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Determining structure and function of steroid dehydrogenase enzymes by sequence analysis, homology modeling, and rational mutational analysis.

ABSTRACT: The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, approximately 300 have been characterized functionally, and the three-dimensional crystal structures of approximately 40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three-dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30-40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3beta-hydroxysteroid dehydrogenase isomerase (3beta-HSDI) has 30% sequence identity with a human UDP galactose 4-epimerase (UDPGE), a SCOR family enzyme for which an X-ray structure has been reported. Both UDPGE and 3-HSDI appear to trace their origins back to bacterial 3alpha,20beta-HSD. Combining three-dimensional structural information and sequence data on the 3alpha,20beta-HSD, UDPGE, and 3beta-HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3-HSDI. We also identified the residues most probably responsible for the isomerase activity of 3beta-HSDI. We test our predictions by specific mutations based on sequence analysis and our structure-based model.

SUBMITTER: Duax WL

PROVIDER: S-EPMC1635414 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Determining structure and function of steroid dehydrogenase enzymes by sequence analysis, homology modeling, and rational mutational analysis.

Duax William L WL Thomas James J Pletnev Vladimir V Addlagatta Anthony A Huether Robert R Habegger Lukas L Weeks Charles M CM

Annals of the New York Academy of Sciences 20051201

The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, approximately 300 have been characterized functionally, and the three-dimensional crystal structures of approximately 40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological fu ...[more]

PMID: 16467263

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Project description:BackgroundFucosidosis is an autosomal recessive lysosomal storage disease caused by defective alpha-L-fucosidase (FUCA1) activity, leading to the accumulation of fucose-containing glycolipids and glycoproteins in various tissues. Clinical features include angiokeratoma, progressive psychomotor retardation, neurologic signs, coarse facial features, and dysostosis multiplex.MethodsAll exons and flanking intron regions of FUCA1 were screened by direct sequencing to identify mutations and polymorphisms in three unrelated families with fucosidosis. Bioinformatics tools were then used to predict the impacts of novel alterations on the structure and function of proteins. Furthermore, the identified mutations were localized onto a 3D structure model using the DeepView Swiss-PdbViewer 4.1 software, which established a function-structure relationship of the FUCA1 proteins.ResultsFour novel mutations were identified in this study. Two patients (P1 and P2) in Families 1 and 2 who had the severe phenotype were homoallelic for the two identified frameshift mutations p.K57Sfs*75 and p.F77Sfs*55, respectively. The affected patient (P3) from Family 3, who had the milder phenotype, was heterozygous for the novel missense mutation p.G332E and the novel splice site mutation c.662+5g>c. We verified that this sequence variation did not correspond to a polymorphism by testing 50 unrelated individuals. Additionally, 16 FUCA1 polymorphisms were identified. The structure prediction analysis showed that the missense mutation p.G332E would probably lead to a significant conformational change, thereby preventing the expression of the FUCA1 protein indeed; the 3D structural model of the FUCA1 protein reveals that the glycine at position 332 is located near a catalytic nucleophilic residue. This makes it likely that the enzymatic function of the protein with p.G332E is severely impaired.ConclusionThese are the first FUCA1 mutations identified in Tunisia that cause the fucosidosis disease. Bioinformatics analysis allowed us to establish an approximate structure-function relationship for the FUCA1 protein, thereby providing better genotype/phenotype correlation knowledge.

Project description:Human P2Y receptors encompass at least eight subtypes of Class A G protein-coupled receptors (GPCRs), responding to adenine and/or uracil nucleotides. Using a BLAST search against the Homo sapiens subset of the SWISS-PROT and TrEMBL databases, we identified 68 proteins showing high similarity to P2Y receptors. To address the problem of low sequence identity between rhodopsin and the P2Y receptors, we performed a multiple-sequence alignment of the retrieved proteins and the template bovine rhodopsin, combining manual identification of the transmembrane domains (TMs) with automatic techniques. The resulting phylogenetic tree delineated two distinct subgroups of P2Y receptors: Gq-coupled subtypes (e.g., P2Y1) and those coupled to Gi (e.g., P2Y12). On the basis of sequence comparison we mutated three Tyr residues of the putative P2Y1 binding pocket to Ala and Phe and characterized pharmacologically the mutant receptors expressed in COS-7 cells. The mutation of Y306 (7.35, site of a cationic residue in P2Y12) or Y203 in the second extracellular loop selectively decreased the affinity of the agonist 2-MeSADP, and the Y306F mutation also reduced antagonist (MRS2179) affinity by 5-fold. The Y273A (6.48) mutation precluded the receptor activation without a major effect on the ligand-binding affinities, but the Y273F mutant receptor still activated G proteins with full agonist affinity. Thus, we have identified new recognition elements to further define the P2Y1 binding site and related these to other P2Y receptor subtypes. Following sequence-based secondary-structure prediction, we constructed complete models of all the human P2Y receptors by homology to rhodopsin. Ligand docking on P2Y1 and P2Y12 receptor models was guided by mutagenesis results, to identify the residues implicated in the binding process. Different sets of cationic residues in the two subgroups appeared to coordinate phosphate-bearing ligands. Within the P2Y1 subgroup these residues are R3.29, K/R6.55, and R7.39. Within the P2Y12 subgroup, the only residue in common with P2Y1 is R6.55, and the role of R3.29 in TM3 seems to be fulfilled by a Lys residue in EL2, whereas the R7.39 in TM7 seems to be substituted by K7.35. Thus, we have identified common and distinguishing features of P2Y receptor structure and have proposed modes of ligand binding for the two representative subtypes that already have well-developed ligands.

Dataset Information

Determining structure and function of steroid dehydrogenase enzymes by sequence analysis, homology modeling, and rational mutational analysis.

Publications

Determining structure and function of steroid dehydrogenase enzymes by sequence analysis, homology modeling, and rational mutational analysis.

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