Characterization of Disease-related 5β-Reductase (AKR1D1) Mutations Reveals Their Potential to Cause Bile Acid Deficiency

Abstract

Bile acid deficiency is a serious syndrome in newborns that can result in death if untreated. 5␤-Reductase deficiency is one form of bile acid deficiency and is characterized by dramatically decreased levels of physiologically active 5␤-reduced bile acids. AKR1D1 (aldo-keto reductase 1D1) is the only known human enzyme that stereo-specifically reduces the ⌬ 4 double bond in 3-keto steroids and sterols to yield the 5␤-hydrogenated product. Analysis of the AKR1D1 gene in five patients with 5␤-reductase deficiency revealed five different mutations resulting in an amino acid substitution in the protein. To investigate a causal role for these observed point mutations in AKR1D1 in 5␤-reductase deficiency, we characterized their effect on enzymatic properties. Attempts to purify mutant enzymes by overexpression in Escherichia coli only yielded sufficient amounts of the P133R mutant for further characterization. This enzyme displayed a highly reduced K m and V max reminiscent of uncompetitive kinetics with 4-cholesten-7␣-ol-3-one as substrate. In addition, this mutant displayed no change in cofactor affinity but was more thermolabile in the absence of NADPH as judged by CD spectroscopy. All mutants were compared following expression in HEK 293 cells. Although these enzymes were equally expressed based on mRNA levels, protein expression and functional activity were dramatically reduced. Cycloheximide treatment also revealed that several of the expressed mutants were less stable. Our findings show that the reported mutations in AKR1D1 in patients with 5␤-reductase lead to significantly decreased levels of active enzyme and could be causal in the development of bile acid deficiency syndrome.AKR1D1 (aldo-keto reductase 1D1) is implicated as one of the key enzymes in bile acid biosynthesis. The enzyme is the only known human ⌬ 4 -3-ketosteroid 5␤-reductase and catalyzes the reduction of the ⌬ 4 -3-ketosteroid to form the A/B cis ring structure, utilizing NADPH as a cofactor (1). The ⌬ 4 -3-ketosteroid functionality is common to all steroid hormones except the estrogens, and the C4-C5 double bond can be further reduced in a stereo-specific manner. 5␣-Reduction of testosterone to 5␣-dihydrotestosterone results in increased androgen receptor activation (2, 3), whereas 5␤-reduction of progesterone to 5␤-pregnane-3,20-dione results in activation of the pregnane X receptor (4) and constitutive active/androstane receptor (5). In bile acid biosynthesis, AKR1D1 reduces ⌬ 4 -cholesten-

Genetic Variation in Aldo-Keto Reductase 1D1 (AKR1D1) Affects the Expression and Activity of Multiple Cytochrome P450s

“…The absence of AKR1D1 coding variation likely reflects the important function of this gene in regulating bile acid levels. Human AKR1D1 deficiency is rare and patients with AKR1D1 deficiency accumulate bile acid intermediates and lack downstream functional bile acids, and leads to hepatotoxicity (Drury et al, 2010). For example, AKR1D1 sequence variation was reported in infant patients presenting with primary bile acid deficiency (Gonzales et al, 2004) who had a compound heterozygous status represented by two nonconservative missense mutations, P133R (C467G) in exon 4 and R261C (C850T) in exon 7.…”

Rate of steroid double-bond reduction catalysed by the human steroid 5β-reductase (AKR1D1) is sensitive to steroid structure: implications for steroid metabolism and bile acid synthesis

“…The critical involvement of AKR1D1 in bile acid synthesis is highlighted by the clinical observation that a disorder in bile acid synthesis is associated with congenital deficiencies in the AKR1D1 gene [44,45]. We have shown five AKR1D1 mutations identified in patients to be causal for the condition as the mutations strongly affected AKR1D1 function [46]. With a low k cat value of 2 min −1 , AKR1D1 is estimated to be abundantly expressed as 1 % of soluble protein in the liver to satisfy the demand of synthesizing several hundred milligrams of bile acids daily.…”

Advances in Evaluation of Chronic Diarrhea in Infants

“…Certainly, inherited and non-inherited conditions associated with cholestatic liver disease and surgical resection of the distal bowel may result in steatorrhea, and as many as 6 rare selective autosomal recessive congenital defects of bile acid synthesis have been described. 98 …”

“…98,99 Patients with STXBP2 mutations also display hemophagocytic lympho-histiyocytosis (HLH) type 5, requiring bone marrow transplantation (BMT) to treat their HLH. However, BMT has no effect on the intestinal phenotype and PN is still required post-transplantation.…”