3UZZ

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

Summary for 3UZZ

• Entry DOI: 10.2210/pdb3uzz/pdb
• Related: 3UZW 3UZX 3UZY
• Descriptor: 3-oxo-5-beta-steroid 4-dehydrogenase, NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, TESTOSTERONE, ... (6 entities in total)
• Functional Keywords: aldo-keto reductase, steroid and bile acid metabolism, catalytic tetrad mutant, tim barrel, oxidoreductase
• Biological source: Homo sapiens (human)
• Cellular location: Cytoplasm: P51857
• Total number of polymer chains: 2
• Total formula weight: 81313.67

Authors

Chen, M.,Christianson, D.W.,Penning, T.M. (deposition date: 2011-12-07, release date: 2012-03-21, Last modification date: 2023-09-13)

Primary citation

Chen, M.,Drury, J.E.,Christianson, D.W.,Penning, T.M.
Conversion of Human Steroid 5beta-Reductase (AKR1D1) into 3β-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: EXAMPLE OF PERFECT ENZYME ENGINEERING.
J.Biol.Chem., 287:16609-16622, 2012

PubMed Abstract: Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(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 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(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 Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-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.

PubMed: 22437839
DOI: 10.1074/jbc.M111.338780

Experimental method

X-RAY DIFFRACTION (1.82 Å)