3UZZ: Crystal structure of 5beta-reductase (AKR1D1) E120H mutant in complex with NADP+ and delta4-androstenedione

Citation:
Abstract
Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5beta-reduction of Delta(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5beta-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5alpha-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3beta-HSD as opposed to a 3alpha-HSD. The catalytic efficiency achieved for 3beta-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5beta-dihydrotestosterone, and Delta(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Delta(4)-double bond and confers 3beta-HSD activity on the 5beta-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its alpha-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.
PDB ID: 3UZZDownload
MMDB ID: 98195
PDB Deposition Date: 2011/12/7
Updated in MMDB: 2017/11
Experimental Method:
x-ray diffraction
Resolution: 1.82  Å
Source Organism:
Similar Structures:
Biological Unit for 3UZZ: monomeric; determined by author and by software (PISA)
Molecular Components in 3UZZ
Label Count Molecule
Protein (1 molecule)
1
3-oxo-5-beta-steroid 4-dehydrogenase(Gene symbol: AKR1D1)
Molecule annotation
Chemicals (3 molecules)
1
1
2
1
3
1
* Click molecule labels to explore molecular sequence information.

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