Document Detail


Engineering steroid hormone specificity into aldo-keto reductases.
MedLine Citation:
PMID:  11306084     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), 20alpha-HSDs, as well as the 5beta-reductases. To elucidate the molecular determinants of steroid hormone recognition we used rat liver 3alpha-HSD (AKR1C9) as a starting structure to engineer either 5beta-reductase or 20alpha-HSD activity. 5beta-Reductase activity was introduced by a single point mutation in which the conserved catalytic His (H117) was mutated to Glu117. The H117E mutant had a k(cat) comparable to that for homogeneous rat and human liver 5beta-reductases. pH versus k(cat) profiles show that this mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH(2)(+) character facilitates enolization of the Delta(4)-3-ketosteroid and subsequent hydride transfer to C5. Since 5beta-reductase precedes 3alpha-HSD in steroid hormone metabolism it is likely that this metabolic pathway arose by gene duplication and point mutation. 3alpha-HSD is positional and stereospecific for 3-ketosteroids and inactivates androgens. The enzyme was converted to a robust 20alpha-HSD, which is positional and stereospecific for 20-ketosteroids and inactivates progesterone, by the generation of loop-chimeras. The shift in log(10)(k(cat)/K(m)) from androgens to progestins was of the order of 10(11). This represents a rare example of how steroid hormone specificity can be changed at the enzyme level. Protein engineering with predicted outcomes demonstrates that the molecular determinants of steroid hormone recognition in AKRs will be ultimately rationalized.
Authors:
T M Penning; H Ma; J M Jez
Related Documents :
21527634 - Structural and morphological characterization of aggregated species of α-synuclein ind...
7945794 - Primate relaxin: synthesis of gorilla and rhesus monkey relaxins.
2950274 - Isolation and structural analysis of the circulating human cardiodilatin (alpha anp).
2862084 - Amino acids release somatostatin-like immunoreactivity from isolated rat glomeruli.
18584594 - Production of biosurfactants from sugar alcohols and vegetable oils catalyzed by lipase...
23787314 - The variation of volatile fatty acid compositions in sewer length, and its effect on th...
Publication Detail:
Type:  In Vitro; Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Chemico-biological interactions     Volume:  130-132     ISSN:  0009-2797     ISO Abbreviation:  Chem. Biol. Interact.     Publication Date:  2001 Jan 
Date Detail:
Created Date:  2001-04-18     Completed Date:  2001-05-10     Revised Date:  2014-03-18    
Medline Journal Info:
Nlm Unique ID:  0227276     Medline TA:  Chem Biol Interact     Country:  Ireland    
Other Details:
Languages:  eng     Pagination:  659-71     Citation Subset:  IM    
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
20-Hydroxysteroid Dehydrogenases / chemistry,  genetics,  metabolism
20-alpha-Hydroxysteroid Dehydrogenase
3-Hydroxysteroid Dehydrogenases / chemistry,  genetics,  metabolism
3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
Alcohol Oxidoreductases / chemistry*,  genetics*,  metabolism
Animals
Catalytic Domain / genetics
Humans
Hydrogen-Ion Concentration
Kinetics
Liver / enzymology
Models, Molecular
Oxidoreductases / chemistry,  genetics,  metabolism
Point Mutation
Protein Conformation
Protein Engineering
Rats
Recombinant Fusion Proteins / chemistry,  genetics,  metabolism
Steroids / metabolism*
Substrate Specificity
Grant Support
ID/Acronym/Agency:
DK47015/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Recombinant Fusion Proteins; 0/Steroids; EC 1.-/Oxidoreductases; EC 1.1.-/3-Hydroxysteroid Dehydrogenases; EC 1.1.-/Alcohol Oxidoreductases; EC 1.1.1.-/20-Hydroxysteroid Dehydrogenases; EC 1.1.1.149/20-alpha-Hydroxysteroid Dehydrogenase; EC 1.1.1.21/carbonyl reductase (NADPH); EC 1.1.1.50/3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); EC 1.3.99.6/3-oxo-5 beta-steroid delta 4-dehydrogenase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for sub...
Next Document:  Functional genomic studies of aldo-keto reductases.