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. ? ? ?
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