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Determining Structure and Function of Steroid Dehydrogenase Enzymes by Sequence Analysis, Homology Modeling, and Rational Mutational Analysis
Author(s) -
DUAX WILLIAM L.,
THOMAS JAMES,
PLETNEV VLADIMIR,
ADDLAGATTA ANTHONY,
HUETHER ROBERT,
HABEGGER LUKAS,
WEEKS CHARLES M.
Publication year - 2005
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1336.015
Subject(s) - homology modeling , biology , computational biology , sequence alignment , isomerase , sequence analysis , genetics , oxidoreductase , enzyme , biochemistry , peptide sequence , gene
A bstract : The short‐chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, ∼300 have been characterized functionally, and the three‐dimensional crystal structures of ∼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 3β‐hydroxysteroid dehydrogenase isomerase (3β‐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 3α,20β‐HSD. Combining three‐dimensional structural information and sequence data on the 3α,20β‐HSD, UDPGE, and 3β‐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 3β‐HSDI. We test our predictions by specific mutations based on sequence analysis and our structure‐based model.