Human Aldo-Keto Reductase (AKR) 1D1 (steroid 5?-reductase) catalyzes the conversion of all ?4-3- ketosteroids to form 5? -dihydrosteroids, a first step in the elimination of steroid hormones, and an essential step in the synthesis of all bile-acids. Point mutations in AKR1D1 have been associated with 5?-reductase deficiency, which results in fatal neonatal hepatitis and cholestasis. Whether these mutations are the molecular basis of this disease has not been established. AKR1D1 then couples with a series of AKR1C isoforms (3-ketosteroid reductases) to yield 5?,3a - and 5?,3? - tetrahydrosteroids (THS). The 5?,3a-configuration common to all bile-acids is likely formed when AKR1D1 couples to AKR1C4. Whether these two genes from the AKR superfamily are responsible for bile acid formation remains to be formally proven. AKR1C isoforms are also highly polymorphic and single nucleotide polymorphisms (SNPs) that impact enzyme activity or the stereochemistry of product formation could adversely affect bile-acid biosynthesis and hence liver function.
In Aim#1, we will elucidate the mechanism of steroid double-bond reduction catalyzed by homogeneous recombinant AKR1D1 using kinetic isotope effects and transient state kinetics. We will test the hypothesis that a single amino acid substitution in the conserved AKR catalytic tetrad shifts the pKb of the catalytic tyrosine and this is responsible for steroid double-bond reduction.
In Aim#2, we will use site-directed mutagenesis to test the hypothesis that inherited AKR1D1 mutations affect structure (CD-spectra), stability (CD-melt curves) or function (steady state kinetics) and are causal in 5? - reductase deficiency.
In Aim#3, we will test whether AKR1D1 preferentially couples to a specific AKR1C isoform to catalyze the formation of 5?,3a-THS bile acid precursors using reconstituted systems in vitro, transfection studies in AKR null cells, and by phenotyping reactions in human hepatocytes.
In Aim#4, we will conduct site-directed mutagenesis informed by crystallographic structures to identify the discrete amino acids in AKR1C4 that determine its preference for C27 5? - dihydrosteroid bile-acid precursors observed in vitro. Non-synonymous high penetrance SNPs will be introduced into AKR1C4 to determine whether these allelic variants affect enzyme function or stereochemical outcome, and pre-dispose individuals to attenuated or aberrant bile-acid formation. These studies will advance our knowledge of hepatic steroid hormone metabolism and bile-acid biosynthesis, and could impact the diagnosis and therapy of bile-acid deficiencies. The production of bile-acids is essential for normal liver function and absorption of fats and fat-soluble vitamins from the G.I. tract. Individual susceptibility to bile-acid deficiency may result from inherited mutations or common variants in Aldo-Keto Reductase genes involved in bile-acid synthesis. This application will establish whether these genetic changes result in abnormal bile-acid production and are causal in these deficiencies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK047015-18
Application #
8131701
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Sechi, Salvatore
Project Start
1994-05-01
Project End
2013-08-31
Budget Start
2011-09-01
Budget End
2013-08-31
Support Year
18
Fiscal Year
2011
Total Cost
$262,974
Indirect Cost
Name
University of Pennsylvania
Department
Pharmacology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Penning, Trevor M (2016) Single-molecule enzymology of steroid transforming enzymes: Transient kinetic studies and what they tell us. J Steroid Biochem Mol Biol 161:5-12
Penning, Trevor M (2015) The aldo-keto reductases (AKRs): Overview. Chem Biol Interact 234:236-46
Chen, Mo; Jin, Yi; Penning, Trevor M (2015) In-Depth Dissection of the P133R Mutation in Steroid 5?-Reductase (AKR1D1): A Molecular Basis of Bile Acid Deficiency. Biochemistry 54:6343-51
Chen, Mo; Jin, Yi; Penning, Trevor M (2015) The rate-determining steps of aldo-keto reductases (AKRs), a study on human steroid 5?-reductase (AKR1D1). Chem Biol Interact 234:360-5
Penning, Trevor M; Chen, Mo; Jin, Yi (2015) Promiscuity and diversity in 3-ketosteroid reductases. J Steroid Biochem Mol Biol 151:93-101
Chen, Mo; Penning, Trevor M (2014) 5?-Reduced steroids and human ?(4)-3-ketosteroid 5?-reductase (AKR1D1). Steroids 83:17-26
Penning, Trevor M (2014) Androgen biosynthesis in castration-resistant prostate cancer. Endocr Relat Cancer 21:T67-78
Rižner, Tea Lanišnik; Penning, Trevor M (2014) Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism. Steroids 79:49-63
Jin, Yi; Chen, Mo; Penning, Trevor M (2014) Rate of steroid double-bond reduction catalysed by the human steroid 5?-reductase (AKR1D1) is sensitive to steroid structure: implications for steroid metabolism and bile acid synthesis. Biochem J 462:163-71
Barski, Oleg A; Mindnich, Rebekka; Penning, Trevor M (2013) Alternative splicing in the aldo-keto reductase superfamily: implications for protein nomenclature. Chem Biol Interact 202:153-8

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