Chemico-Biological Interactions p. 250 - 254 (2011)
Update date:2022-09-26
Topics:
Mindnich, Rebekka
Drury, Jason E.
Penning, Trevor M.
The stereospecific 5β-reduction of Δ4-3-ketosterols is very difficult to achieve chemically and introduces a 90° bend between ring A and B of the planar steroid. In mammals, the reaction is catalyzed by steroid 5β-reductase, a member of the aldo-keto reductase (AKR) family. The human enzyme, AKR1D1, plays an essential role in bile-acid biosynthesis since the 5β-configuration is required for the emulsifying properties of bile. Deficient 5β-reductase activity can lead to cholestasis and neo-natal liver failure and is often lethal if it remains untreated. In five patients with 5β-reductase deficiency, sequencing revealed individual, non-synonymous point mutations in the AKR1D1 gene: L106F, P133R, G223E, P198L and R261C. However, mapping these mutations to the AKR1D1 crystal structure failed to reveal any obvious involvement in substrate or cofactor binding or catalytic mechanism, and it remained unclear whether these mutations could be causal for the observed disease. We analyzed the positions of the reported mutations and found that they reside in highly conserved portions of AKR1D1 and hypothesized that they would likely lead to changes in protein folding, and hence enzyme activity. Attempts to purify the mutant enzymes for further characterization by over-expression in Escherichia coli yielded sufficient amounts of only one mutant (P133R). This enzyme exhibited reduced Km and kcat values with the bile acid intermediate Δ4-cholesten-7α- ol-3-one as substrate reminiscent of uncompetitive inhibition. In addition, P133R displayed no change in cofactor affinity but was more thermolabile as judged by CD-spectroscopy. When all AKR1D1 mutants were expressed in HEK 293 cells, protein expression levels and enzyme activity were dramatically reduced. Furthermore, cycloheximide treatment revealed decreased stability of several of the mutants compared to wild type. Our data show, that all five mutations identified in patients with functional bile acid deficiency strongly affected AKR1D1 enzyme functionality and therefore may be causal for this disease.
View MoreShandong Zhongcheng Barium Salt Co., Ltd
Contact:+86-15725732638
Address:No.29 baoxi road, hi-tech zone, zibo, shandong
Shandong Ailitong New Material Co.,Ltd
Contact:+86-536-3226266
Address:zhongjia village, putong town , qingzhou city,Shandong Province,China
Tianjin Ingenochem Technology Co.,Ltd
Contact:+86-22-23677060
Address:Hitech Green Industry Park K2-9-602, Nankai district
JIN TAN CHENG'EN CHEMICAL CO.,LTD.
Contact:86-519-82116250
Address:NO.102,village Dongfang,conomic development zone
Shao Xing Empire Import&Export CO.,ltd
Contact:86-575-82127757
Address:11#, Weiwu Road, Shangyu Industrial Park, Hangzhou Bay, Hangzhou, Zhejiang Province, China
Doi:10.1021/acs.orglett.8b02233
(2018)Doi:10.1021/ac60295a019
(1970)Doi:10.1016/S0040-4039(01)98221-8
(1970)Doi:10.1021/jo01322a017
(1979)Doi:10.1007/s00706-014-1368-5
(2015)Doi:10.1016/S0040-4020(00)00403-8
(2000)