Unitat de Biotecnologia Computacional, Departament de Bioquímica i Biotecnologia, Universitat Rovira i Virgili, Catalonia, Spain
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Abstract |
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Introduction |
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Materials and Methods |
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In a second stage, all the A+B segments were used to produce two different sets of sequences, one corresponding to the strict domain A and the other corresponding to domain B. Once again, the boundary between domains was obtained by visually inspecting all crystallized AAMYs. The sequence for the B domain goes from the highly conserved His residue located after ßA3 to four residues downstream of the Asp that binds to Ca2+ (just before A3, the
-helix preceding the highly conserved ßA4). Domain A was obtained by joining the two subsegments on the left and right of domain B. For some sequences (e.g., Q05884 [StrLi]), the C-terminal boundary of domain B was difficult to find. In these special cases, we used the PSIpred server (http://insulin.brunel.ac.uk/psipred) to predict the secondary structure for the corresponding A+B sequences and identify where
A3 began. This server was chosen because at the latest Critical Assessment of Techniques for Protein Structure Prediction (CASP3; http://PredictionCenter.llnl.gov/casp3/), it was the most accurate method of secondary-structure prediction tested, achieving an overall three-state accuracy of 77% across 24 prediction targets. The PSIpred server was used (1) to find the locations of ß-strands in domain A of sequences with an overall low similarity with crystallized AAMYs and (2) to confirm the locations of secondary structures inferred from alignments with sequences from crystallized structures. Two prediction methods offered by the server were used extensively to test the coherence of the secondary-structure assignments. These were PSIpred for predicting protein secondary structure and GenTHREADER for predicting protein tertiary structure by fold recognition.
To avoid bias in our results due to highly similar isozymes of a given species in the full sample, we defined an AAMY "representative sample." The new sample therefore contained 7 sequences from Archaea, 44 from Bacteria, and 61 from Eukaryota and was made up of AAMYs from different biological species and isozymes with similarity indices (SI) below 95% for both the A and the B domains. The results presented in this paperwhen not specifically indicatedare from the representative sample. The set of AAMY sequences that form this sample is shown in figure 2
, in which sequences may be identified by their Swiss-All accession numbers and the abbreviations for the names of the species are made up of the first three letters of the genus name followed by the first two letters of the species name (e.g., AerHy for Aeromonas hydrophila). Results from the full sample may be obtained as supplementary material from our web site (http://argo.urv.es/pujadas/AAMY/AAMY_01).
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Crystallographic data retrieval, as well as sequence- and structure-derived information, were taken from the database and links in the Structure Explorer (http://www.rcsb.org/pdb/). The PDB entries for AAMY were as follows: 1BSI (Rydberg et al. 1999
), 1B2Y (Qian et al. 1994
), 1CPU (G. D. Brayer et al., personal communication), 1HNY (Brayer, Luo, and Withers 1995
), and 1SMD (Ramasubbu et al. 1996
) from Homo sapiens; 1BVN (Wiegand, Epp, and Huber 1995
), 1DHK (Bompard-Gilles et al. 1996
), 1JFH (Qian et al. 1997
), 1OSE (Gilles et al. 1996
), 1PIF (Machius et al. 1996
), 1PIG (Machius et al. 1996
), and 1PPI (Qian et al. 1994
) from Sus scrofa; 1JAE (Strobl et al. 1998a
), 1TMQ (Strobl et al. 1998b
), and 1VIW (Nahoum et al. 1999
) from Tenebrio molitor; 1AMY (Kadziola et al. 1994
), 1AVA (Vallee et al. 1998
), and 1BG9 (Kadziola et al. 1998
) from Hordeum vulgare; 2AAA (Boel et al. 1990
) from Aspergillus niger; 2TAA (Matsuura et al. 1984
) and 6TAA and 7TAA (Brzozowski and Davies 1997
) from Aspergillus oryzae; 1BLI (Machius et al. 1998
), 1BPL (Machius, Wiegand, and Huber 1995
), and 1VJS (Hwang et al. 1997
) from Bacillus licheniformis; 1BAG (Fujimoto et al. 1998
) from Bacillus subtilis; and 1AQH, 1AQM (Aghajari et al. 1998a
) and 1B0I (Aghajari et al. 1998b
) from Pseudoalteromonas haloplanctis. X-ray diffraction resolutions and R factors for these structures ranged from 1.6 to 3.2 Å and from 0.151 to 0.208, respectively. Although 1BVZ from Thermoactinomyces vulgaris (Kamitori et al. 1999
) is considered an AAMY in its PDB file, a FASTA search (http://www2.ebi.ac.uk/fasta3/) showed that this enzyme is a neopullulanase (E.C. 3.2.1.135) whose sequence matches the NEPU_THEVU (Q08751) SwissProt entry exactly.
Multiple-sequence alignments were carried out for protein sequences of the working database with the CLUSTAL V algorithm (Higgins and Sharp 1989
) and the commercial program MEGALIGN, version 3.16, from the Lasergene software package (1997; DNASTAR, Inc., London, England) running in a Power Macintosh. Initial dendrograms and SIs were calculated by applying available MEGALIGN subroutines that calculated the SI parameter between two sequences (i and j) based on the method of Wilbur and Lipman (1983)
with a gap penalty of 3, a K-tuple of 1, five top diagonals, and a window size of 5. The SI was calculated as the number of exactly matching residues in this alignment minus a "gap penalty" for every gap introduced. The result was then expressed as a percentage of the length of the shorter sequence. Multiple-alignment parameters (fixed and floating gap penalties) both had a value of 10. The protein weight matrix was PAM 250. We calculated the phylogenetic trees by the neighbor-joining method (Saitou and Nei 1987
) with 1,000 bootstrap replicates and a seed value of 111 with the CLUSTAL X program, version 1.8 (Thompson et al. 1997
). Unrooted trees were drawn with NJPLOT (Perrière and Gouy 1996
).
Hydrogen bonds involved in helix-capping interactions at the N- and C-terminal ends of A2, along with distances between donors and acceptors, were analyzed using HBPLUS (McDonald and Thornton 1994
). The capping interactions were visually analyzed with the program Rasmol (Sayle and Milner-White 1995
) using a Silicon Graphics Indigo2 XZ workstation. The DSSP algorithm included in Rasmol was used to determine the limits of ß-strands which were not included in the PDB files (e.g., some of the ß-strands in the TIM barrel of 1AQH), although their presence was obvious in the visualization.
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Results and Discussion |
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Taxonomic Distribution of AAMYs in the Clusters
Figure 2A
shows that AAMYs from Archaea are distributed in two clusters: AI, which contains all of the Crenarchaeota sequences, and AII, which contains all of the Euryarchaeota sequences. The cluster distribution of the Archaea sequences is therefore closely related to the taxonomic grouping. At this point, we would also like to mention that considering sequences from cluster AI "true" AAMYs is controversial (S. Janecek, personal communication). Although it appears that they are able to attack the starch (Kato et al. 1996
), their investigated substrate/product specificity and mode of action seem different from those found in ordinary AAMYs. Moreover, GenBank (Benson et al. 2000) provides two sequences under two different accession numbersone designated as AAMY and equivalent to SwissProt entry Q53641 (D64131), and the second designated as maltooligosyltrehalose trehalohydrolase TreZ gene (D83245)whose complete alignment shows that they are 100% identical. Despite these contradictory facts, we included the three Sulfolobus sequences in our study because it seems clear that to some extent they show 3.2.1.1 activity (Kato et al. 1996
).
The tree for Bacteria displays eight clusters and one sequence that appear to be solitary from an evolutionary point of view (P14898[DicTh]; see fig. 2B
). In contrast to the Archaea results, there is no general correspondence between cluster distribution and taxonomy for Bacteria (see table 3
). In this sense, the distribution of AAMY sequences within clusters appears to be rather scattered. For instance, AAMYs from Firmicutes Actinobacteria Actinobacteridae may be found in clusters BI (2 out of 2) and BVIII (9 out of 13); Firmicutes Actinobacteria Thermoactinomyces are distributed between clusters BII (1 out of 5) and BIV (1 out of 2); Firmicutes from the bacillus/clostridium group may be found in clusters BII (4 out of 5), BIV (1 out of 2), BV (7 out of 9), BVI (7 out of 7), and BVIII (1 out of 13) and outside any cluster (1). Moreover, Proteobacteria AAMYs from the gamma subdivision are scattered between clusters BIII (1 out of 2), BV (2 out of 9), BVII (3 out of 3), and BVIII (3 out of 13). The only Thermotogales AAMY (P96107) falls inside cluster BIII. As we can see in table 3
, if a more extended taxonomic classification is used, the scattering between clusters remains (e.g., Bacillaceae/Bacillus AAMYs are scattered among clusters BV and BVI). There is some scattered clustering in the unrooted evolutionary tree for Bacteria described by Janecek (1994)
(i.e., the clustering of Proteobacteria and Firmicutes [EscCo and SalTy with BacSt, BacAm, and BacLi; PseHa with StrHy, StrTl, StrVl, StrLi, StrGr, and TheCu]), but this phenomenon is described more accurately in this study. Moreover, cluster BV simultaneously contains AAMYs from Firmicutes and Proteobacteria species (this is also true for cluster BVIII; see table 3
). Figure 2B
also shows that poorly related AAMY sequences frequently coexist in the same species: (1) P22630 (cluster BVII) and P41131 (cluster BVIII) from AerHy; (2) Q52413 (cluster BVIII) and Q52414 (cluster BVII) from PseSp; (3) Q56791 (cluster BVII) and Q60102 (cluster BIII) from XanCa; (4) Q05884 (cluster BI) and P97179 (cluster BVIII) from StrLi; (5) Q60051 (cluster BIV) and Q60053 (cluster BII) from TheVu; and (6) O50583/Q53786 (cluster BV) and O50582 (cluster BVI) from StrBo.
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Interkingdom Relationships Between Clusters
Long et al. (1987)
, Janecek (1994)
, and Janecek et al. (1999)
also discovered the linkage between some of the AAMYs that belong to the cluster pairs BVIII/EIII, AII/BV, and AII/EI. Linkage of the BVIII/EIII pair is confirmed by our results with domain A sequences (SI ranges from 27% to 38%), although both clusters significantly increased their members (see fig. 4A
). With respect to the AII/BV relationship described by Janecek et al. (1999)
, we found that the SI values of some members of clusters BV and AII were also around 30% when their domain A sequences were compared (e.g., the SI value for O50200 [TheSp] and P06278 [BacLi] was 32.7%; see fig. 4B
). Moreover, one of the residues considered by Janecek et al. (1999)
to be highly characteristic of Archaea AAMYs from cluster AIIi.e., Trp in position i+2 from the ßA5 catalytic Gluwas also found in all BV members. This confirms Janecek et al.'s (1999)
results for EscCo (P26612) and BacLi (P06278). These authors have suggested that this Trp plays the same role in the active site of AAMYs from cluster AII as the equivalent Trp in the HorVu AAMY (it forms a stacking interaction with one of the acarbose rings bound in the active site; Kadziola et al. 1998
). Therefore, by drawing a parallel with Janecek et al.'s conclusions, we may expect the same role for the equivalent Trp in BV AAMYs. On the other hand, we did not detect a set of homogeneous SI values when comparing domain A sequences from clusters AII and EI (SI ranges from 27.2% to 17.1%). Nevertheless, it is clear that this relationship exists at a more localized level (see fig. 1
in Janecek et al. 1999
). By comparing all clusters, we also detected a strong similarity between domain A sequences from clusters BIV and EII (see fig. 4C
). The range for the SI values obtained from all of the comparisons between members of BIV and EII is 27%38%. The relationship between BIV and EII involves one newly described cluster (BIV) and therefore cannot be inferred from Janecek's et al. (1999)
results. Consequently, AII, BV, and EI would appear to share the same common ancestor (and the same is also true for BVIII and EIII and for BIV and EII). Moreover, the common ancestor for AII/BV/EI, the one for BVIII/EIII, and the one for BIV/EII are different. This would suggest that the divergence between these three ancestors was a very early event in AAMY evolution. Interkingdom multialignments for the relationships between clusters that are discussed in this section may be obtained from our website or from the EMBL Sequence Alignment database. The codes in this database are DS43802 for AII/BV/EI, DS43803 for BIV/EII, and DS43804 for BVIII/EIII multialignments.
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We have therefore made a classification of known AAMYs which is based on an objective definition of a cluster (see above). This definition describes the most important known AAMY relationships (Long et al. 1987
; Janecek 1994
; Janecek et al. 1999
) and is useful for finding, in a systematic way, (1) new clusters (AI, BII, and BIV), (2) undescribed relationships between clusters (BIV/EII), and (3) scattering of bacteria between different clusters irrespective of the taxonomy.
Conserved Sequence Segments in the A Domain and Structural Analysis of the N- and C-Terminal Capping of Helix A2
Table 4
shows four motifswritten in PROSITE syntax (Hofmann et al. 1999
)corresponding to the four most conserved sequence segments in the A domain of AAMYs. The motifs were found by ocular inspection of the regions of the three multialignments (Archaea, Bacteria, and Eukaryota) where the sequences were highly similar. The multialignments were constructed in this case with the full sample (144 sequences), and the numbers show the residue occurrence in the representative sample (112 sequences). Note that the four conserved segments bundle at the C-terminal end of the barrel scaffold (where the core of the enzyme activity is located; see fig. 1
).
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All noncrystallized AAMYs show sequences that are compatible with a capping box and a Schellman motif for the N- and C-terminal ends of A2, respectively. From a sequence point of view, the
A2 Schellman motif found in AAMYs is more heterogeneous than the capping box that begins at the same helix (e.g., the C' position is occupied by eight different residues in our AAMY sample). The length of
A2 defined by both capping motifs is 15 residues for all AAMYs except Q60053 (TheVu). The structural preservation of this helix throughout evolution is interesting and requires new experimental approaches to determine more precisely why this helix is so important in AAMYs.
Using various (ß/)8 barrel enzymes, Janecek (1996)
defined the concept of "hidden homology" as a conserved region that is more or less preserved throughout evolution in the equivalent part of the structure of the other enzymes that share this folding motif. Following this definition, we studied whether the same type of capping box and Schellman motif were also found in the other crystallized TIM barrels from GH-13 (Pujadas and Palau 1999
; http://argo.urv.es/
pujadas/TIM). The structures analyzed have the following enzyme activities: 2.4.1.19 (cyclomaltodextrin glucanotransferase), 3.2.1.10 (oligo-1,6-glucosidase), 3.2.1.60 (glucan 1,4-
-maltotetrahydrolase), 3.2.1.68 (isoamylase), and 3.2.1.135 (neopullulanase). Our results demonstrate that in all cases, the structure of both capping motifs, the length of the helix
A2, and the number of residues between the invariable Gly and Asp are the same as those found in AAMYs. The numberings for GlyN' in the capping box, for the C' residue in the Schellman motif, and for the highly conserved Asp (ßA3) in some PDB sequences which are representative of the above-mentioned enzyme activities are Gly114-Asn130-Asp136 (1CIU; Knegtel et al. 1996
), Gly76-Asn92-Asp98 (1UOK; Watanabe et al. 1997
), Gly90-Gly106-Asp112 (2AMG; Morishita et al. 1997
), Gly270-Gly286-Asp292 (1BF2; Katsuya et al. 1998
), and Gly217-Gly233-Asp239 (1BVZ; Kamitori et al. 1999
). We may therefore conclude that the first part of the L2H2L'2ß3L3 motiffrom Gly to Aspmay be considered a strictly conserved structural motif in GH-13. Whether this motif is a "hidden homology" sensu stricto must be corroborated by finding other TIM barrel proteins which have no apparent sequence relationship with those from GH-13 but have (1) an
-helix 15 residues long that is limited at its N-terminal end by a capping box with an invariant Gly in N' position and at its C-terminal end by a Schellman motif and (2) an invariant Asp located at a constant distance from GlyN'.
Rasmol scripts that focus on the above-mentioned capping arrangements for AAMYs and other crystallized TIM barrels from GH-13 can be found on our website.
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Speculations and Conclusions |
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Our results suggest a general hypothesis for the evolution of domain A in AAMYs which involves two waves of evolutionary events. In the first wave, a limited set of genes strongly diverged from their common ancestral, or first AAMY, gene. These "first-generation" AAMYs probably had very low SI between them, had different lengths, and were the precursors of some of the unrelated clusters shown in figure 2
. Therefore, the only characteristics that the first-generation AAMYs preserved from the original gene were (1) the four segments indicated in table 4
, (2) the 3-D structure, and (3) the characteristics of helix A2. Some examples of these first-generation AAMYs are the common ancestor for AII/BV/EI, that for BVIII/EIII, and that for BIV/EII. Each of these first-generation AAMYs would further evolve in a second wave to produce the sequences that form each cluster (each first-generation AAMY gives a different cluster). These "second-generation" sequences diverge only slightly when compared with those of the first wave and make up the contemporaneous AAMYs. During this second wave, HGT could generate the interkingdom similarities between clusters (AII/BV/EI, BIV/EII, and BVIII/EIII). In this sense, Mazodier and Davies (1991)
suggested that the sequence similarity between the AAMY from StrLm (P09794; cluster BVIII) and those from mammalian and invertebrates (cluster EIII) found by Long et al. (1987)
may be proof of natural gene transfer between distantly related organisms. Mazodier and Davies (1991)
suggested that the HGT direction would be from Eukaryota to Streptomyces.
The clustering tree for Bacteria shows two characteristics that are not found in the trees for Archaea and Eukaryota: (1) the scattering in different clusters of AAMY sequences either from the same species or from closely related species (based on taxonomic grounds) and its opposite (the grouping of AAMYs from very distantly related organisms), and (2) that poorly related AAMY sequences frequently coexist in the genomes of some species and are included in different clusters. Following the above-mentioned general hypothesis for the evolution of domain A in AAMYs, we suggest that the evolutionary events that gave rise to this special distribution of bacterial AAMYs occurred during the first wave. This hypothesis is based on the fact that the sequences affected by these evolutionary phenomena are fully coherent with the characteristics of the rest of the sequences in the cluster. Therefore, it proves that they share the same common ancestor.
We may wonder which evolutionary events are responsible for the fact that the classification of the bacterial AAMY genes is not coherent with the current classification of the species. Probably, the first AAMY gene evolved to give rise to the limited set of first-generation AAMYs in two different ways: (1) gene duplications followed by independent parsimonious evolution, and (2) HGT. In addition to AAMY, the GH-13 superfamily comprises another 18 enzyme activities. There is functional (Kuriki and Imanaka 1999
) and sequence-based evidence (del-Rio, Morett, and Soberon 1997; Garcia-Vallvé, Palau, and Romeu 1999
) that the "first" GH-13 in a genome can give rise to a set of paralogs through massive gene duplication. A posteriori, these sequences can evolve by independent parsimonious evolution and acquisition of subtly different specificities to obtain the rest of GH-13 in the genome. Nevertheless, new glycoside hydrolase genes may also be acquired by a genome in a radically different way: by HGT from an exogen organism (Mazodier and Davies 1991
; Garcia-Vallvé, Palau, and Romeu 1999
; Garcia-Vallvé, Romeu, and Palau 2000
). Figure 2B
shows that poorly related AAMY sequences coexist in the genomes of some bacterial species. Most probably, the same is also true for other bacteria, but for the moment, only one gene has been characterized. Therefore, the use of completed bacterial genomes could help us to discover if there is more than one AAMY gene and to test their mutual evolutionary relationships, i.e., to determine whether they are paralogs or one of them has arrived by HGT. Since function assignment for genome-derived sequences is usually obtained by sequence comparison with proteins of known function and not from biochemical analysis, such a study should not be restricted to AAMYs and therefore should consider all GH-13 genes. Only 11 out of 25 completed bacterial genomes (Aquifex aeolicus, Bacillus subtilis, Chlamydia muridarum, Chlamydia pneumoniae, Chlamydia trachomatis, Deinococcus radiodurans, Escherichia coli, Haemophilus influenzae, Mycobacterium tuberculosis, Synechocystis sp., and Thermotoga maritima) have at least one GH-13 (93 genes). According to Garcia-Vallvé, Palau, and Romeu (1999)
, GH-13 sequences in E. coli and B. subtilis seem to be the product of the gene duplication of a common ancestor not arrived at by HGT to their genomes (and the same is valid for the GH-13 of the rest of completed bacterial genomes; S. Garcia-Vallvé, personal communication). The bacterial genomes, where poorly related AAMY sequences coexist (i.e., AerHy, PseSp, XanCa, StrLi, TheVu, and StreBo), have not yet been completed, and therefore only partial information is known. Once completed, it would be of interest to study whether these genes are paralogs or the result of HGT. Such information would allow us to fill in the gaps of the story of AAMY's evolution.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Abbreviations: 3-D, three-dimensional; AAMY, -amylase; GH-13, glycoside hydrolase from family 13; HGT, horizontal gene transfer; PDB, Protein Data Bank; SI, similarity index.
2 Keywords: -amylase
TIM barrel
protein evolution
helix capping
structural phylogeny
3 Address for correspondence and reprints: Gerard Pujadas, Unitat de Biotecnologia Computacional, Departament de Bioquímica i Biotecnologia, Universitat Rovira i Virgili, Tarragona 43005, Catalonia, Spain. E-mail: pujadas{at}quimica.urv.es
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