Download MendeleyConversion of Human Steroid 5beta-Reductase (AKR1D1) into 3¦Â-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: EXAMPLE OF PERFECT ENZYME ENGINEERING.
Chen, M., Drury, J.E., Christianson, D.W., Penning, T.M.(2012) J Biol Chem 287: 16609-16622
- PubMed: 22437839
- DOI: https://doi.org/10.1074/jbc.M111.338780
- Primary Citation of Related Structures:
3UZW, 3UZX, 3UZY, 3UZZ - PubMed Abstract:
Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5¦Â-reduction of ¦¤(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 5¦Â-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5¦Á-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3¦Â-HSD as opposed to a 3¦Á-HSD. The catalytic efficiency achieved for 3¦Â-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5¦Â-dihydrotestosterone, and ¦¤(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 ¦¤(4)-double bond and confers 3¦Â-HSD activity on the 5¦Â-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its ¦Á-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.
Organizational Affiliation:
Department of Pharmacology and Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
