Institute of Experimental Genetics, Genome Analysis Center, GSF-National Research Center for Environment and Health, Neuherberg, Germany
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Abstract |
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Introduction |
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The low degree of amino acid conservation is, however, a major obstacle for reconstructing evolutionary relationships within the SCAD family. Attempts to identify a robust deep-branching pattern by analyzing multiple-sequence alignments were unsuccessful (Persson et al. 1999
). To escape from the twilight zone of protein sequence alignments (Rost 1999
) we decided to exploit the large numbers of atomic coordinates available for SCAD family members, assuming that 3D structures are more conserved than primary sequence and should also reflect phylogenetic patterns. This approach has been pioneered by Rossmann et al. (Eventoff and Rossmann 1975
; Matthews and Rossmann 1985
), as well as by Johnson, Sutcliffe, and Blundell (1990), and more recently by Bujnicki (Bujnicki 1999, 2000
), who used 3D comparisons to determine the phylogeny of protein families. Examination of a limited number of Rossmann-fold dehydrogenases (but no SCAD protein) demonstrated the general feasibility and reliability of the method, but because of the small data set, only very general conclusions were possible at that time. Since then the number of available 3D structures has expanded rapidly, including a large number of SCAD proteins, beginning with rat dihydropteridine reductase in 1992 (Varughese et al. 1992
). We used these data to determine a reliable evolutionary tree of the SCAD proteins. In this way it was possible for the first time to get a comprehensive structure-based insight into structural relationships within this important family.
An important further objective was to develop a method to integrate proteins with unknown 3D structure into the structure-based scaffold, using multiple-sequence alignments. This approach was then applied to the 17beta-hydroxysteroid dehydrogenases (17beta-HSDs), one of the best characterized subgroups of the SCAD family that activates and inactivates vertebrate sex hormones by reduction and oxidation at position 17 of the steroid backbone (Peltoketo et al. 1999
; Duax, Ghosh, and Pletnev 2000
; Labrie et al. 2000b;
Adamski and Jakob 2001
). Ten different isoforms of 17beta-HSDs have been described so far in vertebrates, and the number is still growing. Isotypes of 17beta-HSDs differ in tissue distribution, substrate specificity, and preferred direction of catalysis. They generally show a low level of sequence conservation (<30% identity). Dysregulation of several members of this subfamily has been implicated in a variety of human diseases, such as pseudohermaphroditism (17beta-HSD3: Geissler et al. 1994
), Zellweger-like syndrome (17beta-HSD4 = MFP2: van Grunsven et al. 1998
), polycystic kidney disease (17beta-HSD8 = HKE6: Fomitcheva et al. 1998
), Alzheimer disease (17beta-HSD10: He et al. 1999
), osteoporosis (17beta-HSD4: Jakob et al. 1997
; Janssen et al. 1999
), and a wide variety of endocrine related cancers (17beta-HSD1, 17beta-HSD2, and others: English et al. 2000
; Labrie et al. 2000a;
Sasano et al. 2000
).
Phylogenetic analysis of the 17beta-HSDs has so far been limited in scope and included only the closest relatives of some isozymes (retinol dehydrogenases, 11beta-hydroxysteroid dehydrogenases: Krozowski 1992, 1994
; Baker 1996, 1998, 2001
; Lanisnik Rizner et al. 1999
) or an arbitrary selection of other SCAD proteins (Grundy et al. 1997
). The small number of proteins surveyed and the lack of an obvious outgroup for the 17beta-HSDs seriously confounded the analysis, hampering any reliable evolutionary reconstruction. As a detailed understanding of the relationships of different 17beta-HSDs is a prerequisite for functional assignments of new HSDs (Breitling et al. 2001a, 2001b
) and for the rational design of drugs targeting selected isoforms (Tremblay and Poirier 1998
; Ngatcha, Luu-The, and Poirier 2000
; Penning et al. 2001
; Poirier et al. 2001
), we decided to use the structural data for a comprehensive reevaluation of the evolutionary history of 17beta-HSDs.
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Materials and Methods |
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Results |
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Discussion |
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Integration of Multiple-sequence Alignments and the Structure-based Tree
The comparison of atomic coordinates of members of the SCAD family yielded not only a phylogenetic tree but also a protein sequence alignment that reliably identified equivalent amino acid residues. Our results show that a maximum parsimony analysis of this alignment determined a phylogenetic pattern that was largely congruent with the structure-based tree. Because of this, it was possible to use sequence-based algorithms to integrate proteins with unknown 3D structure into the evolutionary scenario. The placement of the 17beta-HSDs within the SCAD tree (which in every case reflects the placement of a large number of homologous sequences examined in parallel) indicates that the results are most likely not distorted by long-branch attraction, the most common problem of parsimony tree reconstructions (Kuhner and Felsenstein 1994
). This might be because of the uniform rate of evolution on all branches as indicated by the structure-based trees, as well as by the use of a large number of homologous sequences that are expected to disrupt very long branches. The obtained topology is therefore likely to be very reliable.
Reclassification of 17beta-HSDs
From previous sequence-based analysis it was obvious that 17beta-HSD activity arose convergently in all 17beta-HSD isoforms (Baker 1996
). Thus, the subsumption of 17beta-HSDs in two subgroups arising independently in different regions of the SCAD tree was unexpected. It indicates that the aptitude to develop an activity toward position 17 of steroid substrates arose only twice within the SCAD family. Both groups of 17beta-HSDs include oxidative and reductive enzymes. In both the cases the oxidative (steroid inactivating) enzymes form a separate subgroup. This agrees well with the hypothesis of a shared ancestral substrate for these proteins: retinols in the case of 17beta-HSD type 2, 6, and 9 (Baker 1998
) and fatty-acids in the case of 17beta-HSD type 4, 8, and 10 (Baker 2001
). The functional and structural connection between the oxidative subgroups and their respective reductive paralogues has yet to be determined.
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Conclusions |
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Abbreviations: 17beta-HSD, 17beta-hydroxysteroid dehydrogenase; SCAD, short-chain alcohol dehydrogenase.
Keywords: short-chain alcohol dehydrogenases
17beta-hydroxysteroid dehydrogenases
protein structure
phylogeny
Address for correspondence and reprints: Jerzy Adamski, Institute of Experimental Genetics, Genome Analysis Center, GSF-National Research Center for Environment and Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany. adamski{at}gsf.de
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References |
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