cysteines in the respective positions for all bacterial PGs. Bacterial EPGDs contain an even smaller number of cysteine residues which are also without specific arrangement (Figure 1
).
On the other hand, the cysteines among fungal PGs are very well conserved. Aspergillus niger PG II (Van Santen et al., 1999) has four disulfides: Cys30Cys45, Cys203Cys219, Cys329Cys334 and Cys353Cys362. While the first two bridges should be present in all fungal PGs, the one corresponding with Cys329Cys334 is missing in the PGs from yeasts (Cys
Val and Cys
Ala substitutions). With regard to the fourth SS bridge, the corresponding cysteines are absent in both PGs from Claviceps purpurea and the one from Chondrostereum purpureum. The insect PG from P.cochleariae has all the cysteines in accordance with those present in the group of fungal PGs. Based on the alignment shown in Figure 1
it is possible to suppose that the fungal EPGs could contain all the four disulfides present in fungal PGs. However, the position of the first cysteine from the first disulfide (Cys30Cys45 in the fungal PGs) is shifted and corresponds to Cys50 of A.tubigensis EPG, and there is Cys
Ala substitution in EPG from Fusarium oxysporum in the position corresponding to the first cysteine of the third disulfide (Cys329; Aspni2.pg numbering). Fungal EPGs possess two additional conserved cysteines, Cys348Cys357 (A.tubigensis EPG numbering), forming probably an extra disulfide bridge.
Since the three-dimensional structure of a plant polygalaturonase has still not been determined and the presence of disulfides in these enzymes has not been experimentally proved, the eventual SS bridges can be proposed by analogy with fungal PGs only. Thus, plant enzymes could contain the three disulfides corresponding with the second, third and fourth disulfides of fungal PGs, positioned at Cys272Cys289, Cys399Cys405 and Cys427Cys442, respectively (Lycopersicon esculentum PG A numbering). There are two exceptions, the pollen PG from Nicotiana tabacum with the CysArg substitution in the position corresponding with the Cys399 and the one from L.esculentum (TAPG3) with a shorter polypeptide chain. With regard to the first disulfide present in fungal PGs, all plant enzymes contain only the second cysteine, Cys103, which corresponds to Cys45 of A.niger PG II. However, there is a strictly conserved cysteine residue in all plant enzymes, Cys130 (LycesA.pg numbering), which could eventually form the SS bridge equivalent to the first disulfide of fungal PGs. Most of the plant polygalacturonases have further cysteines, Cys186, Cys194, Cys229, Cys362 and Cys368 (LycesA.pg numbering), four of which (except for the Cys229) are absent in the PGs from clade D. Clade D, on the other hand, contains an extra cysteine corresponding with Ala317 in the LycesA.pg, which was proposed as a pollen-specific cysteine residue (Tebbutt et al., 1994
; Petersen et al., 1996
). This cysteine is further present in most members of clade C and in the PGs from Cucumis melo (Cucme3.pg) and Medicago sativa (Medsa.pg). Cys186 is not present in clades D, E and in the pollen PG from M.sativa (Medsa.pp). This is also the case for Cys194, which is absent in two more pollen PGs, those from Gossypium barbadense and Gossypium hirsutum. Cys229 was not observed in the sequences of PGs from A.thaliana (Arath2.pg, Arath4.pg and Arath11.pg) and Prunus persica (Prupe1.pg) as well as of EPGs from A.thaliana (Arath3.epg, Arath4.epg, Arath5.epg and Arath7.epg) and Brassica napus (Brana.pep). Both Cys362 and Cys368 are present in all plant polygalacturonases except for those from clade D and Arath4.epg, thus indicating the possibility of forming a disulfide bridge.
As far as the aromatic amino acid residues are concerned, those characteristic for the individual groups of polygalacturonases (PGs, EPGs and EPGDs as well as bacteria, fungi and plants) were briefly described above. The interest in these residues is due to the fact that they may be involved in binding of substrate not only in polygalacturonases (Rao et al., 1996) but also in other glycoside hydrolases, e.g. in amylases (Clarke and Svensson, 1984
; Gibson and Svensson, 1986
; Williamson et al., 1997
). The invariantly conserved tyrosine, Tyr291 (Stratilová et al., 1993
, 1998
), to which also the function has been proposed (Stratilová et al., 1996
), was recently confirmed by site-directed mutagenesis to be indispensable for effective catalysis constituting the subsite +1 (Pagès et al., 2000
). There are two further aromatic positions conserved among the different groups of polygalacturonases. These are not conserved strictly but only aromatic residues (Trp, Phe and Tyr) occur there. The first one is at Trp115, which is in all bacterial EPGDs, most fungal EPGs and three plant pollen PGs replaced by tyrosine and phenylalanine (Figure 1
). The only exception is the PG from Agrobacterium tumefaciens (with a shorter polypeptide chain) that evidently does not possess an aromatic residue equivalent to Trp115. The second aromatic position corresponds with Phe271 which alternates with tyrosine (60:40%, respectively) only. The A.tumefaciens PG with a methionine residue equivalent to Phe271 exhibits an exceptional behaviour again.
Evolutionary tree of all endo- and exo-polygalacturonases
The evolutionary tree showing for the first time the relationships of a complete as possible set of sequenced polygalacturonases belonging to bacteria, fungi, plants and animals (represented by an insect) is presented in Figure 2A. The tree is based on the sequence alignment shown in Figure 1
and thus reflects the sequence similarities and differences discussed above.
Basically, the tree manifests that there are three main groups: bacteria, fungi and plants (Figure 2A), bacteria being positioned, however, between the fungal PGs and fungal EPGs. The only one representative of the animal kingdom, the insect PG from P.cochlearie, is included in the cluster of fungal PGs. Following this basic division of the tree, one can further see the clustering according to the endo- and exo-mode of action of these enzymes especially among the bacterial and fungal polygalacturonases and into the plant clades.
While bacterial EPGDs form their own cluster, the group of bacterial PGs is not so homogeneous reflecting the lower degree of mutual sequence similarity. The two PGs from E.carotovora are very closely related to each other and are located next to the pair of PGs from Agrobacterium vitis and Ralstonia solanacearum. There are three further PGs located on long branches. Two of them, those from A.tumefaciens and T.maritima, have been determined as putative PGs only (Rong et al., 1991; Nelson et al., 1999
) so that it is not possible to correlate their position in the tree with their exact enzyme specificity. The last bacterial PG from Burkholderia cepacia is even more isolated which may reflect the fact that this PG is a plasmid-encoded protein (Gonzalez et al., 1997
).
The consequence of clear sequence differences between fungal PGs and EPGs (Figure 1) is that these two groups of polygalacturonases are well separated in the evolutionary tree (Figure 2A
), both forming quite homogeneous and isolated groups. The most remarkable feature of the fungal PG part of the tree is the location of the insect PG from P.cochlearie (representing the animal kingdom) directly among the fungal PGs. However, this is based on the resemblance of the insect sequence to the fungal ones described above. The PG from C.purpureum seems to be the most distantly related member of the fungal PG group (Figure 2A
) in agreement with several non-conservative substitutions in characteristic positions (Figure 1
). The two PGs from yeasts are both positioned adjacent to each other thus indicating that all eventual yeast PGs would form their own separate yeast cluster in the frame of all fungal PGs. Several sub-clusters or sub-groups can be found in the fungal PG part of the tree. Based on the analysis of 35 sequences of fungal PGs Wubben et al. (Wubben et al., 1999
) have proposed five monophyletic groups of closely related PGs. The present study covering more fungal PGs (43 sequences) indicates that the number of the so-called monophyletic groups is probably higher and will even rise as more sequences become available.
As mentioned in the section dealing with the sequence comparison of plant PGs and EPGs, these enzymes have been classified into the five clades (Hadfield and Bennett, 1998; Torki et al., 2000
) plus the PG from cedar (Table II
). This division can also be seen from the tree (Figure 2A
) where the plant polygalacturonases form clusters according to their clades and one long isolated branch leading to the cedar PG (Cryja.pp), which is the only plant gymnosperm PG. The detailed analysis of the branch arrangement in the plant angiosperm part of the tree suggested that the `plant' branch leads to the node separating clades E, D and B on the one side from those of C and A on the other side. The only `exception' is the clustering of the five PGs from A.thaliana (Arath1.pg-Arath5.pg) from clade A together with the three further PGs from this plant that should form clade D (Torki et al., 2000
). The largest clade C (Table II
) contains polygalacturonases expressed mainly in flower buds, flowers and pollen that are thought to encode the EPGs (Torki et al., 1999
, 2000
) with one exception, the PG from M.sativa induced by a Rhizobium strain, which was, however, originally revealed to exhibit extremely high sequence similarity to its pollen counterpart (Muñoz et al., 1998
).
In order to re-analyse the plant part of the evolutionary tree with respect to the plant clades, a further tree was constructed (based on the alignment shown in Figure 1), however, excluding the positions with gaps. The simplified version of this tree is shown in Figure 2B
. The detailed analysis of the branch arrangement in the plant part of the tree in this case suggested that the `plant' branch leads to the node separating clade D (three PGs from A.thaliana) from the rest of the plant enzymes. The rest was then divided into well separated clades (without dividing any of them) so that clades C and A were on the one side and clades E and B were on the other side. The only gymnosperm PG from cedar was on its own long branch (adjacent to clade B) which reflected its higher dissimilarity (discussed above) with the other plant PGs which are angiosperm. With regard to bacterial and fungal parts of the tree, in the case when the positions with gaps were excluded, taxonomy was fully respected so that there were two separate branches in the tree (Figure 2B
): one leading to fungal PGs and EPGs, and the other one leading to bacterial PGs and EPGDs.
Rhamnogalacturonases
The alignment of all sequenced rhamnogalacturonases is presented in Figure 3. As is well known (Coutinho and Henrissat, 2000
) the RGs belong to glycoside hydrolase family 28 together with polygalacturonases although the conserved active-site regions of PGs are slightly modified in the sequences of RGs. Thus, 193_GLD (A.aculeatus RG numbering) corresponds with 178_NTD (A.niger PG II numbering), 215_DE with 201_DD, 237_SGG with 222_GHG, and 269_MIK with 256_RIK. It means that the most significant amendments in the sequences of RGs in comparison with polygalacturonases are the lack of His223 in the third region (SGG/GHG) and hydrophilic
hydrophobic substitution in the fourth region (MIK/RIK). With regard to the invariant tyrosine residue, Tyr291, present in polygalacturonases (Figure 1
), this residue may have its equivalent in RGs (Tyr301; A.aculeatus RG numbering); however, it seems that there is no corresponding tyrosine in the RG from Botryotinia fuckeliana (Figure 3
).
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Endoxylogalacturonan hydrolase
The sequence of this new member of the glycoside hydrolase family 28 was determined only recently (Van der Vlugt-Bergmans et al., 2000). In contrast to RGs, the XGH sequence exhibits better similarity in the four active-site segments to polygalacturonases (205_NTD, 228_DD, 250_SHG and 284_GIK; A.tubigensis XGH numbering). It has the active-site DD dipeptide as well as the His251 equivalent to His223 of A.niger PG II. It also possesses the tyrosine residue corresponding with the Tyr291 present in polygalacturonases. All this can be supported by the model of the three-dimensional structure of XGH (Figure 4
) constructed using the X-ray co-ordinates of A.niger PG II (van Santen et al., 1999
; Protein Data Bank code: 1CZF) as template.
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In order to find the most closely related family 28 sequence to that of XGH the BLAST search was used (Altschul et al., 1990). It was found that the sequence of XGH exhibits the highest similarity to that of EPG from Cochliobolus carbonum. These two sequences have 39.9% identity and 55.4% similarity (the alignment not shown). In general, the sequence of A.tubigensis XGH exhibits higher similarity to the sequences of fungal EPGs than to those of fungal PGs (Van der Vlugt-Bergmans et al., 2000
). Remarkably, the similarity is lower to the EPG from the same organism A.tubigensis (53.5%) than to the taxonomically more distantly related EPG from C.carbonum (55.4%). Despite this pronounced sequence similarity to fungal EPGs, the XGH sequence does not contain most of the conserved regions characteristic of fungal EPGs, thus indicating its enzymatic uniqueness.
All glycoside hydrolase family 28 enzymes
Based on the analysis of available amino acid sequences of PGs, EPGDs, EPGs, RGs and XGH discussed above, a set of sequences of the family 28 members representing all the individual groups was aligned (Figure 5). It is evident that despite the overall rather low sequence similarity, each representative contains its functionally important residues in the segments equivalent to the four conserved active-site segments of PGs (178_NTD, 201_DD, 222_GHG and 256_RIK) as well as at least the conservative substitution of the Tyr291. This makes from them a common family in the frame of all glycoside hydrolases (Coutinho and Henrissat, 2000
). On the other hand, there are some important changes of the residues adjacent to the residues constituting the active site, especially in RGs. In fact there are only three strictly conserved residues in common that could be functionally important in the family 28, i.e. Asp180, Asp201, Lys258. This reflects very probably the fact that even closely related fungal PGs from A.niger have different specific kinetic parameters on polygalacturonic acid and a specific mode of action (Pavenicová, 2000). Therefore, for example, the Met150 of PG II from A.niger located at the subsite 2 has no strictly conserved equivalents in the frame of the entire family 28 (Figure 5
) although its mutation to glutamine affected catalysis (Pagès et al., 2000
).
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As far as the cysteine residues are concerned there is only one cysteine, Cys45, conserved invariantly throughout the family 28 (Figure 5). Except for the bacterial PGs and EPGDs, all the members of this family could have six cysteine residues in common corresponding with three of the four disulfide bridges (Cys203Cys219, Cys329Cys334 and Cys353Cys362) present in the PG II from A.niger.
Based on the alignment of representative members an evolutionary tree was constructed (Figure 6) showing the mutual relationships in the frame of the entire family 28. The long branches reflect the overall rather low sequence similarity among the groups of bacterial, fungal, plant and insect PGs, EPGDs and EPGs as well as RGs and XGH. However, the taxonomy is respected so that bacterial PGs and EPGDs as well as plant PGs and EPGs are on the bacterial and plant nodes, respectively, on the neighbouring branches. As was discussed above, the insect PG is most closely related to fungal PGs represented by the PG II from A.niger and the XGH to fungal EPGs represented by the EPG from A.tubigensis. The RG A from A.aculaetus positioned on the longest isolated branch manifests the amendments in the sequences of RGs even in the four active-site segments.
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Notes |
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Acknowledgments |
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References |
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Received December 15, 2000; revised March 20, 2001; accepted June 15, 2001.