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Molecular Mechanism Study on Stereo-Selectivity of Α Or Β Hydroxysteroid Dehydrogenases

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Molecular Mechanism Study on Stereo-Selectivity of Α Or Β Hydroxysteroid Dehydrogenases crystals Article Molecular Mechanism Study on Stereo-Selectivity of α or β Hydroxysteroid Dehydrogenases Miaomiao Gao, Kaili Nie, Meng Qin, Haijun Xu, Fang Wang and Luo Liu * Beijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing 100029, China; [email protected] (M.G.); [email protected] (K.N.); [email protected] (M.Q.); [email protected] (H.X.); [email protected] (F.W.) * Correspondence: [email protected] Abstract: Hydroxysteroid dehydrogenases (HSDHs) are from two superfamilies of short-chain de- hydrogenase (SDR) and aldo–keto reductase (AKR). The HSDHs were summarized and classified according to their structural and functional differences. A typical pair of enzymes, 7α–hydroxysteroid dehydrogenase (7α–HSDH) and 7β–hydroxysteroid dehydrogenase (7β–HSDH), have been reported before. Molecular docking of 7-keto–lithocholic acid(7–KLA) to the binary of 7β–HSDH and nicoti- namide adenine dinucleotide phosphate (NADP+) was realized via YASARA, and a possible binding model of 7β–HSDH and 7–KLA was obtained. The α side of 7–KLA towards NADP+ in 7β–HSDH, while the β side of 7–KLA towards nicotinamide adenine dinucleotide (NAD+) in 7α–HSDH, made the orientations of C7–OH different in products. The interaction between Ser193 and pyrophosphate of NAD(P)+ [Ser193–OG··· 3.11Å··· O1N–PN] caused the upturning of PN–phosphate group, which formed a barrier with the side chain of His95 to make 7–KLA only able to bind to 7β–HSDH with + α side towards nicotinamide of NADP . A possible interaction of Tyr253 and C24 of 7–KLA may contribute to the formation of substrate binding orientation in 7β–HSDH. The results of sequence Citation: Gao, M.; Nie, K.; Qin, M.; alignment showed the conservation of His95, Ser193, and Tyr253 in 7β–HSDHs, exhibiting a sig- Xu, H.; Wang, F.; Liu, L. Molecular nificant difference to 7α–HSDHs. The molecular docking of other two enzymes, 17β–HSDH from Mechanism Study on Stereo- the SDR superfamily and 3(17)α–HSDH from the AKR superfamily, has furtherly verified that the Selectivity of α or β Hydroxysteroid stereospecificity of HSDHs was related to the substrate binding orientation. Dehydrogenases. Crystals 2021, 11, 224. https://doi.org/10.3390/ Keywords: hydroxysteroid dehydrogenases; short-chain dehydrogenase superfamily; aldo–keto cryst11030224 reductases superfamily; stereospecificity Academic Editor: Borislav Angelov Received: 29 January 2021 1. Introduction Accepted: 21 February 2021 Published: 25 February 2021 Hydroxysteroid dehydrogenases (HSDHs) are a type of nicotinamide adenine dinu- cleotide (phosphate) NAD(H)/NADP(H)-dependent oxidoreductase that can specifically Publisher’s Note: MDPI stays neutral catalyze the reduction of carbonyl to hydroxy of steroid-skeleton structures such as steroid with regard to jurisdictional claims in hormones and bile acids, in addition to a reversible reaction. All HSDHs can be divided published maps and institutional affil- into two different protein superfamilies—the short-chain dehydrogenase/reductase (SDR) iations. superfamily and aldo–keto reductase (AKR) superfamily [1,2]. A variety of HSDHs from different species have been reported. Most of them belong to the SDR superfamily, such as 7α–HSDH (EC 1.1.1.159), 7β–HSDH (EC 1.1.1.201), and 11β–HSDH (EC 1.1.1.146), which have been identified in Escherichia coli, Collinsella aerofaciens, and Homo sapiens, respectively [3–5], usually with a length of 250–350 amino acids [6]. They all have a Copyright: © 2021 by the authors. β α Licensee MDPI, Basel, Switzerland. core composed of seven parallel -sheets and several -helices wraps around the core This article is an open access article (Figure1 ), which form two typical domains named Rossmann fold (each composed of distributed under the terms and two βαβ units) for coenzyme binding [6,7]. Aldo–keto reductases (AKRs) catalyze the conditions of the Creative Commons carbonyl reduction reaction with a wide range of substrate selectivity, including sugar Attribution (CC BY) license (https:// aldehydes, keto-steroids, keto-prostaglandins, and retinals [8]. These enzymes are usually creativecommons.org/licenses/by/ with a length of 320 residues on average, forming an (α/β)8-barrel fold [8,9]. According 4.0/). to the differences of sources, HSDHs such as 3α–HSDH (EC 1.1.1.145), 17α–HSDH (EC Crystals 2021, 11, 224. https://doi.org/10.3390/cryst11030224 https://www.mdpi.com/journal/crystals Crystals 2021, 11, 224 2 of 25 differences of sources, HSDHs such as 3α–HSDH (EC 1.1.1.145), 17α–HSDH (EC 1.1.1.148) and 17β–HSDH (EC 1.1.1.51, EC 1.1.1.357, EC 1.1.1.270, etc.) usually belong to two super- families. For example, 3α–HSDHs from Comamonas testosteron [10] and Pseudomonas sp. [11] belong to the SDR superfamily, while from Rattus norvegicus [12] and Homo sapiens [13] belong to the AKR superfamily (Figure 2). This article reviews the HSDHs from SDR and AKR superfamilies that were identi- Crystals 2021, 11, 224 fied in mammals and bacteria and then classifies the HSDHs according to their functions2 of 24 in steroid hormone metabolism or bile acid biosynthesis. The differences of NAD(P)(H)- binding models to enzymes and enzymatic reaction characteristics between SDR and AKR 1.1.1.148)superfamilies and 17wereβ–HSDH also summarized. (EC 1.1.1.51, AEC semi-flexible 1.1.1.357, ECmolecular 1.1.1.270, docking etc.) usually was used belong for fur- to twother superfamilies.exploration of differences For example, of substrat 3α–HSDHse binding from modelComamonas to enzyme–coenzyme testosteron [10] and complexPseu- domonasbetweensp. SDR [11 ]and belong AKR to superfamilies, the SDR superfamily, in additi whileon to from theRattus reasons norvegicus of stereospecificity[12] and Homo of sapienssubstrate[13 or] belong product to in the HSDHs. AKR superfamily (Figure2). FigureFigure 1.1. ComparisonComparison of of structures structures (A ,(CA),C and) and folding folding topologies topologies (B,D )(B of,D hydroxysteroid) of hydroxysteroid dehydrogenases dehydro- (HSDHs)genases (HSDHs) from short-chain from short-chain dehydrogenase/reductase dehydrogenase/reductase (SDR) superfamily(SDR) superfamily (7β–HSDH(PDB:5GT9)) (7β– andHSDH(PDB:5GT9)) aldo–keto reductase and aldo–keto (AKR) superfamily reductase (3(AKR)α–HSDH(PDB:4L1X)). superfamily (3α–HSDH(PDB:4L1X)). A–helixes are represented Α–he- as cylinderslixes are represented (in red) and asβ-sheets cylinders are (in represented red) and β as-sheets arrows are (in represented blue). Several as arrows important (in blue). loops Several related important loops related to coenzyme or substrate binding between α-helixes and β–sheets are la- to coenzyme or substrate binding between α-helixes and β–sheets are labeled, which are loop1–6 beled, which are loop1–6 between β(A–F)–α(A–F) in 7β–HSDH and loop1 (βA–αA), loop2 (βG– between β(A–F)–α(A–F) in 7β–HSDH and loop1 (βA–αA), loop2 (βG–αG1) and loop3 (βH–αH1) in αG1) and loop3 (βH–αH1) in 3α–HSDH. Figures A and C were created by Pymol [14]. 3α–HSDH. Figures A and C were created by Pymol [14]. This article reviews the HSDHs from SDR and AKR superfamilies that were identified in mammals and bacteria and then classifies the HSDHs according to their functions in steroid hormone metabolism or bile acid biosynthesis. The differences of NAD(P)(H)- binding models to enzymes and enzymatic reaction characteristics between SDR and AKR superfamilies were also summarized. A semi-flexible molecular docking was used for further exploration of differences of substrate binding model to enzyme–coenzyme complex between SDR and AKR superfamilies, in addition to the reasons of stereospecificity of substrate or product in HSDHs. Crystals 2021, 11, Crystals224 2021, 11, 224 3 of 25 3 of 24 Figure 2. Classification of common HSDHs from SDR and AKR superfamilies according to their source differences. HSDHs Figure 2. Classification of common HSDHs from SDR and AKR superfamilies according to their related to secondarysource differences. bile acids biosynthesisHSDHs related are to mainly secondary from bile bacteria, acids biosynthesis existing in the are SDR mainly superfamily, from bacteria, whereas HSDHs related to steroidexisting hormone in the SDR biosynthesis superfamily, are mainlywhereas from HSDHs mammals, related existing to steroid in hormone both SDR biosynthesis and AKR superfamilies. are More detailed informationmainly from is shown mammals, in Table existing S3. in both SDR and AKR superfamilies. More detailed information is shown in Table S3. 2. Classification and Functions of HSDHs 2. Classification andHSDHs Functions can be of classified HSDHs according to their structural or functional differences. From HSDHs cana structural be classified perspective, according HSDHs to their belong structural to two or functional protein superfamilies—short-chain differences. From dehy- a structural perspective,drogenase/reductase HSDHs belong (SDR) to and two aldo–keto protein superfamilies—short-chain reductase (AKR) superfamily dehy- [1,2]. Most SDRs drogenase/reductaseare active (SDR) as dimersand aldo–keto or tetramers. reductase They (AKR) usually superfamily have a core [1,2]. composed Most SDRs of seven parallel are active as dimersβ-sheets, or tetramers. with several Theyα–helixes usually wrapped have a core around, composed which of form seven two parallel typical domainsβ- named sheets, with severalRossmann α–helixes fold (eachwrapped composed around, of which two βαβ formunits) two fortypical coenzyme domains binding named [7 ]. In contrast, Rossmann foldmost (each AKRs composed are soluble, of two monomeric βαβ units) proteins for coenzyme and have binding an architecture [7]. In contrast, of (α/β )8-barrel fold most AKRs arecalled soluble, TIM-barrel, monomeric which proteins was named and have after an a prototypicalarchitecture memberof (α/β)8 of-barrel AKRs, fold triosephosphate called TIM-barrel,isomerase which (TIM) was [15 named,16]. after a prototypical member of AKRs, tri- osephosphate isomeraseAll HSDHs (TIM) can[15,16].
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