Dhagat Urmi, Endo Satoshi, Mamiya Hiroaki, Hara Akira, El-Kabbani Ossama
Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):198-204. doi: 10.1107/S0907444909051464. Epub 2010 Jan 22.
Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo-keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17)alpha-hydroxysteroids. The Y224D mutation of AKR1C21 reduced the K(m) value for NADP(H) by up to 80-fold and completely reversed the 17alpha stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3 A resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3alpha-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type.
小鼠3(17)α-羟基类固醇脱氢酶(AKR1C21)是唯一一种催化3-和17-酮类固醇立体特异性还原为相应3(17)α-羟基类固醇的醛酮还原酶。AKR1C21的Y224D突变使NADP(H)的K(m)值降低了80倍，并完全逆转了该酶的17α立体特异性。Y224D突变体在2.3 Å分辨率下的晶体结构表明，该突变导致柔性环B的构象发生变化，包括V形凹槽，这是野生型AKR1C21活性位点结构的独特特征，由Tyr224和Trp227的侧链形成。此外，参与形成辅酶结合安全带的残基的突变(Y224F和Q222N)与野生型相比，在3α-羟基/3-酮类固醇和17-羟基/酮类固醇的动力学常数上表现出类似的变化。
