Giménez-Dejoz Joan, Weber Susanne, Barski Oleg A, Möller Gabriele, Adamski Jerzy, Parés Xavier, Porté Sergio, Farrés Jaume
Department of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain.
Institute of Experimental Genetics, Genome Analysis Center, Helmholtz Zentrum Muenchen, 85764 Neuherberg, Germany.
Chem Biol Interact. 2017 Oct 1;276:182-193. doi: 10.1016/j.cbi.2017.03.007. Epub 2017 Mar 18.
Aldo-keto reductases (AKRs) are distributed in three families and multiple subfamilies in mammals. The mouse Akr1b3 gene is clearly orthologous to human AKR1B1, both coding for aldose reductase, and their gene products show similar tissue distribution, regulation by osmotic stress and kinetic properties. In contrast, no unambiguous orthologs of human AKR1B10 and AKR1B15.1 have been identified in rodents. Although two more AKRs, AKR1B7 and AKR1B8, have been identified and characterized in mouse, none of them seems to exhibit properties similar to the human AKRs. Recently, a novel mouse AKR gene, Akr1b16, was annotated and the respective gene product, AKR1B16 (sharing 83% and 80% amino acid sequence identity with AKR1B10 and AKR1B15.1, respectively), was expressed as insoluble and inactive protein in a bacterial expression system. Here we describe the expression and purification of a soluble and enzymatically active AKR1B16 from E. coli using three chaperone systems. A structural model of AKR1B16 allowed the estimation of its active-site pocket volume, which was much wider (402 Å) than those of AKR1B10 (279 Å) and AKR1B15.1 (60 Å). AKR1B16 reduced aliphatic and aromatic carbonyl compounds, using NADPH as a cofactor, with moderate or low activity (highest k values around 5 min). The best substrate for the enzyme was pyridine-3-aldehyde. AKR1B16 showed poor inhibition with classical AKR inhibitors, tolrestat being the most potent. Kinetics and inhibition properties resemble those of rat AKR1B17 but differ from those of the human enzymes. In addition, AKR1B16 catalyzed the oxidation of 17β-hydroxysteroids in a NADP-dependent manner. These results, together with a phylogenetic analysis, suggest that mouse AKR1B16 is an ortholog of rat AKR1B17, but not of human AKR1B10 or AKR1B15.1. These human enzymes have no counterpart in the murine species, which is evidenced by forming a separate cluster in the phylogenetic tree and by their unique activity with retinaldehyde.
醛酮还原酶（AKRs）在哺乳动物中分布于三个家族和多个亚家族。小鼠Akr1b3基因与人AKR1B1明显是直系同源基因，二者均编码醛糖还原酶，其基因产物表现出相似的组织分布、受渗透应激调控以及动力学特性。相比之下，在啮齿动物中尚未鉴定出与人AKR1B10和AKR1B15.1明确的直系同源基因。尽管在小鼠中又鉴定并表征了另外两种AKRs，即AKR1B7和AKR1B8，但它们似乎均未表现出与人类AKRs相似的特性。最近，一个新的小鼠AKR基因Akr1b16被注释，其相应的基因产物AKR1B16（分别与AKR1B10和AKR1B15.1共享83%和80%的氨基酸序列同一性）在细菌表达系统中以不溶性和无活性蛋白形式表达。在此，我们描述了使用三种伴侣系统从大肠杆菌中表达和纯化可溶性且具有酶活性的AKR1B16。AKR1B16的结构模型使我们能够估计其活性位点口袋体积，该体积比AKR1B10（279 Å）和AKR1B15.1（60 Å）的活性位点口袋体积宽得多（402 Å）。AKR1B16以NADPH作为辅因子，还原脂肪族和芳香族羰基化合物，活性适中或较低（最高k值约为5 min⁻¹）。该酶的最佳底物是吡啶 - 3 - 醛。AKR1B16对经典的AKR抑制剂表现出较弱的抑制作用，托瑞司他是最有效的抑制剂。其动力学和抑制特性类似于大鼠AKR1B17，但与人类酶不同。此外，AKR1B16以NADP依赖的方式催化17β - 羟基类固醇的氧化。这些结果连同系统发育分析表明，小鼠AKR1B16是大鼠AKR1B17的直系同源基因，而非人类AKR1B10或AKR1B15.1的直系同源基因。这些人类酶在鼠类物种中没有对应物，这在系统发育树中形成一个单独的簇以及它们对视黄醛具有独特活性中得到证明。
