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]. specifically catalyze the redox of steroid-skeleton substrates, such as All HSDHsbile can acids specifically and a series catalyze of steroid the redox hormones of steroid-skeleton [17,18]. According substrates, to the differencessuch as of reacting bile acids and positionsa series of andsteroid hydroxyl hormones orientations [17,18]. According of substrates to the or differences products, of HSDHs reacting can be divided positions and intohydroxyl 11 types orientat at least,ions which of substrates are 3α–HSDH or products, (EC 1.1.1.50,HSDHs ECcan 1.1.1.51,be divided EC into 1.1.1.53, etc.) and 11 types at least,3β–HSDH which are (EC1.1.1.145, 3α–HSDH (EC EC 1.1.1.270,1.1.1.50, EC EC 1.1.1.51, 1.1.1.391, EC etc.),1.1.1.53, 7α–HSDH etc.) and (EC 3β– 1.1.1.159) and HSDH (EC1.1.1.145,7β–HSDH EC (EC1.1.1.270, 1.1.1.201), EC 1.1.1.391, 11β–HSDH etc.), (EC 7α 1.1.1.146),–HSDH (EC 12α 1.1.1.159)–HSDH (EC and 1.1.1.176) 7β– and 12β– HSDH (EC 1.1.1.201),HSDH (EC 11 1.1.1.238),β–HSDH 17(ECα–HSDH 1.1.1.146), (EC 12 1.1.1.148)α–HSDH and (EC 17 β1.1.1.176)–HSDH (EC and 1.1.1.51, 12β– EC 1.1.1.357, HSDH (EC 1.1.1.238),EC 1.1.1.270, 17α–HSDH etc.) and (EC 20 1.1.1.148)α–HSDH and (EC 17 1.1.1.149)β–HSDH and (EC 20 1.1.1.51,β–HSDH EC (EC 1.1.1.357, 1.1.1.53), catalyzing EC 1.1.1.270, etc.)the reductionand 20α–HSDH of carbonyl (EC 1.1.1.149) on C3, C7, and C11, 20 C12,β–HSDH C17, and(EC C201.1.1.53), of steroid-backbone catalyzing substrates the reduction (producingof carbonyl hydroxyl on C3, C7, with C11, an αC12,or βC17,orientation) and C20 andof steroid-backbone their reverse reactions, sub- respectively. strates (producingA part hydroxyl of HSDHs with only an existsα or β in orientation) SDR or AKR and superfamilies their reverse (Table reactions, S1 and re- Table S2). All spectively. A part7α–HSDHs, of HSDHs 7β –HSDHs,only exists and in 11SDRβ–HSDHs or AKR weresuperfamilies reported (Table to exist S1 in and the SDRTa- superfamily, bleS2). All 7αwhile–HSDHs, most 7β 17–HSDHs,α–HSDHs and and 11β 20–HSDHsα–HSDHs were belong reported to AKRs. to exist However, in the SDR enzymes such as superfamily, while3α/β –HSDHsmost 17α and–HSDHs 17β–HSDHs and 20α could–HSDHs be foundbelong in to both AKRs. superfamilies. However, en- Numerous 3α– α β α zymes such asHSDHs 3α/β–HSDHs of AKRs and have 17β–HSDHs functions could of 17 be–HSDH, found in 17 both–HSDH superfamilies. or 20 –HSDH Nu- [12,19]. In α merous 3α–HSDHsparticular, of AKRs 3 –HSDHs have functions (AKR1C31, of AKR1C32, 17α–HSDH, AKR1C33) 17β–HSDH from orOryctolagus 20α–HSDH cuniculus (rabbit) β α [12,19]. In particular,have other 3α–HSDHs two functions (AKR1C31, of 17 –HSDHAKR1C32, and AKR1C33) 20 –HSDH from [20 ].Oryctolagus cu- niculus (rabbit)2.1. have Characteristics other two functions and Functions of 17β of–HSDH HSDHs and from 20 SDRsα–HSDH [20].

2.1. Characteristics andConsisted Functions of of more HSDHs than from 163,120 SDRs members [21], SDR enzymes are found in differ- ent classes of oxidoreductases, isomerases, and lyases [21,22], with multiple functions

Crystals 2021, 11, 224 4 of 24

such as carbonyl–alcohol oxidoreduction, steroid isomerase, enoyl–CoA reduction, de- carboxylase, dehalogenase, dehydratase, C=N reduction and epimerase [22–24], covering broad substrates such as sugars, steroids, retinoids, lipids, polyols, prostaglandins and androstenedione [24,25]. SDRs could be further classified into six types at least according to their sequence differences, i.e., “Classical,” “Extended,” “Atypical,” “Intermediate,” “Divergent,” “Complex” and some members “Unassigned” (Table1)[ 21,22,26]. Sequence alignment was used to analyze structural differences among SDR–HSDHs (HSDHs from SDR superfamily), demonstrating that most SDR–HSDHs are of the “Classical” type except 3β–HSDH, which is more similar to the “Intermediate” type (Table1 , Figure S1). Even though SDR–HSDHs share a low sequence identity of 23–53% (https://blast.ncbi.nlm.nih. gov/Blast.cgi?CMD=Get&RID=3AGHXAGR114 (accessed on 29 January 2021)), all SDR– HSDHs are conservative in structures which composed of a core of Rossmann fold, with several α–helixes wrapped around it (Figure1). SDR–HSDHs are mainly able to catalyze the dehydrogenation/hydrogenation of substrates with steroid-skeleton, participating in the pathways such as steroid hormone biosynthesis, steroid degradation, and secondary bile acid biosynthesis in organisms (Table S3) [27].

Table 1. Features of six subfamilies of the SDR superfamily.

Coenzyme HSDHs Monomer Size Coenzymes Catalytic Reaction Active Site Sequence Identity Binding Divergent NAD(H) Enoyl reductases GxxxxxSxA YxxMxxxK 28–99% Classical ~250 aa NAD(P)(H) Oxidation/reduction TGxxxGxG YxxxK 8–99% Intermediate ~250 aa Drosophila ADH G/AxxGxxG/A 27–99% Extended ~350 aa NAD(P)(H) Epimerases Dehydratases TGxxGxxG 10–99% Complex NADP(H) β-ketoacyl reduction YxxxN 20–74% Atypical NmrA-like Lacks catalytic Tyr “x” signalizes any amino acid residues.

2.1.1. HSDHs in Primary or Secondary Bile Acid Biosynthesis Bile acids serve significant roles in cholesterol metabolism and the digestion of lipids for mammals [28]. The composition of bile acids pools is different in different species. For example, the human bile acids pool is usually composed of hydrophobic bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA), whereas the mice bile acids pool mainly consists of hydrophilic bile acids, muricholic acids(β–MCA) and ursodeoxycholic acid (UDCA) [17,29,30]. All these bile acids can be divided into two forms-primary bile acids (CA and CDCA) and secondary bile acids (DCA, LCA, UDCA, etc.), whose production is related to the activities of the liver and intestinal microbiota, respectively [31]. A number of SDR–HSDHs related to secondary bile acid synthesis were found in intestinal bacteria. The metabolic pathway of secondary bile acids was shown in Figure3. 3α–HSDHs found in species Pseudomonas sp. [11], Comamonas testosterone [32], Corynebac- terium glutamicum, etc. [33] are able to catalyze the hydrogenation/dehydrogenation of carbonyl and hydroxyl on C3 position of bile acids, participating in the conversation of deoxycholate and lithocholate to isodeoxycholate and isolithocholate (https://biocyc.org/ META/NEW-IMAGE?type=PATHWAY&object=PWY-7755 (accessed on 29 January 2021)). A pair of epimerases, 7α–HSDH and 7β–HSDH, could specifically catalyze the reduction of C7=O or the oxidation of C7–OH on steroid-skeleton [34,35], with a substrate or prod- uct of CDCA(7α–OH) and UDCA(7β–OH). These two epimerases work together for the conversion of CDCA to UDCA in industry, with an intermediate 7-keto–lithocholic acid (7–KLA). UDCA has significant pharmaceutical applications in the treatment of gallstones, biliary cirrhosis, and even liver cancers [36]. Crystals 2021, 11, 224 5 of 25

Crystals 2021, 11, 224 5 of 24 acid (7–KLA). UDCA has significant pharmaceutical applications in the treatment of gall- stones, biliary cirrhosis, and even liver cancers [36].

Figure 3. HSDHs-relatedHSDHs-related bile acids synthesis and metabolism. Prim Primaryary bile bile acids acids (cholic (cholic acid acid (CA) (CA) and chenodeoxycholic acid (CDCA)) (CDCA)) in in the the liver liver are are usually usually conjugated conjugated with with glycine glycine (G (G),), which which can can be be deconjugated deconjugated by by bile bile salt salt hydrolases hydrolases (BSH). (BSH). Primary bile acids can be biotransformed to secondary bile acids (deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA), etc.) through the 7 α–dehydroxylation pathway and the ox oxidationidation and epimerization of 3α–,– ,7 7αα–– and and 12 12αα–HSDHs.–HSDHs.

2.1.2.2.1.2. HSDHs HSDHs in in Steroi Steroidd Hormone Hormone Biosynthesis Biosynthesis 33ββ–HSDHs–HSDHs (EC (EC 1.1.1.145) 1.1.1.145) have have been been identified identified in in HomoHomo sapiens sapiens (type(type 1–2), 1–2), MusMus mus- mus- culusculus (type(type 1–6), 1–6), RattusRattus norvegicus norvegicus (type(type 1–4), 1–4), MycobacteriumMycobacterium tuberculosis, tuberculosis, etc., usuallyetc., usually with activitieswith activities of dehydrogenase of dehydrogenase and isomerase( and isomeraseΔ5–Δ4) ([37].∆5–∆ Both4) [37 Human]. Both type Human 1 3β–HSD1/iso- type 1 3β– meraseHSD1/isomerase and type 2 and3β–HSD2/isomerase type 2 3β–HSD2/isomerase are NAD(H)-dependent are NAD(H)-dependent enzymes, catalyzing enzymes, the cat- conversionalyzing the of conversion 3β-hydroxy-5-ene-steroids of 3β-hydroxy-5-ene-steroids to 3-oxo-4-ene-steroids to 3-oxo-4-ene-steroids with two sequential with twoen- zymaticsequential reactions. enzymatic Firstly, reactions. 3β–HSDH Firstly, catalyzes 3β–HSDH the dehydrogenation catalyzes the dehydrogenation of 3β-hydroxysteroid of 3β- tohydroxysteroid a 3-oxo intermediate, to a 3-oxo and intermediate, then the Δ and5-form then intermediate the ∆5-form intermediatewas isomerized was to isomerized Δ4-form viato ∆their4-form isomerase via their activity isomerase (Figure activity 4). This (Figure catalytic4). This function catalytic of 3 functionβ–HSDH ofis 3usuallyβ–HSDH re- latedis usually to the related conversation to the conversation of dehydroepian of dehydroepiandrosteronedrosterone (DHEA) to androstenedione, (DHEA) to androstene- 17α– hydroxypregnenolonedione, 17α–hydroxypregnenolone to 17α–hydroxyprogesterone to 17α–hydroxyprogesterone and pregnenolone and pregnenolone to progesterone to pro- [38–40].gesterone In [humans,38–40]. In 3 humans,β–HSD1 3(typeβ–HSD1 1) and (type 3β–HSD2 1) and 3 (typeβ–HSD2 2) are (type encoded 2) are encodedby two distinct by two genesdistinct which genes are which expressed are expressed in a tissue-specific in a tissue-specific pattern pattern[41]. 3β [–HSD141]. 3β –HSD1is expressed is expressed in pe- ripheralin peripheral tissues tissues such as such breast as, breast,placenta, placenta, prostate, prostate, and endome and endometrium,trium, with roles with of produc- roles of tionproduction of some of downstream some downstream steroids steroids such as such testosterone, as testosterone, dihydrotestosterone dihydrotestosterone (DHT), (DHT), and estrogensand estrogens [42]. [The42]. testosterone The testosterone and estradiol and estradiol will promote will promote the growth the growth of breast of breast tumors tu- [43]mors and [43 prostate] and prostate tumors tumors [44]; therefore, [44]; therefore, human human 3β–HSD1 3β–HSD1 is a potential is a potential target target for the for thein- hibitioninhibition of ofestradiol estradiol production production in in breast breast tu tumorsmors and and testosterone testosterone production production in in prostate prostate tumors.tumors. 3 3ββ–HSD2–HSD2 is is expressed expressed in in adrenals, adrenals, ovaries, ovaries, and and testis, testis, which which are are related related to to the synthesissynthesis of steroidsteroid precursorsprecursors required required for for the the production production of of cortisol, cortisol, aldosterone, aldosterone, and and the thefemale female and and male male sex sex hormones hormones [17 ,[17,29,31].29,31]. 11β–HSDHs (EC 1.1.1.146) in humans can be generally divided into two types, 11β–HSD1 and 11β–HSD2, working together on glucocorticoid metabolism to regulate the availability of active glucocorticoids [45]. 11β–HSD1 is NADP(H)-dependent en- zyme, facilitating the conversion of inactive glucocorticoids, cortisone in human and 11-deoxycorticosterone in rodents, to their active 11-keto metabolites, cortisol, and corticos- terone, respectively [46] (Figure5). 11 β–HSD1 is mainly expressed in liver, adipose tissues, brain and skeletal muscles [47], whose function associated with insulin resistance, dyslipi- demia, obesity, hypertension, and other features of mineralocorticoid excess; hence, the

Crystals 2021, 11, 224 6 of 25

Crystals 2021, 11, 224 6 of 24 Figure 4. 3β–HSDHs (3β–HSD1 and 3β–HSD2) from the SDR superfamily have both activities of dehydrogenase and isomerase, catalyzing the oxidation of 3β–hydroxy and the isomerization of Δ5inhibition to Δ4-form. of 11β–HSD1 is a potential method for the treatment of glucocorticoid (active)- related disorders [48,49]. 11β–HSD2 was found primarily in mineralocorticoid target tissues,11β–HSDHs such as (EC kidneys, 1.1.1.146) sweat in glands, humans salivary can be glands,generally and divided colonic into mucosa two types, [50], catalyz-11β– HSD1 and 11β–HSD2, working together on glucocorticoid metabolism to regulate the Crystals 2021, 11, 224 ing the conversion of glucocorticoids to inactive 11-ketosteroids in a NAD(H)-dependent6 of 25 availabilityactivity, in of contrast active glucocorticoids to 11β–HSD1 [51[45].]. Another11β–HSD1 type is ofNADP(H)-de 11β–HSDH,pendent 11β–HSD3, enzyme, has fa- been cilitatingidentified the inconversion zebrafish, of fathead inactive minnow, glucocorticoids, and some cortisone paralogs in amphioxus human and [52 11-deoxycor-]. ticosterone in rodents, to their active 11-keto metabolites, cortisol, and corticosterone, re- spectively [46] (Figure 5). 11β–HSD1 is mainly expressed in liver, adipose tissues, brain and skeletal muscles [47], whose function associated with insulin resistance, dyslipidemia, obesity, hypertension, and other features of mineralocorticoid excess; hence, the inhibition of 11β–HSD1 is a potential method for the treatment of glucocorticoid (active)-related dis- orders [48,49]. 11β–HSD2 was found primarily in mineralocorticoid target tissues, such as kidneys, sweat glands, salivary glands, and colonic mucosa [50], catalyzing the conversion of glucocorticoids to inactive 11-ketosteroids in a NAD(H)-dependent activity, in contrast toFigure Figure11β–HSD1 4. 4. 33ββ–HSDHs–HSDHs [51]. (3Another (3β–HSD1β–HSD1 typeand and 3 β 3of–HSD2)β –HSD2)11β–HSDH, from from the the SDR11 SDRβ–HSD3, superfamily superfamily has have been have both bothidentified activities activities ofin of zebrafish,dehydrogenasedehydrogenase fathead and and minnow, isomerase, isomerase, and ca catalyzingtalyzing some paralogs the the oxidation oxidation amphioxus of of 3 3ββ–hydroxy–hydroxy [52]. and and the the isomerization isomerization of of ∆5 Δto5 to∆4-form. Δ4-form.

11β–HSDHs (EC 1.1.1.146) in humans can be generally divided into two types, 11β– HSD1 and 11β–HSD2, working together on glucocorticoid metabolism to regulate the availability of active glucocorticoids [45]. 11β–HSD1 is NADP(H)-dependent enzyme, fa- cilitating the conversion of inactive glucocorticoids, cortisone in human and 11-deoxycor- ticosterone in rodents, to their active 11-keto metabolites, cortisol, and corticosterone, re- spectively [46] (Figure 5). 11β–HSD1 is mainly expressed in liver, adipose tissues, brain and skeletal muscles [47], whose function associated with insulin resistance, dyslipidemia, obesity, hypertension, and other features of mineralocorticoid excess; hence, the inhibition of 11β–HSD1 is a potential method for the treatment of glucocorticoid (active)-related dis- Figure 5. Human 11β–HSD1 from the SDR superfamily is able to catalyze the activation of glucocor- orders [48,49]. 11β–HSD2 was found primarily in mineralocorticoid target tissues, such as Figureticoids, 5. Human while 11 11ββ–HSD2–HSD1 catalyzes from the aSDR reverse superfamily reaction. is able to catalyze the activation of gluco- corticoids,kidneys, while sweat 11 glands,β–HSD2 salivary catalyzes glands, a reverse and reaction. colonic mucosa [50], catalyzing the conversion of glucocorticoids17β–HSDHs to are inactive encoded 11-ketosteroids by non-homologous in a NAD(H)-dependent genes with different activity, subcellular in contrast local- toizations, 1117β–HSD1–HSDHs coenzymes, [51]. are encodedAnother and substrate by type non-homologous preferencesof 11β–HSDH, [53 genes]. Up11β with to–HSD3, now, different overhas 14 beensubcellular different identified subtypes local- in izations,zebrafish,of 17β –HSDHscoenzymes, fathead (17 minnow, andβ–HSD1–14) substrate and some preferences have paralogs been identified,[5 3].amphioxus Up to mostnow, [52]. over of them 14 different belong tosubtypes the SDR of superfamily17β–HSDHs except(17β–HSD1–14) 17β–HSD5, have which been is identified, a member most of the of AKRthem superfamilybelong to the [ 54SDR,55 ].su- All perfamily17β–HSDHs except are 17 ableβ–HSD5, to catalyze which the is a reduced member or of oxidized the AKR reactions superfamily at position [54,55]. C17 All of17 C18–β– HSDHsor C19–steroids, are able to involvedcatalyze the in thereduced activation or oxid andized inactivation reactions ofat steroidposition hormones C17 of C18– (Figure or C19–steroids,6)[54]. 17β–HSD1 involved could in the catalyze activation the activation and inactivation of estrone of to steroid estradiol hormones in humans. (Figure However, 6) [54].the 17 productionβ–HSD1 could of estradiol catalyze is the linked activation to the developmentof estrone to estradiol of diseases in humans. such as breast However, cancer, theovarian production tumor, of estradiol endometriosis, is linked endometrial to the development hyperplasia, of diseases and uterine such leiomyomaas breast cancer, [56]. It ovarianwas demonstrated tumor, endometriosis, that estradiol endometrial can be converted hyperplasia, to genotoxic and uterine metabolites leiomyoma in breast [56]. tissue. It wasConsequently, demonstrated the that inhibition estradiol of can 17 βbe–HSD1 conver isted one to ofgenotoxic the treatments metabolites for breast in breast cancer tis- in sue.clinical Consequently, [57]. Meanwhile, the inhibition the decreasing of 17β–HSD1 of estradiol is one willof the lead treatments to osteoporosis. for breast 17 βcancer–HSD2 in haveclinical opposing [57]. Meanwhile, actions to the 17 decreasingβ–HSD1, catalyzing of estradiol the will inactivation lead to osteoporosis. of estradiol 17β to–HSD2 estrone, havewhich opposing means theactions inhibition to 17β of–HSD1, 17β–HSD2 catalyzing is one ofthe the inactivati therapieson for of osteoporosis estradiol to [58estrone,]. Other- whichFigurewise, means 175. Humanβ–HSD1 the inhibition11 couldβ–HSD1 catalyze offrom 17 βthe–HSD2 the SDR conversion superfamily is one of of the dehydroepiandrosteroneis therapieable to catalyzes for osteoporosis the activation (DHEA) [58]. of gluco- Oth- to ∆5– erwise,corticoids,androstane–3 17β while–HSD1β 11, 17βcould–HSD2β–diol catalyze catalyzes while 17the aβ reverse –HSD2conversion reaction. catalyze of dehydroepiandrosterone a reverse reaction. ∆5–androstane-3 (DHEA) to β, 17β–diol can be converted to estradiol with functions of 3β–HSDH and aromatase [59]. 17β– HSD317β exists–HSDHs predominantly are encoded in by testis non-homologous and catalyzes genes the conversion with different of ∆4–androsenedione subcellular local- izations,to testosterone. coenzymes, It was and confirmed substrate preferences that its mRNA [53]. wasUp to overexpressed now, over 14 different in prostate subtypes cancer oftissue. 17β–HSDHs Testosterone (17β–HSD1–14) is responsible have for been cell identified, proliferation most in of androgen-dependent them belong to the SDR diseases, su- perfamilyand the inhibition except 17 ofβ–HSD5, 17β–HSD3 which is effectiveis a member for theof the treatment AKR superfamily of diseases [54,55]. such as All prostate 17β– HSDHscancer [are60]. able The to functions catalyze of the other reduced subtypes or oxid of 17izedβ–HSDH reactions were at describedposition C17 in Tableof C18– S3. or C19–steroids, involved in the activation and inactivation of steroid hormones (Figure 6) [54]. 17β–HSD1 could catalyze the activation of estrone to estradiol in humans. However, the production of estradiol is linked to the development of diseases such as breast cancer, ovarian tumor, endometriosis, endometrial hyperplasia, and uterine leiomyoma [56]. It was demonstrated that estradiol can be converted to genotoxic metabolites in breast tis- sue. Consequently, the inhibition of 17β–HSD1 is one of the treatments for breast cancer in clinical [57]. Meanwhile, the decreasing of estradiol will lead to osteoporosis. 17β–HSD2 have opposing actions to 17β–HSD1, catalyzing the inactivation of estradiol to estrone, which means the inhibition of 17β–HSD2 is one of the therapies for osteoporosis [58]. Oth- erwise, 17β–HSD1 could catalyze the conversion of dehydroepiandrosterone (DHEA) to

Crystals 2021, 11, 224 7 of 25

Δ5–androstane–3β, 17β–diol while 17β–HSD2 catalyze a reverse reaction. Δ5–androstane- 3β, 17β–diol can be converted to estradiol with functions of 3β–HSDH and aromatase [59]. 17β–HSD3 exists predominantly in testis and catalyzes the conversion of Δ4–androsenedi- one to testosterone. It was confirmed that its mRNA was overexpressed in prostate cancer tissue. Testosterone is responsible for cell proliferation in androgen-dependent diseases, and the inhibition of 17β–HSD3 is effective for the treatment of diseases such as prostate cancer [60]. The functions of other subtypes of 17β–HSDH were described in Table S3.

2.2. Characteristics and Functions of HSDHs from AKRs Most aldo–keto reductases (AKRs) are soluble monomeric NAD(P)(H) oxidoreduc- tases that catalyze a NAD(P)H-dependent reduction of aldehydes and ketones, yielding primary and secondary alcohols [61]. Over 190 members belonging to AKRs have been reported, with a high sequence identity of 40–60% [2,61,62]. The (α/β)8–TIM barrel pro- vides a common architecture for a NAD(P)(H)-dependent catalytic activity [63]. AKRs have broad substrate specificity, which was determined by the variation of loops on the C-terminal. Their functions were related to the metabolism of sugar, lipid aldehydes, ster- oids, and prostaglandins, etc. [61]. All AKRs can be further classified into 16 families (AKR1–AKR16) [61,62] and HSDHs reported from AKRs (AKR–HSDHs) belong to AKR1[63]. Up to now, four isoforms of human 3α–HSDHs and one isoform of rat 3α– HSDH belonging to AKRs have been reported, which are AKR1C1 (human 3α(20α)– HSDH), AKR1C2 (human type 3), AKR1C3 (human type 2), AKR1C4 (human type 1) and AKR1C9 (rat liver), respectively [64–67]. The human type 2 3α–HSDH (AKR1C3) is also Crystals 2021, 11, 224known as type 5 17β–HSDH, while type 3 (AKR1C2) is also known as bile acid-binding 7 of 24 protein [64–66].

Figure 6. HSDHs-relatedFigure 6. HSDHs-related steroid hormones steroid hormones synthesis synthesis and metabolism. and metabolism. “3βDH/iso” “3βDH/iso” signalizes signalizes the dehydrogenase the and isomerasedehydrogenase activities of 3β–HSDH. and isomerase activities of 3β–HSDH. – – Human aldo–keto2.2. Characteristics reductase andAKR1C3 Functions (type of 2 HSDHs 3α HSDH/type from AKRs 5 17β HSDH) is also known as prostaglandin F synthase [68] and possesses activities in the reduction of Δ4– Most aldo–keto reductases (AKRs) are soluble monomeric NAD(P)(H) oxidoreduc- androstene-3,17-dione to testosterone, 5α–dihydrotestosterone(5α–DHT) to 3α– and 3β– tases that catalyze a NAD(P)H-dependent reduction of aldehydes and ketones, yielding androstanediol, and estrone to 17β–estradiol, respectively (Figure 6) [69], which is ex- primary and secondary alcohols [61]. Over 190 members belonging to AKRs have been pressed in sex hormone-dependent tissues (testis, breast, endometrium, and prostate) and reported, with a high sequence identity of 40–60% [2,61,62]. The (α/β) –TIM barrel pro- sex hormone-independent tissues (kidney and urothelium) [70], whose functions are as-8 vides a common architecture for a NAD(P)(H)-dependent catalytic activity [63]. AKRs sociated with breast cancer, endometrial cancer and the development of endometriosis have broad substrate specificity, which was determined by the variation of loops on the C- and dysmenorrhea [71]. AKR1C2 is another AKR1C isozyme that is highly expressed in terminal. Their functions were related to the metabolism of sugar, lipid aldehydes, steroids, and prostaglandins, etc. [61]. All AKRs can be further classified into 16 families (AKR1– AKR16) [61,62] and HSDHs reported from AKRs (AKR–HSDHs) belong to AKR1 [63]. Up to now, four isoforms of human 3α–HSDHs and one isoform of rat 3α–HSDH belonging to AKRs have been reported, which are AKR1C1 (human 3α(20α)–HSDH), AKR1C2 (human type 3), AKR1C3 (human type 2), AKR1C4 (human type 1) and AKR1C9 (rat liver), respec- tively [64–67]. The human type 2 3α–HSDH (AKR1C3) is also known as type 5 17β–HSDH, while type 3 (AKR1C2) is also known as bile acid-binding protein [64–66]. Human aldo–keto reductase AKR1C3 (type 2 3α–HSDH/type 5 17β–HSDH) is also known as prostaglandin F synthase [68] and possesses activities in the reduction of ∆4– androstene-3,17-dione to testosterone, 5α–dihydrotestosterone(5α–DHT) to 3α– and 3β– androstanediol, and estrone to 17β–estradiol, respectively (Figure6)[ 69], which is ex- pressed in sex hormone-dependent tissues (testis, breast, endometrium, and prostate) and sex hormone-independent tissues (kidney and urothelium) [70], whose functions are associated with breast cancer, endometrial cancer and the development of endometriosis and dysmenorrhea [71]. AKR1C2 is another AKR1C isozyme that is highly expressed in the prostate and catalyzes the oxidation of 3α–androstanediol to the active hormone 5α–DHT, opposite to AKR1C3 [66,72]. AKR1C4 can work in concert with steroid 5β–reductase to produce the 5β,3α– tetrahydrocholestanes in the liver, play a pivotal role in the bile acid biosynthesis, and steroid hormone metabolism [66]. AKR1C1 is usually expressed in lung, liver, testis, mam- mary gland, endometrium, brain, kidney, adipose cells, skin, osteoblasts, and optic nerve head astrocytes [73,74], exhibiting both 3α/β– and 20α–HSD activities and mainly reduc- ing progesterone and 5α–pregnan–3α–ol–20-one to their corresponding inactive metabolite 20a-hydroxyprogesterone with a function of 20-ketosteroid reductase [73,74]. Progesterone plays a crucial role in preventing the onset of premature birth, blunting the estrogenic Crystals 2021, 11, 224 8 of 24

effects in endometriosis and endometrial cancer [75,76]. Thus, the inhibition of AKR1C1 is a potential agent for the treatment of these disorders [77,78].

3. Coenzyme Binding Modes of HSDHs Nearly 150,000 different sequences were annotated as or predicted to be NAD(H)/NADP(H)- dependent oxidoreductases/reductases [79]. The binding model of NAD(H)/NADP(H) in the enzyme is significant for the proton transfer. Several structures were reported for coenzyme binding, which are Rossmann fold, (α/β)8–TIM–barrel, dihydroquinoate synthase-like, and FAD/NAD-binding folds [79–81].

3.1. Coenzyme Binding Modes of the HSDHs from SDRs The sequence alignment of HSDHs in the SDR superfamily shows a low sequence iden- tity of 23–53% (https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=3AGHXAGR114 (accessed on 29 January 2021)). However, all SDR–HSDHs share similar and conservative motifs for NAD(P)(H) binding. The basic structure of SDRs is composed of two Rossman folds, which is stable and no loss of function occurs even with mutations at some sites of the folding and that is why SDRs retain similar catalytic functions even with such low sequence identities [82–84]. The binding sites of the adenosine ring of coenzyme to enzyme are on the loop1, loop2, and loop3 (Figure1), close to the C-terminal part of SDRs, while the nicotinamide- binding region is on loop 4 and loop 6, close to the N-terminal part. A conserved glycine- rich pattern GxxxGxG between the first β–strand and α–helix is usually interacted with pyrophosphate [84–86]. The individual subfamilies of the SDR superfamily differ regarding the spacing of the three glycine residues [87]. For example, it was demonstrated that 3β– HSDHs have a motif of X1–Gly–X2–X3–Gly–X4–X5–Gly (Figure S1), which is similar to the “Intermediate” type [88], while most SDR–HSDHs have a motif of X1–Gly–X2–X3–X4– Gly–X5–Gly and belong to the “Classical” type. In the meantime, the sequence alignment of SDR–HSDHs from different sources showed that the residue X1 in 7β–HSDHs is L/C, which is obviously different from the residue T/S in other SDR–HSDHs (Figure S1). The features among different subfamilies of SDRs were described in Table1. The motif YxxxK on αE-helix has two functions of coenzyme binding and catalysis [89], showing differences among different types of SDRs as well, which will be described in detail below. There is no doubt that NAD(H)/NADP(H) can act as a proton donor or acceptor during redox reactions [90] and can participate in the remodeling of the substrate-binding pocket. The common belief is that the broad substrate specificity of HSDHs is related to the substrate-binding loop in previous studies, but are the interactions between NAD(P)(H) and the enzyme also responsible for the substrate specificity, especially the stereospecificity? The distance between C6 of adenine and C2 of nicotinamide in a syn conformation is fre- quently used to express the degree of extension of coenzyme molecule [91], which is 13.4 Å, 13.7 Å, 14.1 Å, and 14.3Å in 7α–HSDH (PDB: 1FMC) [92], 7β–HSDH (PDB: 5GT9) [93], 11β– HSDH (PDB: 3G49) and 17β–HSDH (PDB: 3QWH), respectively, showing little difference (Figure7). The dihedral angles of PA–O3–PN–O5D (pyrophosphate group), C4B–C2B– C1B–N9A (adenosine ring), and C4D–C2D–C2N–C5N (nicotinamide ring) were used for a further study on conformation change of NAD(P)(H) (Table2). In the pyrophosphate binding region, Ile23 and Thr194 of 7α–HSDH, Val20 and Thr191 of 7β–HSDH, Ile21 and Thr195 of 11β–HSDH, in addition to Ile30 and Thr202 of 3α(17β)–HSDH, are equivalent in function. However, the dihedral angle PA–O3–PN–O5D (–80.6◦) in the 7β–HSDH binary is apparently different compared with others, which may lead to the difference of substrate binding pocket. Another crystal structure of 7α–HSDH from Clostridium absonum was obtained as a tetramer in the study by Lou et al., which exhibited a coenzyme-dependent of NADP(H) [94]. The dihedral angle PA–O3–PN–O5D in the pyrophosphate region is −143.4◦ (Figure8), which is similar to 7 α–HSDH–NAD+ complex (–147.8◦) from E. coli, although the coenzymes of these two crystals are completely different. Both of these two dihedral angles PA–O3–PN–O5D are different from 7β–HSDH–NADP+ complex (–80.6◦), Crystals 2021, 11, 224 9 of 25

2). In the pyrophosphate binding region, Ile23 and Thr194 of 7α–HSDH, Val20 and Thr191 of 7β–HSDH, Ile21 and Thr195 of 11β–HSDH, in addition to Ile30 and Thr202 of 3α(17β)– HSDH, are equivalent in function. However, the dihedral angle PA–O3–PN–O5D (–80.6°) in the 7β–HSDH binary is apparently different compared with others, which may lead to the difference of substrate binding pocket. Another crystal structure of 7α–HSDH from Clostridium absonum was obtained as a tetramer in the study by Lou et al., which exhibited a coenzyme-dependent of NADP(H) [94]. The dihedral angle PA–O3–PN–O5D in the py- rophosphate region is −143.4° (Figure 8), which is similar to 7α–HSDH–NAD+ complex (– 147.8°) from E. coli, although the coenzymes of these two crystals are completely different. Both of these two dihedral angles PA–O3–PN–O5D are different from 7β–HSDH–NADP+ complex (–80.6°), and this is probably able to prove that the stereospecificity of sub- Crystals 2021, 11, 224strate/product is related to the coenzyme conformation. 9 of 24 The pyrophosphate-interacted residues, Ser193 in 7β–HSDH, Thr197 in 11β–HSDH and Ser11 in 3α(17β)–HSDH, are quite special compared with 7α–HSDH ternary. The in- teraction [Thr194–OG1···2.79 Å···O1N–PN] in 7α–HSDH ternary was replaced by [Ser193– OG···3.11Å···O1N–PN]and this isin probably 7β–HSDH, able leading to prove to thata rotation the stereospecificity of the phosphate of substrate/product group PN to is related the outside, whichto the made coenzyme the coenzyme conformation. conformation different (Figure 7).

Figure 7. ResiduesFigure 7. that Residues interacted that withinteracted NAD(P)(H) with NAD(P)(H) in the SDR in superfamily, the SDR superfamily, in which the in which residues the in residues yellow, green, and red color interactin yellow, with green, the adenosine and red color region, interact pyrophosphate with the adenosine region, and region, nicotinamide pyrophosphate region region, of coenzyme, and respectively. The distancenicotinamide between C6 ofregion adenine of coenzyme, and C2 of respectively. nicotinamide The and distance the dihedral between angles C6 ofof PA–O3–PN–O5Dadenine and C2 of (pyrophosphate group), C4B–C2B–C1B–N9Anicotinamide and (adenosine the dihedral ring), angles and of C4D–C2D–C2N–C5N PA–O3–PN–O5D (pyrophosphate (nicotinamide group), ring) in C4B–C2B–C1B– blue color are also shown. This figure wasN9A created (adenosine by Pymol ring), [ 14and]. C4D–C2D–C2N–C5N (nicotinamide ring) in blue color are also shown. This figure was created by Pymol [14]. Table 2. Residues that interacted with NAD(P)(H) in the SDR superfamily and the NAD(P)(H)-conformation differences among them.

HSDHs Interaction between NAD(P)(H) and HSDHs Distance/Å Dihedral Angle/◦ Adenosine Ring Pyrophosphate Nicotinamide Ring C6A–C2N A B C Nicotinamide: Ile192, Thr194; 7α–HSDH Adenine: Asp68, Ile69 Ile23, Thr194 13.4 138.7 47.8 116.5 Ribose: Tyr159, Lys163 Adenine: Phe67, Asp66; Nicotinamide: Thr189, Thr191; 7β–HSDH Ribose: Thr17, Glu18, Val20, Thr191, Ser193 13.7 143.4 80.6 146.2 Ribose: Tyr156, Lys160 Arg40, Argr41 Adenine: Met68; Ribose: Ser18, Nicotinamide: Ile193, Thr195; 11β–HSDH Ile21, Thr197 14.1 134.6 156.5 142.6 Arg41, Ser42 Ribose: Tyr158, Lys162 Adenine: Arg78, Asp76, Ile77, Nicotinamide: Thr200, Thr202; 3α(17β)–HSDH Ser104; Ribose: Gly25, Gly27, Arg28, Ile30, Thr202, Asp203 14.3 143.7 130.6 125.6 Ribose: Asn103, Tyr167, Lys171 Arg28, Ala50, Asn51, Ser52 Dihedral angles A, B and C represents PA–O3–PN–O5D (pyrophosphate group), C4B–C2B–C1B–N9A (adenosine ring), and C4D–C2D– C2N–C5N (nicotinamide ring), respectively. Crystals 2021, 11, 224 10 of 24 Crystals 2021, 11, 224 10 of 25

Figure 8. TheFigure structural 8. The alignment structural (A alignment) of 7α–HSDH (A) of (PDB: 7α–HSDH 5EPO) (PDB: and 7 β5EPO)–HSDH and (PDB: 7β–HSDH 5GT9) and(PDB: the 5GT9) NADP(H) and conforma- tion differencesthe NADP(H) between them conformation (dihedral differences angles of PA–O3–PN–O5D between them (dihedral of pyrophosphate angles of PA–O3–PN–O5D group) (B). Figure of ( C) exhibits the NADP(H)-interactedpyrophosphate residues group) in the (B 7).α –HSDHFigure (C complex.) exhibits This the figureNADP(H)-interacted was created by residues Pymol [14 in]. the 7α–HSDH complex. This figure was created by Pymol [14]. The pyrophosphate-interacted residues, Ser193 in 7β–HSDH, Thr197 in 11β–HSDH In the adenosineand Ser11 ring in binding 3α(17β )–HSDH,region, the are existence quite special of O2′ comparedribose phosphate with 7 αgroup–HSDH in ternary. The NADP(H) makesinteraction the interactions [Thr194–OG1 between··· 2.79 the Åadenosine··· O1N–PN] ribose in 7regionα–HSDH and ternaryβ–HSDHs was replaced by complicated. A[Ser193–OG hydrogen-bond··· 3.11Å network··· O1N–PN] betweenin coenzyme, 7β–HSDH, residues, leading and to asoluble rotation mole- of the phosphate cules maintainsgroup the adenosine PN to the outside,ribose conformation which made theof NADP coenzyme+ in binary conformation or ternary different com- (Figure7). 0 plexes. In the adenosine ring binding region, the existence of O2 ribose phosphate group in These residuesNADP(H) in 7β makes–HSDH the are interactions Arg40–Arg41, between in 11 theβ–HSDH adenosine are ribose Arg41–Ser42, region and andβ –HSDHs com- in 3α(17β)–HSDHplicated. are Ala50–Asn51–Ser52 A hydrogen-bond network (Table 2). between In the nicotinamide coenzyme, residues, ring binding and soluble re- molecules + gion, the interactionsmaintains between the adenosine nicotinamide ribose conformationribose and residues of NADP seemin to binary be conserved. or ternary complexes. Motifs Tyr–X–X–X–LysThese [89–94] residues and in 7 βThr/Ile––HSDHX–Thr are Arg40–Arg41, have conserved in 11β –HSDHinteractions are Arg41–Ser42,with and in NAD(P)(H) (Figure3α(17β 7)–HSDH and Table are 2). Ala50–Asn51–Ser52 The hydrogen bonds (Table existed2). In theamong nicotinamide PN–phosphate ring binding region, group of pyrophosphatethe interactions region between, nicotinamide, nicotinamide and Thr ribose contribute and residues to maintaining seem to the be conserved. ori- Motifs entation of pro-STyr–X–X–X–Lys side of nicotinamide [89–94] towards and Thr/Ile–X–Thr catalytic sites, have for conserved example, the interactions interactions with NAD(P)(H) [Thr189–N···2.9(Figure Å···O7 and7N–Nicotinamide], Table2). The hydrogen [Thr189–O··· bonds3.2 existed Å···N7N–Ni among PN–phosphatecotinamide], group of [Thr189–O···2.9pyrophosphate Å···N–Thr191], region, [Thr191–OG1·· nicotinamide,·3.0 Å···N7N–Nicotinamide], and Thr contribute to maintaining [Thr191– the orienta- tion of pro-S side of nicotinamide towards catalytic sites, for example, the interactions OG1···2.7 Å···O2N–PN], and [Nicotinamide–O7N···2.9 Å···O2N–PN] in the holo-form of [Thr189–N··· 2.9 Å··· O7N–Nicotinamide], [Thr189–O··· 3.2 Å··· N7N–Nicotinamide], 7β–HSDH, which making the happen of reaction possible. [Thr189–O··· 2.9 Å··· N–Thr191], [Thr191–OG1··· 3.0 Å··· N7N–Nicotinamide], [Thr191– ··· ··· ··· ··· Table 2. Residues that interacted with NAD(P)(H)OG1 in2.7 the Å SDRO2N–PN], superfamily and and [Nicotinamide–O7N the NAD(P)(H)-conformation2.9 Å differencesO2N–PN] in the holo-form among them. of 7β–HSDH, which making the happen of reaction possible.

3.2. NAD(H)/NADP(H) Binding Modes of the HSDHs from AKRsDihedral An- HSDHs Interaction between NAD(P)(H) and HSDHs Distance/Å HSDHs from AKRs (AKR–HSDHs) are not only conservedgle/° in structure, but also Adenosine Ring share aPyrophosphate high sequence identityNicotinamide of 64–93% Ring (https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD= C6A–C2N A B C 13 Get&RID=3AH34HGD114Nicotinamide: (accessed Ile192, on 29 Thr194; January 2021)). The basic structure of AKRs is 7α–HSDH Adenine: Asp68, Ile69 Ile23, Thr194 13.4 8. 47.8 116.5 composed of eight β-sheets.Ribose: At least Tyr159, one Lys163α–helix exists between every two β-sheets, which surround the barrel around outside [95,96]. This structure was7 named after TIM-barrel fold, 14 Adenine: Phe67, Asp66; consistedVal20, of anThr191, eightfold Nicotinamide: repeat of (Thr189,α/β) units Thr191; ((α /β)8), was first seen in triosephosphate 7β–HSDH 13.7 3. 80.6 146.2 Ribose: Thr17, Glu18, Arg40, Argr41isomerase Ser193 (TIM) [97,98]. Ribose: Tyr156, Lys160 4 Loops between β-sheets and α-helixes are used for the binding of the coenzyme 13 11β– Adenine: Met68; Ribose: Ser18, Arg41,and substrate. In contrastNicotinamide: with SDR–HSDHs, Ile193, Thr195; the residues that interacted with NADP(H) Ile21, Thr197 14.1 4. 156.5 142.6 HSDH Ser42 adenosine are closer to theRibose: N-terminal, Tyr158, Lys162 whereas residues that interacted with NADP(H) 6 nicotinamide are closer to the C-terminal [99]. The NAD(P)H binds AKRs in an extended anti-conformation [61], stretching through the channel formed by Van der Waals con-

Crystals 2021, 11, 224 11 of 25

Adenine: Arg78, Asp76, Ile77, Ser104; Ri- 14 3α(17β)– Arg28，Ile30, Nicotinamide: Thr200, Thr202; bose: Gly25, Gly27, Arg28, Ala50, Asn51, 14.3 3. 130.6 125.6 HSDH Thr202, Asp203 Ribose: Asn103, Tyr167, Lys171 Ser52 7 Dihedral anglesCrystals A,2021 B and, 11, 224C represents PA–O3–PN–O5D (pyrophosphate group), C4B–C2B–C1B–N9A (adenosine ring), 11 of 24 and C4D–C2D–C2N–C5N (nicotinamide ring), respectively.

3.2. NAD(H)/NADP(H) Binding Modes of the HSDHs from AKRs tacts [100], which composed of loop 1(βA–αA, residues 22–31), loop 2(βG–αG, residues HSDHs from216–235), AKRs and (AKR–HSDHs) loop 3(βH-α H,are residues not only 269–274) conserved (Figure in 1structure,). but also share a Thehigh interactions betweensequence AKR–HSDHs identity and coenzymesof in the binary64–93% or ternary struc- (https://blast.ncbi.nlm.nih.gov/Blast.cgi?tures of 3α–HSDH (PDB:CMD=Get&RID=3AH34HGD114 1J96), 3(17)α–HSDH (PDB: 2P5N), (accessed 17β–HSDH on 29 (PDB: 1ZQ5), January 2021)).and The 20basicα–HSDH structure (PDB: of AKRs 1Q5M) is werecomposed analyzed of eight using β-sheets. Pymol At (distributed least one α by– Schrödinger, helix exists betweenNew york,every NY,two US)β-sheets, (Figure which9)[ 14 surround], to explore the thebarrel conformation around outside changes [95,96]. of NADP(H) after This structure wasbinding named to the after enzyme TIM-barrel and whether fold, consisted these changes of an eightfold are conservative repeat of in(α/ allβ) AKR–HSDHs. units ((α/β)8), wasThe first conformation seen in triosephos of NADP(H)phate isomerase is highly conserved(TIM) [97,98]. in AKRs, showing little difference. Loops betweenThe distance β-sheets between and α-helixes C6 of adenineare used andfor the C2 binding of nicotinamide of the coenzyme is 17.0 Å, and which means the substrate. In contrastoccurrence with SDR–HSDHs, of structural extension the residues of that NADPH interacted (15.6 with Å) due NADP(H) to the interactions aden- between osine are closerresidues to the N-terminal, and coenzyme, whereas such re assidues hydrogen that bonds,interactedπ–π withinteractions, NADP(H) and nico- hydrophobic inter- tinamide are closeractions to the [101 C-terminal], and it is definitely[99]. The NAD(P)H different from binds SDR–HSDHs, AKRs in an extended which is roughlyanti- 14 Å. Other conformation [61],structural stretching changes through and interacted the channel residues formed are describedby Van der in TableWaals S4, contacts which demonstrated [100], which composedhigh similarity of loop and 1( conservationβA–αA, residues in 3α –HSDH,22–31), loop 3(17) 2(αβ–HSDH,G–αG, residues 3α(17β)–HSDH, 216– 17β–HSDH, 235), and loop and3(βH- 20ααH,–HSDH. residues 269–274) (Figure 1).

Figure 9. ResiduesFigure 9. that Residues interacted that with interacted NAD(P)(H) with inNAD(P)(H) AKR superfamily, in AKR insuperfamily, which the residues in which in the green, residues red, and in yellow color interact withgreen, the adenosine red, and yellow region, color pyrophosphate interact with region, the ad andenosine nicotinamide region, pyrophos region, respectively.phate region, The and distance nico- between C6 of adenine andtinamide C2 of nicotinamideregion, respectively. and the The dihedral distance angles between of PA–O3–PN–O5D C6 of adenine (pyrophosphate and C2 of nicotinamide group), C4B–C2B–C1B–N9A and the dihedral angles of PA–O3–PN–O5D (pyrophosphate group), C4B–C2B–C1B–N9A (adenosine (adenosine ring), and C4D–C2D–C2N–C5N (nicotinamide ring) in blue color are also shown. This figure was created by ring), and C4D–C2D–C2N–C5N (nicotinamide ring) in blue color are also shown. This figure was Pymol [14]. created by Pymol [14]. Taking the 3α–HSDH holo-form (PDB: 1J96), for example, the adenine ribose group in The interactions between AKR–HSDHs and coenzymes in the binary or ternary struc- AKR–HSDHs is embedded between αG1-helix and αH-helix, interacted with residues of tures of 3α–HSDH (PDB: 1J96), 3(17)α–HSDH (PDB: 2P5N), 17β–HSDH (PDB: 1ZQ5), and Ala253 from αG1-helix, Arg276, and Gln279 from αH-helix, while the O2’ ribose phosphate 20α–HSDH (PDB: 1Q5M) were analyzed using Pymol (distributed by Schrödinger, New group was contained in the cavity formed by Lys70–Ser271–Tyr272 on the loop 1 and york, NY, US) (FigureArg276 9) on [14],αH-helix. to explore These the residuesconformation and thechanges water of molecules NADP(H) formed after bind- a hydrogen bond ing to the enzymenetwork and whether with the these adenosine change riboses are group,conservative which helpsin all AKR–HSDHs. to fix the adenosine The ribose group conformation ofat theNADP(H) N-terminal. is highly Allthese conserved interactions in AKRs, are highlyshowing conserved little difference. in AKR–HSDHs, The especially the interactions between Ala253, Arg276, and coenzymes, for example, the interactions [Ala253–N··· 3.1Å··· H2O··· 3.1Å··· N1A–adenosine] and [Arg276–NH2··· 3.0Å··· O2X– Phosphate group] [102–104]. The pyrophosphate group crosses through the tunnel composed of loop 1, loop 2, and loop 3, connecting the adenosine ribose at the outside of the barrel and nicotinamide ribose Crystals 2021, 11, 224 12 of 24

in the center. On the inner wall of the tunnel, the residues Ser217–Ala218–Leu219–Ser221 on loop 2, Leu268, Lys270 on loop 3, and Tyr24 on loop 1 formed a hydrogen bond network with water molecules and pyrophosphate groups and maintained the conformation of this region (Figure9). Among them, Ser/Thr217–Ala218–Leu219 and Leu268 are highly conserved. The residue Tyr24 has hydrogen-bond linkage to nicotinamide ribose while Leu219 has hydrogen-bond linkage to adenosine ribose, respectively.

4. Catalytic Mechanism of HSDHs 4.1. Catalytic Mechanism of HSDHs from SDRs It is recognized that the motif Tyr–X–X–X–Lys is associated with the catalytic function of SDRs. This conserved motif attracted much attention as early as 1981 [26,105], and its function was reported in the studies of 3α(20β)–HSDH from Streptomyces hydrogenans [106] and 7α–HSDH from E. coli [92]. However, the conservative motif shows differences in different types of SDRs. The most common motif Tyr–X–X–X–Lys exists in “Classical” SDRs, while the motif in the “Divergent” type is Tyr–x–x–Met–x–x–x–Lys [26], the features of these six types of SDRs were shown in Table1. Tyr is strictly conserved even in the whole SDR superfamily, while Lys and Ser are only conserved in most SDRs. With the assistance of Lys–NH2 to decreases the pKa of Tyr–OH, the side chain phenolic group of Tyr acts as a general acid/base catalyst. The hydride of the substrate is released to the pro-S side of the nicotinamide group of NAD(P)(H) in an oxidated reaction [107]. Apart from being a proton donor or accepter, NAD(P)(H) plays a vital role in the formation and stability of the substrate-binding pocket. A strict binding order of substrate and coenzyme to drosophilid alcohol dehydrogenase (DADH) was reported [108]. DADH could convert alcohol to aldehyde/ketone. In an oxidated reaction, the coenzyme is bound to a free enzyme to form a binary complex firstly and then the substrate binds to the binary complex and the rate-limiting step is the release of the coenzyme product (NADH) [109–111]. This mechanism is similar to the “bi–bi” mechanism of AKRs in which coenzyme binds first and leaves last [16] and probably applies to all SDRs. The catalytic mechanism (Figure 10) of SDRs was investigated through X-ray crys- tallographic structure determination of 3α(20β)–HSDH in a study by Ghosh et al. [106]. It was proposed that the catalytic function of 3α(20β)–HSDH was realized by a triad Ser–Tyr–Lys. Tyr–OH acts as a proton donor applying for an electrophilic attack on the + 20-keto oxygen in the reduction of 20-keto [106]. Lys–NH3 is close to Tyr, facilitating the transformation of the proton. Ser participates in catalysis through direct interaction with Tyr–OH or just as part of a proton-relay network [106]. Tanaka et al. proposed a three-step catalytic mechanism for 7α–HSDH and confirmed it through the mutation of Tyr159, Lys163, and Ser146 [92]. Firstly, the Lys163 binds to NAD(H) and lowers the pKa value of Tyr159 and then Tyr159 was deprotonated and interacted with the C7 position of 7-oxoglycochenodeoxycholic acid (7-oxo–GCDCA), the intermediate was stabilized by Ser146 with a hydrogen bond to C7–O. Secondly, the deprotonated Tyr acts as a catalytic base and extracts the hydrogen from C7–OH. NAD+ accepts another hydrogen released from position C7 in the same time [92,112]. In the studies of DADH, the theoretical calcu- lations suggested that the proton was released from a coupled irregular ionization of the active site groups Tyr151 and Lys155 instead of a single residue Tyr151 [91,108]. Other amino acid residues participating in the catalytic reactions were proposed. Lou et al. elaborated the mechanism in the study of 7α–HSDH from Clostridium absonum [94], it was found that the serine in Ser–Tyr–Lys triad was replaced by threonine, without a complete activity loss of 7α–HSDH. In the study of 3(20)β–HSDH, it was found that the Asp participated in the catalysis, which formed an active site tetrad Ser–Tyr–Lys–Asp. Crystals 2021, 11, 224Crystals 2021, 11, 224 13 of 25 13 of 24

Figure 10. TheFigure catalytic 10. mechanismThe catalytic of mechanismHSDHs from of the HSDHs SDR superfamily. from the SDR superfamily.

The catalytic4.2. mechanism Catalytic Mechanism (Figure 10) of of HSDHs SDRs was from investigated AKRs through X-ray crystal- lographic structure determinationAll HSDHs catalyze of 3α(20 theβ oxidations)–HSDH in of a hydroxyl study by to Ghosh ketone/aldehyde et al. [106]. on It steroid-skeleton was proposed thatsubstrates the catalytic or their function reversible of 3α reaction,(20β)–HSDH with was the assistancerealized by of a NAD(P)(H)triad Ser– and sharing a Tyr–Lys. Tyr–OHsimilar acts as catalytic a proton mechanism donor applying no matter for an in electrophilic SDR or in AKR. attack However, on the 20- unlike in SDRs, it keto oxygen in thewas reduction a tetrad Asp–Tyr–Lys–His of 20-keto [106]. insteadLys–NH of3+ a is triad close Ser–Tyr–Lys to Tyr, facilitating play a catalytic the role in AKRs. transformation ofAKR–HSDHs the proton. Ser accept participates 4-pro-R hydridein catalysis from through A-face ofdirect NADPH, interaction while with SDR–HSDHs accept Tyr–OH or just 4-pro-Sas part hydrideof a proton-relay from B-face network NAD(P)H [106]. [61 Ta,113naka,114 et]. Inal. theproposed verification a three- experiment of these step catalytic mechanismsites, no activityfor 7α–HSDH of the enzyme and confirmed was detected it through with the a mutation mutation of of Y48F, Tyr159, while 1460-fold less Lys163, and Ser146active [92]. in Firstly, K77M mutationthe Lys163 and binds 8000-folds to NAD(H) increasing and lowers of Km inthe H110N pKa value mutation of was observed Tyr159 and thenin Tyr159 aldose was reductase deprotonated (EC 1.1.1.21) and interacted [115–117]. with the C7 position of 7-oxo- glycochenodeoxycholicAn acid ordered (7-oxo–GCDCA), “bi–bi” kinetic th mechanisme intermediate of AKRswas stabilized was proposed, by Ser146 in which coenzyme with a hydrogenbinds bond first to C7–O and leaves. Secondly, last [the16, 118deprotonated]. Cooper et Ty al.r acts described as a catalytic the mechanism base detailed in and extracts the thehydrogen structure from of ratC7–OH. 3α–HSDH NAD (AKR1C9),+ accepts another which containshydrogen nine released discrete from steps in the kinetic position C7 in themechanism same time and[92,112]. is associated In the studies with of 16 DADH, rate constants. the theoretical Firstly, calculations the coenzyme binds to the suggested that theenzyme proton and was forms released a loose from structure a coupled of the irregular coenzyme ionization factor complex, of the active and then the complex site groups Tyr151undergoes and Lys155 two instead conformational of a single changes residue to Tyr151[91,108]. make the binding tighter. It was found that an Other amino86-fold acid residues and 110-fold participating increase in ofthe binding catalytic affinity reactions for were NADPH proposed. and NADP Lou + was realized et al. elaborated afterthe mechanism successive conformationalin the study of 7 changes,α–HSDH via from the verificationClostridium ofabsonum steady-state, [94], transient state it was found thatkinetics, the serine and in kinetic Ser–Tyr isotope–Lys effectstriad was [103 ,replaced117]. by threonine, without a complete activity lossA of “push–pull” 7α–HSDH. In catalytic the study mechanism of 3(20)β– wasHSDH, proposed it was tofound explanate that the the reduced and Asp participatedoxidated in the catalysis, reactions which of AKRs formed (Figure an active 11)[ 119site]. tetrad In a reduction Ser–Tyr– directionLys–Asp. at pH 6.0, the side chain amino group(–NH2) of Lys was protonated, which was then interacted with the 4.2. Catalytic Mechanismphenolic of groupHSDHs (–OH) from AKRs of Tyr. In the meantime, the imidazole group of His attracted hydrogen at 3-N position, which was also interacted with the phenolic group (–OH) of Tyr. All HSDHs catalyze the oxidations of hydroxyl to ketone/aldehyde on steroid-skele- These two interactions ultimately reduce the pKa value of Tyr and promote the transfer of ton substrates or their reversible reaction, with the assistance of NAD(P)(H) and sharing protons. Meanwhile, the Asp also participates in the reaction through a salt bridge to Lys. a similar catalytic mechanism no matter in SDR or in AKR. However, unlike in SDRs, it In an oxidation direction, the interaction between –COO– of Asp and –NH of Lys promotes was a tetrad Asp–Tyr–Lys–His instead of a triad Ser–Tyr–Lys play a catalytic role in2 the abstraction of Tyr phenolate anion to hydrogen from the steroid alcohol [113–120]. AKRs. AKR–HSDHs accept 4-pro-R hydride from A-face of NADPH, while SDR–HSDHs accept 4-pro-S hydride from B-face NAD(P)H [61,113,114]. In the verification experiment of these sites, no activity of the enzyme was detected with a mutation of Y48F, while 1460- fold less active in K77M mutation and 8000-folds increasing of Km in H110N mutation was observed in aldose reductase (EC 1.1.1.21) [115–117]. An ordered “bi–bi” kinetic mechanism of AKRs was proposed, in which coenzyme binds first and leaves last [16,118]. Cooper et al. described the mechanism detailed in the structure of rat 3α–HSDH (AKR1C9), which contains nine discrete steps in the kinetic mechanism and is associated with 16 rate constants. Firstly, the coenzyme binds to the

Crystals 2021, 11, 224 14 of 25

enzyme and forms a loose structure of the coenzyme factor complex, and then the complex undergoes two conformational changes to make the binding tighter. It was found that an 86-fold and 110-fold increase of binding affinity for NADPH and NADP+ was realized after successive conformational changes, via the verification of steady-state, transient state kinetics, and kinetic isotope effects [103,117]. A “push–pull” catalytic mechanism was proposed to explanate the reduced and ox- idated reactions of AKRs (Figure 11) [119]. In a reduction direction at pH 6.0, the side chain amino group(–NH2) of Lys was protonated, which was then interacted with the phenolic group (–OH) of Tyr. In the meantime, the imidazole group of His attracted hy- drogen at 3-N position, which was also interacted with the phenolic group (–OH) of Tyr. These two interactions ultimately reduce the pKa value of Tyr and promote the transfer of protons. Meanwhile, the Asp also participates in the reaction through a salt bridge to Lys. In an oxidation direction, the interaction between –COO– of Asp and –NH2 of Lys Crystals 2021, 11, 224 promotes the abstraction of Tyr phenolate anion to hydrogen from the steroid alcohol14 of 24 [113–120].

FigureFigure 11. 11. TheThe catalytic catalytic mechanism mechanism of of HS HSDHsDHs from from the the AKR AKR superfamily. superfamily.

5.5. Stereospecificity Stereospecificity Study Study of of HSDHs HSDHs through through Molecular Molecular Docking Docking InIn the the reduction reduction direction direction of ofHSDHs, HSDHs, the the carbonyl carbonyl is reduced is reduced to α–OH to α–OH or β–OH. or β–OH. The stereoisomericThe stereoisomeric differences differences at different at different positions positions will lead will to leadfunctional to functional divergences divergences of ster- oidof steroidmolecules. molecules. For example, For example, both UDCA both and UDCA CDCA and are CDCA reduction are reduction products products of 7-keto- of LCA7-keto-LCA (7–KLA), (7–KLA), with an withα–OH an inα CDCA–OH in and CDCA β–OH and inβ UDCA.–OH in However, UDCA. However, UDCA exhibits UDCA muchexhibits better much performance better performance than CDCA than in CDCA dissolvin in dissolvingg cholesterol, cholesterol, with less with toxicity. less toxicity.In the processIn the process of UDCA of UDCA synthesis synthesis with a with substrate a substrate of CDCA, of CDCA, 7–KLA 7–KLA (C7=O) (C7=O) usually usually occurs occurs as anas intermediate. an intermediate. The Thetwo-step two-step proc processess including including the oxidization the oxidization of 7α of–OH 7α –OHfirstly firstly and then and thethen reduction the reduction of 7-keto of 7-keto group group [121–123]. [121–123 Both]. Both reactions reactions require require NAD(P)(H) NAD(P)(H) as as a aproton proton acceptoracceptor or or donor donor to to participate. participate. Other Other crys crystaltal structures structures of of HSDHs HSDHs were were reported, reported, such such asas 3 3αα–HSDH,–HSDH, 3β 3–HSDH,β–HSDH, 17 17α–HSDH,α–HSDH, 17β 17–HSDH,β–HSDH, etc. etc. In the In ster theeospecificity stereospecificity study study of 7β of– HSDH,7β–HSDH, Savino Savino et al. etproposed al. proposed a possibilit a possibilityy that the that substrates the substrates were bound were boundto 7α–HSDH to 7α– andHSDH 7β–HSDH and 7β –HSDHin opposite in opposite orientations, orientations, which caused which causedopposite opposite stereoselectivity stereoselectivity of prod- of ucts,products, based basedon a structural on a structural alignment alignment of 7β–HSDH of 7β–HSDH (apo-form) (apo-form) [124] and [124 7]α and–HSDH 7α–HSDH (holo- form)(holo-form) [92]. [92]. OtherOther SDRs, SDRs, such such as as tropinon tropinonee reductase reductase (EC (EC 1.1.1.236) 1.1.1.236) TR TR-I-I and and TR-II, TR-II, are are able able to to stereospecificallystereospecifically reduce reduce the the 3-keto 3-keto group group of of tropinone tropinone to to stereoisomeric stereoisomeric forms forms the the enan- enan- tiomerstiomers tropine tropine ( α-hydroxy) andand pseudotropinepseudotropine ( β(β-hydroxy).-hydroxy). It It is is believed believed that that the the reaction reac- tionstereospecificities stereospecificities of TR-Iof TR-I and and TR-II TR-II are are related related to to the the electrostaticelectrostatic environmentenvironment of of the the substrate-bindingsubstrate-binding pocket, pocket, whic whichh is is caused caused by by differently differently charged charged residues residues (positively (positively charged His112 in TR-I while negatively charged Glu156 in TR-II). The substrate-binding directions are determined by charged residues in the pocket, which lead to the stereospeci- ficity of reduction products [125,126]. (R)-hydroxypropyl-coenzyme M dehydrogenase (R-HPCDH) and (S)-hydroxypropyl-coenzyme M dehydrogenase (S-HPCDH) existing in epoxide metabolism of Xanthobacter autotrophicus Py2, usually catalyze the oxidization of (R)- and (S)-enantiomers of 2-hydroxypropyl coenzyme to the achiral product 2-ketopropyl- coenyme M, respectively. A negatively charged sulfonate group, participating in the reaction as a convenient “handle” for substrate binding, was considered related to the substrate binding orientation [127,128]. Other enzymes, such as naphthalene dioxygenase (NDO) from Pseudomonas sp. are able to catalyze cis-dihydroxylation of multiple substrates, whose stereospecificity is related to Phe-352 [129–131].

5.1. The Methods of Molecular Docking In order to explore the stereoisomeric characteristics of HSDHs, the holo-form of 7β–HSDH (PDB: 5GT9), 17β–HSDH (PDB: 3QWH) [132] from the SDR superfamily and 3(17)α–HSDH (PDB: 2P5N) [133] from AKR superfamily were used as receptors for molec- ular docking. The 7β–HSDH (PDB: 5GT9) structure contains two monomers (A and B), Crystals 2021, 11, 224 15 of 24

with each one binding one NADP+ molecule. The 17β–HSDH (PDB: 3QWH) structure contains four monomers (A, B, C, and D), and each one is complexed with a NADP+, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one and di(hydroxyethyl)ether. The 3(17)α–HSDH (PDB: 2P5N) contains two crystallo-graphically independent monomers, with each one binding an NADPH and (4S)-2-methyl-2,4-pentanediol (Protein Data Bank: https://www.rcsb.org/structure/3QWH (accessed on 29 January 2021) and https://www. rcsb.org/structure/2P5N (accessed on 29 January 2021)). For simplifying the docking, we used a single subunit as the receptor. In 5GT9, the conformations of NADP+ in subunits A and B are quite different, and subunit B was finally used as receptor after structure alignment or comparison with 7α–HSDH (PDB:1FMC) and 11β–HSDH (PDB:3G49), for they share similar NAD(P)(H) conformation. In 3QWH and 2P5N, the NADP(H) conformations are similar between subunits, and other molecules except NADP(H) were removed. Two ligands, 7–KLA and androstenedione, were downloaded from ZINC (http://zinc.docking.org/substances/ZINC000004428526/ (accessed on 29 January 2021) and http://zinc.docking.org/substances/ZINC0000135 47326/ (accessed on 29 January 2021)). Both ligands and receptors were processed for energy minimization before docking. A suitable cell around the substrate-binding pocket of receptors was set. The files should be named X_receptor and X_ligand (X represents the same letters or numbers) and the files were placed in the same folder. The docking was realized via YASARA (distributed by YASARA Biosciences GmbH, Vienna, VIE, AUT. http://www.yasara.org/ (accessed on 29 January 2021)) under pH 7.4 with a forcefield of AMBER03. In order to make the substrate bind the binding pocket better, several flexible residues were set, which in 5GT9 are Val 20, His 95, Tyr156, Lys160, Thr189, Thr191, Ser193, and Tyr253; in 3QWH are Val107, Phe109, Ser153, Asn154, Phe159, Tyr167, Pro197, Gly198, Gly199, Thr200, Phe205, His206, Glu207, Val208, Ser209, Tyr212, Ile213, Pro214, Met227, Ala228, Ala231, Asp266, and Ala269; in 2P5N are Lys31, Tyr55, His117, Tyr118, Phe219, Tyr224, Gly225 Gly226, and Trp227. Setting files X_receptor and X_ligand as the targets through YASARA, the docking was realized by dock_run.mcr, and then dock_play. mcr was used for further analysis.

5.2. Results and Discussion of Molecular Docking A crystal structure of the ternary complex of 7α–HSDH, NAD+ and 7-oxo–GCDCA was obtained in a study by Tanaka N et al. [92]. In the crystal structure, 7-oxo–GCDCA is bound to 7α–HSDH–NADH complex in the orientation of α side toward catalytic residue Tyr159 and β side towards NADH. A hydrogen-bond network was constructed among the C24=O of 7-oxo–GCDCA, Gly97, the adenosine ribose region, and the pyrophosphate region of NADH as well as water molecules for substrate binding, with a binding direction of substrate C24=O towards C-terminal and C3–OH toward N-terminal of 7α–HSDH. Hydrogen bonds directed or mediated by water molecules exist among C3–OH, Asn151, and Glu253. At the active sites, hydrogen-bond connections of [Ser146–OH··· 2.6 Å··· O– C7] and [Tyr163–OH··· 2.8 Å··· O–C7] were generated among C7=O of substrate and side chains of Ser146 and Tyr159. The structure binding model is analogous when in a tetramer crystal of 7α–HSDH from Clostridium absonum, even though with a coenzyme of NADP+ and a substrate of taurochenodeoxycholic acid (TCDCA); even the Ser of Tyr–Lys–Ser was changed by Thr [94]. The semi-flexible molecular docking was realized using the holo-form structure of 7β–HSDH–NADP+ as a receptor, and the 7–KLA as a ligand. The residues Val 20, His 95, Tyr156, Lys160, Thr189, Thr191, Ser193, and Tyr253 were set as flexible residues, and the results are shown in Figure 12. The substrate 7–KLA binds to the binary 7β–HSDH–NADP+ in the orientation of β side toward catalytic residue Tyr156 and α side toward NADP+, with a distance of 4.0 Å and 3.8 Å between C7–O and Tyr156–OH and C4–nicotinamide group ([Tyr163–OH··· 3.1 Å··· O–C7] and [C7–O··· 3.4 Å··· C4–nicotinamide]), respec- tively, which is opposite to 7α–HSDH. The binding energy is 7.79 kcal/mol. Three key residues His95, Ser193, and Tyr253 are probably related to the orientation of substrate bind- Crystals 2021, 11, 224 16 of 25

Crystals 2021, 11, 224 16 of 24

region of NADH as well as water molecules for substrate binding, with a binding direc- tion of substrate C24=O towards C-terminal and C3–OH toward N-terminal of 7α–HSDH. ing,Hydrogen which bonds conserved directed in7β or–HSDH mediated and by definitely water molecules different exist from among 7α–HSDHs C3–OH, (Figure Asn151, 13 ). Anand obstruction Glu253. At the formed active by sites, the hydrogen-bond side chain of His95, connections Ser193 of and [Ser146–OH···2.6 the pyrophosphate Å···O–C7] group + ofand NADP [Tyr163–OH···2.8makes the substrateÅ···O–C7] onlywere ablegenerated to bind among to the C7=O pocket of ofsubstrate the nicotinamide and side chains region + forof Ser146 the happening and Tyr159. of reaction,The structure which binding make model the β side is analogous of the substrate when in towarda tetramer the crystal NADP impossible.of 7α–HSDH The from orientation, Clostridium with absonum C3–OH, even toward though C-terminal with a coenzyme (or the adenosine of NADP region+ and a of + + NADPsubstrate) andof taurochenodeoxycholic C24–O towards N-terminal acid (or(TCDCA); nicotinamide even the region Ser ofof NADPTyr–Lys–Ser), helped was to thechanged formation by Thr of [94]. this binding model.

FigureFigure 12.12. TheThe substrate-binding substrate-binding model model in in7α–HSDH 7α–HSDH (PDB: (PDB: 1FMC) 1FMC) and the and molecular the molecular docking docking resultsresults ofof 7–KLA to to 7 7ββ–HSDH–HSDH (PDB: (PDB: 5GT9), 5GT9), androstenedione androstenedione to 17 toβ–HSDH 17β–HSDH (PDB: (PDB: 3QWH) 3QWH) and and 3(17)3(17)αα–HSDH–HSDH (PDB: 2P5N), 2P5N), respective respectively.ly. The The distances distances between between the thereaction reaction position position C7=O C7=O (in (in 77αα–HSDH–HSDH and 7 β–HSDH)–HSDH) and and Tyr–OH, Tyr–OH, C4 C4 of of nicotinamide nicotinamide and and the thereaction reaction position position C17=O C17=O (in (in 17β–HSDH and 3(17)α–HSDH) and Tyr–OH, C4 of nicotinamide were measured using Pymol and shown in blue color.

Crystals 2021, 11, 224 18 of 25

dihydrotestosterone (5α–DHT), catalyzing the reduction of 5α–DHT to produce 3α– and Crystals 2021, 11, 224 3β–androstanediol. The molecular docking was realized using 5α–DHT as a ligand and 17 of 24 AKR1C1 and AKR1C2 as receptors. It was found that the A-ring of steroid 5α–DHT pre- sented its β-face to the 4-pro-R hydrogen when docked into AKR1C2(3β–HSDHs),

Figure 13. Sequence alignment of 7α–HSDHs and 7β–HSDHs. The accession number are: 7β– HSDH from Collinsella aerofaciens, UniProtKB/Swiss-Prot A4ECA9.1; from Ruminococcus gnavus, UniProtKB/Swiss-Prot A7B4V1.1; from Clostridium sardiniense, GenBank AET80684.1; from Ruminococ-

cus torques, GenBank CBL26204.1; from Eubacterium limosum, WP_038354045.1; from Clostridium nige- riense, WP_066892209.1; from Libanicoccus massiliensis, WP_073294202.1. 7α–HSDH from Escherichia coli, UniProtKB/Swiss-Prot P0AET8.1; from Clostridium sardiniense, UniProtKB/Swiss-Prot G9FRD7.1; from Shigella dysenteriae, GenBank ABB61947.1; from Bacteroides fragilis, GenBank AAD49430.2; from Shigella boydii, GenBank ABB66134.1; from Bosea sp., GenBank OYW60865.1; from Kaistia adipate, WP_029074103.1; from Grimontia sp., WP_046304034.1; from Brevundimonas, WP_046653274.1; from Erythrobacter atlanticus, WP_048884278.1; from Shewanella morhuae, WP_076500293.1; from Pseudoruege- ria aquimaris, WP_085868047.1. This figure was created by ESPript on line [134]. Crystals 2021, 11, 224 18 of 24

The differences of substrate binding between SDRs and AKRs were analyzed through a semi-flexible molecular docking of ligand androstenedione to 17β–HSDH-NADP+ (PDB: 3QWH) from SDR superfamily and 3α(17α)–HSDH–NADP+ (PDB: 2P5N) from AKR superfamily, with flexible residues Val107, Phe109, Ser153, Asn154, Phe159, Tyr167, Pro197, Gly198, Gly199, Thr200, Phe205, His206, Glu207, Val208, Ser209, Tyr212, Ile213, Pro214, Met227, Ala228, Ala231, Asp266, and Ala269 in 17β–HSDH, as well as Lys31, Tyr55, His117, Tyr118, Phe219, Tyr224, Gly225 Gly226, and Trp227 in 3α(17α)–HSDH. The results were shown in Figure 12. In AKRs, the substrate-binding pocket is composed of loops between αA-βA (residues 21–30), αB–βB (residues 52–60), and αG–βG (residues 220–228). The binding directions of substrate and coenzyme are vertical in space, while it is parallel in SDRs, which may be the reason why HSDHs with a catalytic function at positions 7, 11, and 12 are mainly present in SDRs. The relative position of substrate binding pocket and coenzyme binding pocket makes it difficult for the positions 7, 11, and 12 of the steroid- skeleton substrates to approach the catalytic center, which impacts the proton transfer among substrate, catalytic residues, and nicotinamide group of NAD(P)(H). The catalytic residues in 17β–HSDH and 3α(17α)–HSDH are Ser–Tyr–Lys and Asp– Tyr–Lys–His, respectively. The hydrogen of hydroxyl (–OH) at substrate reaction position was from the catalytic residue Tyr when in a reduction direction. Androstenedione is bound to 17β–HSDH–NADP+ binary with an orientation of β side towards catalytic residue Tyr and α side towards B-face of nicotinamide group, with a distance of 3.1 Å and 3.4 Å between C17=O and Tyr–OH and C4–nicotinamide group in 17β–HSDH. The ligand androstenedione binds to 3α(17α)–HSDH–NADP+ binary in an opposite orientation to 17β–HSDH, with a distance of 3.8 Å and 3.6 Å between C17=O and Tyr–OH and C4– nicotinamide group in 3α(17α)–HSDH. A docking result of C3=O of the ligand toward catalytic residue Tyr and α side toward A-face of nicotinamide group was obtained as well, with a distance of 6.2 Å and 3.4 Å between C3=O and Tyr–OH and C4–nicotinamide group. Further determination of the binding method may require molecular dynamics simulation. The catalytic mechanism of 3α(17α)–HSDH (AKR1C21) for different reactions on posi- tions C3 and C17 has been studied through molecular docking. 3-keto/3α-hydroxysteroids and the 17-keto steroids were used for docking to 3α(17α)–HSDH. C3 and C17 steroid substrates exhibit different binding orientations due to hydrogen-bonding interactions affected by residues Lys31, Gly225, and Gly226 in the substrate-binding pocket. Human HSDHs of AKR1C1 and AKR1C2 usually act as 3α/3β–HSDHs in the reduction of 5α- dihydrotestosterone (5α–DHT), catalyzing the reduction of 5α–DHT to produce 3α– and 3β–androstanediol. The molecular docking was realized using 5α–DHT as a ligand and AKR1C1 and AKR1C2 as receptors. It was found that the A-ring of steroid 5α–DHT presented its β-face to the 4-pro-R hydrogen when docked into AKR1C2(3β–HSDHs), Whereas A-ring of 5α–DHT presented its α-face to the 4-pro-R hydrogen of coenzyme when docked into AKR1C1(3α–HSDHs). The different orientations may be related to different residues of the steroid-binding pockets, which is Leu of AKR1C1 while Val in AKR1C2 [100,133,135].

6. Conclusions In this article, the HSDHs from the SDR and AKR superfamilies were summarized and classified according to their structural and functional characteristics. The different NAD(P)(H)-binding models of these two types of HSDHs were analyzed, with the pro-S side of nicotinamide group towards catalytic residues in SDR superfamily while pro-R side of nicotinamide group towards catalytic residues in AKR superfamily. It impacts the catalytic mechanisms of SDR–HSDHs and AKR–HSDHs. AKR–HSDHs accept 4-pro-R hydride from NADPH, while SDR–HSDHs accept 4-pro-S hydride from NAD(P)H. The majority of AKR–HSDHs are NADPH-dependent and conserved in coenzyme conforma- tions. However, the conformations of the NAD(P)(H) show large differences in the SDR superfamily, especially between α–HSDHs and β–HSDHs. The different conformation in the pyrophosphate region of NAD(P)(H) between 7α–HSDH and 7β–HSDH attracted Crystals 2021, 11, 224 19 of 24

much attention. The dihedral angle (PA–O3–PN–O5D) of the pyrophosphate region is roughly –140◦ in two structures of 7α–HSDH with different coenzymes, while it is –80.6◦ in 7β–HSDH. A typical pair of stereoisomeric enzymes, 7α–HSDH and 7β–HSDH, were used to analyze the stereospecificity of HSDHs. A semi-flexible molecular docking was realized using the holo-form of 7β–HSDH–NADP+ as a receptor and 7–KLA as a ligand via YASARA. A possible binding mode of 7β–HSDH and 7–KLA was obtained. The result was confirmed by comparison to a ternary complex of 7α–HSDH. A hydrophilic face (α side) of 7–KLA toward NADP+ in 7β–HSDH made the orientation of C7–OH different in the prod- uct, while a hydrophobic face (β side) towards NAD+ in 7α–HSDH. One reason leading to the different substrate orientations is the different conformation of the pyrophosphate region of coenzymes caused by Ser193 in 7β–HSDH. An interaction toward pyrophosphate [Ser193–OG··· 3.11 Å··· O1N–PN] caused the upturning of PN–phosphate, which formed a barrier with the side chain of His95 to prevent 7–KLA from passing through this region and could only be combined with NADP+ as α side towards nicotinamide group. A possi- ble interaction existing in the Tyr253 of 7β–HSDH and C24–O of 7–KLA may contribute to the formation of substrate binding orientation. The results of sequence alignment showed the conservative of these three residues (His95, Ser193, Tyr253) in 7β–HSDH, which had a significant difference to 7α–HSDH. The molecular docking of the other two enzymes, 17β–HSDH from the SDR superfamily and 3(17)α–HSDH from the AKR superfamily, has further verified the relation between stereospecificity of HSDHs and substrate binding orientation. However, the results still need a further mutation to confirm in future studies.

Supplementary Materials: The following are available online at https://www.mdpi.com/2073-435 2/11/3/224/s1. Figure S1: Sequence alignment of HSDHs from SDR superfamily, Table S1: HSDHs identified belonging to SDR superfamily, Table S2: HSDHs identified belonging to AKR superfamily, Table S3: Functions of HSDHs and their related metabolic pathways, Table S4: Residues that interact with NAD(P)(H) in AKR superfamily and their conformation differences of coenzyme. Author Contributions: Conceptualization, L.L. and K.N.; methodology, M.G. and H.X.; validation, M.Q., L.L. and M.G.; resources, H.X.; data curation, L.L. and M.G.; writing—original draft preparation, L.L. and M.G.; writing—review and editing, L.L. and M.Q.; supervision, F.W.; All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (grant number 21978017, 21978020, 21861132017). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Kisiela, M.; Skarka, A.; Ebert, B.; Maser, E. Hydroxysteroid dehydrogenases (HSDs) in bacteria—A bioinformatic perspective. J. Steroid Biochem. Mol. Biol. 2012, 129, 31–46. [CrossRef] 2. Penning, T.M. Human hydroxysteroid dehydrogenases and pre-receptor regulation: Insights into inhibitor design and evaluation. J. Steroid Biochem. Mol. Biol. 2011, 125, 46–56. [CrossRef][PubMed] 3. Yoshimoto, T.; Higashi, H.; Kanatani, A.; Lin, X.S.; Nagai, H.; Oyama, H.; Kurazono, K.; Tsuru, D. Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme. J. Bacteriol. 1991, 173, 2173–2179. [CrossRef][PubMed] 4. Hirano, S.; Masuda, N. Characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens. Appl. Environ. Microbiol. 1982, 43, 1057–1063. [CrossRef] 5. Tannin, G.M.; Agarwal, A.K.; Monder, C.; New, M.I.; White, P.C. The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. J. Biol. Chem. 1991, 266, 16653–16658. [CrossRef] 6. Jörnvall, H.; Höög, J.-O.; Persson, B. SDR and MDR: Completed genome sequences show these protein families to be large, of old origin, and of complex nature. FEBS Lett. 1999, 445, 261–264. [CrossRef] 7. Jörnvall, H.; Persson, B.; Krook, M.; Atrian, S.; Gonzalez-Duarte, R.; Jeffery, J.; Ghosh, D. Short-chain dehydrogenases/reductases (SDR). Biochemistry 1995, 34, 6003–6013. [CrossRef] Crystals 2021, 11, 224 20 of 24

8. Penning, T.M. The aldo-keto reductases (AKRs): Overview. Chem. Interact. 2015, 234, 236–246. [CrossRef] 9. Jez, J.M.; Bennett, M.J.; Schlegel, B.P.; Lewis, M.; Penning, T.M. Comparative anatomy of the aldo–keto reductase superfamily. Biochem. J. 1997, 326, 625–636. [CrossRef][PubMed] 10. Möbus, E.; Maser, E. Molecular Cloning, Overexpression, and Characterization of Steroid-inducible 3α-Hydroxysteroid Dehydro- genase/Carbonyl Reductase from Comamonas testosteroni. J. Biol. Chem. 1998, 273, 30888–30896. [CrossRef] 11. Suzuki, K.; Ueda, S.; Sugiyama, M.; Imamura, S. Cloning and expression of a Pseudomonas 3α-hydroxy steroid dehydrogenase- encoding gene in Escherichia coli. Gene 1993, 130, 137–140. [CrossRef] 12. Bennett, M.J.; Schlegel, B.P.; Jez, J.M.; Penning, T.M.; Lewis, M. Structure of 3α-Hydroxysteroid/Dihydrodiol Dehydrogenase Complexed with NADP+. Biochemistry 1996, 35, 10702–10711. [CrossRef] 13. Deyashiki, Y.; Ogasawara, A.; Nakayama, T.; Nakanishi, M.; Miyabe, Y.; Sato, K.; Hara, A. Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder. Biochem. J. 1994, 299, 545–552. [CrossRef][PubMed] 14. DeLano, W.L. The PyMol Molecular Graphics System. Proteins 2002, 30, 442–454. 15. Bashton, M.; Chothia, C. The geometry of domain combination in proteins 1 1Edited by J. Thornton. J. Mol. Biol. 2002, 315, 927–939. [CrossRef] 16. Hyndman, D.; Bauman, D.R.; Heredia, V.V.; Penning, T.M. The aldo-keto reductase superfamily homepage. Chem. Interact. 2003, 144, 621–631. [CrossRef] 17. Chiang, J.Y.L. Bile acids: Regulation of synthesis. J. Lipid Res. 2009, 50, 1955–1966. [CrossRef] 18. Schiffer, L.; Barnard, L.; Baranowski, E.S.; Gilligan, L.C.; Taylor, A.E.; Arlt, W.; Shackleton, C.H.; Storbeck, K.-H. Human steroid biosynthesis, metabolism and excretion are differentially reflected by serum and urine steroid metabolomes: A comprehensive review. J. Steroid Biochem. Mol. Biol. 2019, 194, 105439. [CrossRef][PubMed] 19. Higaki, Y.; Kamiya, T.; Usami, N.; Shintani, S.; Shiraishi, H.; Ishikura, S.; Yamamoto, I.; Hara, A. Molecular Characterization of Two Monkey Dihydrodiol Dehydrogenases. Drug Metab. Pharmacokinet. 2002, 17, 348–356. [CrossRef] 20. Endo, S.; Matsunaga, T.; Arai, Y.; Ikari, A.; Tajima, K.; El-Kabbani, O.; Yamano, S.; Hara, A.; Kitade, Y. Cloning and Characterization of Four Rabbit Aldo-Keto Reductases Featuring Broad Substrate Specificity for Xenobiotic and Endogenous Carbonyl Compounds: Relationship with Multiple Forms of Drug Ketone Reductases. Drug Metab. Dispos. 2014, 42, 803–812. [CrossRef] 21. Persson, B.; Kallberg, Y. Classification and nomenclature of the superfamily of short-chain dehydrogenases/reductases (SDRs). Chem. Interact. 2013, 202, 111–115. [CrossRef] 22. Maser, E. Xenobiotic carbonyl reduction and physiological steroid oxidoreduction. Biochem. Pharmacol. 1995, 49, 421–440. [CrossRef] 23. Kallberg, Y.; Oppermann, U.; Persson, B. Classification of the short-chain dehydrogenase/reductase superfamily using hidden Markov models. FEBS J. 2010, 277, 2375–2386. [CrossRef] 24. Roth, S.; Stockinger, P.; Steff, J.; Steimle, S.; Sautner, V.; Tittmann, K.; Pleiss, J.; Müller, M. Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases. ChemBioChem 2020, 21, 2615–2619. [CrossRef][PubMed] 25. Beck, K.R.; Kaserer, T.; Schuster, D.; Odermatt, A. Virtual screening applications in short-chain dehydrogenase/reductase research. J. Steroid Biochem. Mol. Biol. 2017, 171, 157–177. [CrossRef][PubMed] 26. Hoffmann, F.; Maser, E. Carbonyl Reductases and Pluripotent Hydroxysteroid Dehydrogenases of the Short-chain Dehydroge- nase/reductase Superfamily. Drug Metab. Rev. 2007, 39, 87–144. [CrossRef][PubMed] 27. Wu, X.; Lukacik, P.; Kavanagh, K.L.; Oppermann, U. SDR-type human hydroxysteroid dehydrogenases involved in steroid hormone activation. Mol. Cell. Endocrinol. 2007, 265, 71–76. [CrossRef] 28. Staley, C.; Weingarden, A.R.; Khoruts, A.; Sadowsky, M.J. Interaction of gut microbiota with bile acid metabolism and its influence on disease states. Appl. Microbiol. Biotechnol. 2017, 101, 47–64. [CrossRef] 29. Fiorucci, S.; Distrutti, E. Bile Acid-Activated Receptors, Intestinal Microbiota, and the Treatment of Metabolic Disorders. Trends Mol. Med. 2015, 21, 702–714. [CrossRef] 30. Li, J.; Dawson, P.A. Animal models to study bile acid metabolism. Biochim. Biophys. Acta BBA Mol. Basis Dis. 2019, 1865, 895–911. [CrossRef] 31. Sivamaruthi, B.S.; Fern, L.A.; Ismail, D.S.N.R.P.H.; Chaiyasut, C. The influence of probiotics on bile acids in diseases and aging. Biomed. Pharmacother. 2020, 128, 110310. [CrossRef] 32. Oppermann, U.C.T.; Maser, E. Characterization of a 3alpha-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from the Gram-Negative Bacterium Comamonas testosteroni. JBIC J. Biol. Inorg. Chem. 1996, 241, 744–749. [CrossRef][PubMed] 33. Yang, J.; Yang, S. Comparative analysis of Corynebacterium glutamicum genomes: A new perspective for the industrial production of amino acids. BMC Genom. 2017, 18, 940. [CrossRef] 34. Liu, L.; Aigner, A.; Schmid, R.D. Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Appl. Microbiol. Biotechnol. 2010, 90, 127–135. [CrossRef] 35. Chen, X.; Cui, Y.; Feng, J.; Wang, Y.; Liu, X.; Wu, Q.; Zhu, D.; Ma, Y. Flavin Oxidoreductase-Mediated Regeneration of Nicotinamide Adenine Dinucleotide with Dioxygen and Catalytic Amount of Flavin Mononucleotide for One-Pot Multi-Enzymatic Preparation of Ursodeoxycholic Acid. Adv. Synth. Catal. 2019, 361, 2497–2504. [CrossRef] 36. He, X.-L.; Wang, L.-T.; Gu, X.-Z.; Xiao, J.-X.; Qiu, W.-W. A facile synthesis of ursodeoxycholic acid and obeticholic acid from cholic acid. Steroids 2018, 140, 173–178. [CrossRef] Crystals 2021, 11, 224 21 of 24

37. Simard, J.; Ricketts, M.-L.; Gingras, S.; Soucy, P.; Feltus, F.A.; Melner, M.H. Molecular Biology of the 3β-Hydroxysteroid Dehydrogenase/∆5-∆4 Isomerase Gene Family. Endocr. Rev. 2005, 26, 525–582. [CrossRef] 38. Thomas, J.L.; Mack, V.L.; Sun, J.; Terrell, J.R.; Bucholtz, K.M. The functions of key residues in the inhibitor, substrate and cofactor sites of human 3β-hydroxysteroid dehydrogenase type 1 are validated by mutagenesis. J. Steroid Biochem. Mol. Biol. 2010, 120, 192–199. [CrossRef][PubMed] 39. Thomas, J.L.; Duax, W.L.; Addlagatta, A.; Brandt, S.; Fuller, R.R.; Norris, W. Structure/Function Relationships Responsible for Coenzyme Specificity and the Isomerase Activity of Human Type 1 3β-Hydroxysteroid Dehydrogenase/Isomerase. J. Biol. Chem. 2003, 278, 35483–35490. [CrossRef][PubMed] 40. Thomas, J.L.; Bose, H.S. Regulation of human 3-beta-hydroxysteroid dehydrogenase type-2 (3βHSD2) by molecular chaperones and the mitochondrial environment affects steroidogenesis. J. Steroid Biochem. Mol. Biol. 2015, 151, 74–84. [CrossRef] 41. Pletnev, V.Z.; Thomas, J.L.; Rhaney, F.L.; Holt, L.S.; Scaccia, L.A.; Umland, T.C.; Duax, W.L. Rational proteomics V: Structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3β-hydroxysteroid dehydrogenase/isomerase type 1. J. Steroid Biochem. Mol. Biol. 2006, 101, 50–60. [CrossRef] [PubMed] 42. Hettel, D.; Sharifi, N. HSD3B1 status as a biomarker of androgen deprivation resistance and implications for prostate cancer. Nat. Rev. Urol. 2017, 15, 191–196. [CrossRef][PubMed] 43. Santen, R.J.; Yue, W.; Wang, J.-P. Estrogen metabolites and breast cancer. Steroids 2015, 99, 61–66. [CrossRef] 44. Duarsa, G.W.K.; Sari, Y.A.; Oka, A.A.G.; Santosa, K.B.; Yudiana, I.W.; Tirtayasa, P.M.W.; Pramana, I.B.P.; Kloping, Y.P. Serum testosterone and prostate-specific antigen levels are major risk factors for prostatic volume increase among benign prostatic hyperplasia patients. Asian J. Urol. 2020, 1–9. [CrossRef] 45. Damiani, F.; Makieva, S.; Rinaldi, S.F.; Hua, L.; Marcolongo, P.; Petraglia, F.; Norman, J.E. 11β-hydroxysteroid dehydrogenase type 1 and pregnancy: Role in the timing of labour onset and in myometrial contraction. Mol. Cell. Endocrinol. 2017, 447, 79–86. [CrossRef][PubMed] 46. Dammann, C.; Stapelfeld, C.; Maser, E. Expression and activity of the cortisol-activating enzyme 11β-hydroxysteroid dehydroge- nase type 1 is tissue and species-specific. Chem. Interact. 2019, 303, 57–61. [CrossRef][PubMed] 47. Valeur, E.; Christmann-Franck, S.; Lepifre, F.; Carniato, D.; Cravo, D.; Charon, C.; Bozec, S.; Musil, D.; Hillertz, P.; Doare, L.; et al. Structure-based design of 7-azaindole-pyrrolidine amides as inhibitors of 11β-hydroxysteroid dehydrogenase type I. Bioorgan. Med. Chem. Lett. 2012, 22, 5909–5914. [CrossRef][PubMed] 48. Goldberg, F.W.; Dossetter, A.G.; Scott, J.S.; Robb, G.R.; Boyd, S.; Groombridge, S.D.; Kemmitt, P.D.; Sjögren, T.; Gutierrez, P.M.; Deschoolmeester, J.; et al. Optimization of Brain Penetrant 11β-Hydroxysteroid Dehydrogenase Type I Inhibitors and in Vivo Testing in Diet-Induced Obese Mice. J. Med. Chem. 2014, 57, 970–986. [CrossRef] 49. Siu, M.; Johnson, T.O.; Wang, Y.; Nair, S.K.; Taylor, W.D.; Cripps, S.J.; Matthews, J.J.; Edwards, M.P.; Pauly, T.A.; Ermolieff, J.; et al. N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11β-hydroxysteroid dehydrogenase type 1: Discovery of PF-915275. Bioorgan. Med. Chem. Lett. 2009, 19, 3493–3497. [CrossRef] 50. Sandeep, T.C.; Walker, B.R. Pathophysiology of modulation of local glucocorticoid levels by 11β-hydroxysteroid dehydrogenases. Trends Endocrinol. Metab. 2001, 12, 446–453. [CrossRef] 51. Zhu, Q.; Ge, F.; Dong, Y.; Sun, W.; Wang, Z.; Shan, Y.; Chen, R.; Sun, J.; Ge, R.-S. Comparison of flavonoids and isoflavonoids to inhibit rat and human 11β-hydroxysteroid dehydrogenase 1 and 2. Steroids 2018, 132, 25–32. [CrossRef][PubMed] 52. Baker, M.E. Evolution of 11β-hydroxysteroid dehydrogenase-type 1 and 11β-hydroxysteroid dehydrogenase-type 3. FEBS Lett. 2010, 584, 2279–2284. [CrossRef][PubMed] 53. Mindnich, R.; Möller, G.; Adamski, J. The role of 17 beta-hydroxysteroid dehydrogenases. Mol. Cell. Endocrinol. 2004, 218, 7–20. [CrossRef][PubMed] 54. Moeller, G.; Adamski, J. Multifunctionality of human 17β-hydroxysteroid dehydrogenases. Mol. Cell. Endocrinol. 2006, 248, 47–55. [CrossRef][PubMed] 55. Moeller, G.; Adamski, J. Integrated view on 17beta-hydroxysteroid dehydrogenases. Mol. Cell. Endocrinol. 2009, 301, 7–19. [CrossRef] 56. Marchais-Oberwinkler, S.; Henn, C.; Möller, G.; Klein, T.; Negri, M.; Oster, A.; Spadaro, A.; Werth, R.; Wetzel, M.; Xu, K.; et al. 17β-Hydroxysteroid dehydrogenases (17β-HSDs) as therapeutic targets: Protein structures, functions, and recent progress in inhibitor development. J. Steroid Biochem. Mol. Biol. 2011, 125, 66–82. [CrossRef] 57. Gangloff, A.; Shi, R.; Nahoum, V.; Lin, S. Pseudo-symmetry of C19-steroids, alternative binding orientations and multispecificity in human estrogenic 17β-hydroxysteroid dehydrogenase. FASEB J. 2002, 17, 274–276. [CrossRef] 58. Zhang, C.-Y.; Calvo, E.-L.; Yang, C.-Q.; Liu, J.; Sang, X.-Y.; Lin, S.-X. Transcriptome of 17β-hydroxysteroid dehydrogenase type 2 plays both hormone-dependent and hormone-independent roles in MCF-7 breast cancer cells. J. Steroid Biochem. Mol. Biol. 2019, 195, 105471. [CrossRef] 59. Abdelsamie, A.S.; Salah, M.; Siebenbürger, L.; Hamed, M.M.; Börger, C.; Van Koppen, C.J.; Frotscher, M.; Hartmann, R.W. Development of potential preclinical candidates with promising in vitro ADME profile for the inhibition of type 1 and type 2 17β-Hydroxysteroid dehydrogenases: Design, synthesis, and biological evaluation. Eur. J. Med. Chem. 2019, 178, 93–107. [CrossRef] 60. Ning, X.; Yang, Y.; Deng, H.; Zhang, Q.; Huang, Y.; Su, Z.; Fu, Y.; Xiang, Q.; Zhang, S. Development of 17β-hydroxysteroid dehydrogenase type 3 as a target in hormone-dependent prostate cancer therapy. Steroids 2017, 121, 10–16. [CrossRef] Crystals 2021, 11, 224 22 of 24

61. Mindnich, R.D.; Penning, T.M. Aldo-keto reductase (AKR) superfamily: Genomics and annotation. Hum. Genom. 2009, 3, 362–370. [CrossRef] 62. Jin, Y.; Penning, T.M. Aldo-keto reductases and bioactivation/detoxication. Annu. Rev. Pharmacol. Toxicol. 2007, 47, 263–292. [CrossRef] 63. Rižner, T.L.; Penning, T.M. Role of aldo–keto reductase family 1 (AKR1) enzymes in human steroid metabolism. Steroids 2014, 79, 49–63. [CrossRef] 64. Rižner, T.L.; Penning, T.M. Aldo-keto reductase 1C3—Assessment as a new target for the treatment of endometriosis. Pharmacol. Res. 2020, 152, 104446. [CrossRef] 65. Penning, T.M.; Jin, Y.; Heredia, V.V.; Lewis, M. Structure–function relationships in 3α-hydroxysteroid dehydrogenases: A compar- ison of the rat and human isoforms. J. Steroid Biochem. Mol. Biol. 2003, 85, 247–255. [CrossRef] 66. Penning, T.M.; Burczynski, M.E.; Jez, J.M.; Hung, C.-F.; Lin, H.-K.; Ma, H.; Moore, M.; Palackal, N.; Ratnam, K. Human 3α- hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: Functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem. J. 2000, 351, 67–77. [CrossRef] 67. Wan, R.; Kong, X.; Yang, Y.; Tao, S.; Chen, Y.; Teichmann, A.T.; Wieland, F.H. Role of human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C3) in the extrahepatic metabolism of the steroidal aromatase inactivator Formestane. J. Steroid Biochem. Mol. Biol. 2020, 198, 105527. [CrossRef][PubMed] 68. Penning, T.M. AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase/prostaglandin F synthase): Roles in malignancy and endocrine disorders. Mol. Cell. Endocrinol. 2019, 489, 82–91. [CrossRef][PubMed] 69. Lin, H.-K.; Steckelbroeck, S.; Fung, K.-M.; Jones, A.N.; Penning, T.M. Characterization of a monoclonal antibody for human aldo-keto reductase AKR1C3 (type 2 3α-hydroxysteroid dehydrogenase/type 5 17β-hydroxysteroid dehydrogenase); immuno- histochemical detection in breast and prostate. Steroids 2004, 69, 795–801. [CrossRef][PubMed] 70. Chang, T.S.; Lin, H.-K.; Rogers, K.A.; Brame, L.S.; Yeh, M.M.; Yang, Q.; Fung, K.-M. Expression of aldo-keto reductase family 1 member C3 (AKR1C3) in neuroendocrine tumors & adenocarcinomas of pancreas, gastrointestinal tract, and lung. Int. J. Clin. Exp. Pathol. 2013, 6, 2419–2429. 71. Šmuc, T.; Rižner, T.L. Aberrant pre-receptor regulation of estrogen and progesterone action in endometrial cancer. Mol. Cell. Endocrinol. 2009, 301, 74–82. [CrossRef][PubMed] 72. Rižner, T.L.; Lin, H.K.; Penning, T.M. Role of human type 3 3α-hydroxysteroid dehydrogenase (AKR1C2) in androgen metabolism of prostate cancer cells. Chem. Interact. 2003, 144, 401–409. [CrossRef] 73. El-Kabbani, O.; Dhagat, U.; Hara, A. Inhibitors of human 20α-hydroxysteroid dehydrogenase (AKR1C1). J. Steroid Biochem. Mol. Biol. 2011, 125, 105–111. [CrossRef] 74. El-Kabbani, O.; Scammells, P.J.; Day, T.; Dhagat, U.; Endo, S.; Matsunaga, T.; Soda, M.; Hara, A. Structure-based optimization and biological evaluation of human 20α-hydroxysteroid dehydrogenase (AKR1C1) salicylic acid-based inhibitors. Eur. J. Med. Chem. 2010, 45, 5309–5317. [CrossRef] 75. Lima, M.A.; Silva, S.V.; Jaeger, R.G.; Freitas, V.M. Progesterone decreases ovarian cancer cells migration and invasion. Steroids 2020, 161, 108680. [CrossRef][PubMed] 76. Di Renzo, G.C.; Tosto, V.; Tsibizova, V. Progesterone: History, facts, and artifacts. Best Pr. Res. Clin. Obstet. Gynaecol. 2020, 69, 2–12. [CrossRef] 77. Brožiˇc, P.; Cesar, J.; Kovaˇc, A.; Davies, M.; Johnson, A.; Fishwick, C.; Rižner, T.L.; Gobec, S. Derivatives of pyrimidine, phthalimide and anthranilic acid as inhibitors of human hydroxysteroid dehydrogenase AKR1C1. Chem. Interact. 2009, 178, 158–164. [CrossRef] 78. Beraniˇc,N.; Gobec, S.; Rižner, T.L. Progestins as inhibitors of the human 20-ketosteroid reductases, AKR1C1 and AKR1C3. Chem. Interact. 2011, 191, 227–233. [CrossRef][PubMed] 79. Vidal, L.S.; Kelly, C.L.; Mordaka, P.M.; Heap, J.T. Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application. Biochim. Biophys. Acta BBA Proteins Proteom. 2018, 1866, 327–347. [CrossRef][PubMed] 80. You, C.; Huang, R.; Wei, X.; Zhu, Z.; Zhang, Y.-H.P. Protein engineering of oxidoreductases utilizing nicotinamide-based coenzymes, with applications in synthetic biology. Synth. Syst. Biotechnol. 2017, 2, 208–218. [CrossRef][PubMed] 81. Iyanagi, T. Molecular mechanism of metabolic NAD(P)H-dependent electron-transfer systems: The role of redox cofactors. Biochim. Biophys. Acta BBA Bioenerg. 2019, 1860, 233–258. [CrossRef] 82. Filling, C.; Berndt, K.D.; Benach, J.; Knapp, S.; Prozorovski, T.; Nordling, E.; Ladenstein, R.; Jörnvall, H.; Oppermann, U. Critical Residues for Structure and Catalysis in Short-chain Dehydrogenases/Reductases. J. Biol. Chem. 2002, 277, 25677–25684. [CrossRef] 83. Kavanagh, K.L.; Jornvall, H.; Persson, B.; Oppermann, U. Medium- and short-chain dehydrogenase/reductase gene and protein families. Cell. Mol. Life Sci. 2008, 65, 3895–3906. [CrossRef] 84. Bhatia, C.; Oerum, S.; Bray, J.; Kavanagh, K.L.; Shafqat, N.; Yue, W.; Oppermann, U. Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure–activity relationships. Chem. Interact. 2015, 234, 114–125. [CrossRef][PubMed] 85. Bray, J.E.; Marsden, B.D.; Oppermann, U. The human short-chain dehydrogenase/reductase (SDR) superfamily: A bioinformatics summary. Chem. Interact 2009, 178, 99–109. [CrossRef][PubMed] 86. Filling, C.; Wu, X.; Shafqat, N.; Hult, M.; Mårtensson, E.; Shafqat, J.; Oppermann, U.C. Subcellular targeting analysis of SDR-type hydroxysteroid dehydrogenases. Mol. Cell. Endocrinol. 2001, 171, 99–101. [CrossRef] Crystals 2021, 11, 224 23 of 24

87. Duax, W.L.; Ghosh, D.; Pletnev, V. Steroid dehydrogenase structures, mechanism of action, and disease. Vitam. Horm. 2000, 58, 121–148. [CrossRef] 88. Grant, G.A. Contrasting catalytic and allosteric mechanisms for phosphoglycerate dehydrogenases. Arch. Biochem. Biophys. 2012, 519, 175–185. [CrossRef] 89. Liu, Y.; Xu, S.; Zhang, C.; Zhu, X.; Hammad, M.A.; Zhang, X.; Christian, M.; Zhang, H.; Liu, P. Hydroxysteroid dehydrogenase family proteins on lipid droplets through bacteria, C. elegans, and mammals. Biochim. Biophys. Acta BBA Mol. Cell Biol. Lipids 2018, 1863, 881–894. [CrossRef] 90. Sherbet, D.P.; Papari-Zareei, M.; Khan, N.; Sharma, K.K.; Brandmaier, A.; Rambally, S.; Chattopadhyay, A.; Andersson, S.; Agarwal, A.K.; Auchus, R.J. Cofactors, redox state, and directional preferences of hydroxysteroid dehydrogenases. Mol. Cell. Endocrinol. 2007, L, 83–88. [CrossRef] 91. Wushur, I.; Sylte, I.; Winberg, J.-O. The catalytic reaction mechanism of drosophilid alcohol dehydrogenases. Perspect. Sci. 2015, 4, 46–54. [CrossRef] 92. Tanaka, N.; Nonaka, T.; Tanabe, T.; Yoshimoto, T.; Tsuru, D.; Mitsui, Y. Crystal Structures of the Binary and Ternary Complexes of 7α-Hydroxysteroid Dehydrogenase from Escherichia coli. Biochemistry 1996, 35, 7715–7730. [CrossRef] 93. Wang, R.; Wu, J.; Jin, D.K.; Chen, Y.; Lv, Z.; Chen, Q.; Miao, Q.; Huo, X.; Wang, F. Structure of NADP+-bound 7β-hydroxysteroid dehydrogenase reveals two cofactor-binding modes. Acta Crystallogr. Sect. F Struct. Biol. Commun. 2017, 73, 246–252. [CrossRef] 94. Lou, D.; Wang, B.; Tan, J.; Zhu, L.; Cen, X.; Ji, Q.; Wang, Y. The three-dimensional structure of Clostridium absonum 7α- hydroxysteroid dehydrogenase: New insights into the conserved arginines for NADP(H) recognition. Sci. Rep. 2016, 6, 22885. [CrossRef][PubMed] 95. Zhang, B.; Zhu, D.-W.; Hu, X.-J.; Zhou, M.; Shang, P.; Lin, S.-X. Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3): The V54L mutation restricting the steroid alternative binding and enhancing the 20α-HSD activity. J. Steroid Biochem. Mol. Biol. 2014, 141, 135–143. [CrossRef][PubMed] 96. Couture, J.-F.; Legrand, P.; Cantin, L.; Labrie, F.; Luu-The, V.; Breton, R. Loop Relaxation, A Mechanism that Explains the Reduced Specificity of Rabbit 20α-Hydroxysteroid Dehydrogenase, A Member of the Aldo-Keto Reductase Superfamily. J. Mol. Biol. 2004, 339, 89–102. [CrossRef][PubMed] 97. Ma, H.; Ratnam, K.; Penning, T.M. Mutation of Nicotinamide Pocket Residues in Rat Liver 3α-Hydroxysteroid Dehydrogenase Reveals Different Modes of Cofactor Binding. Biochemistry 2000, 39, 102–109. [CrossRef] 98. Wierenga, R. The TIM-barrel fold: A versatile framework for efficient enzymes. FEBS Lett. 2001, 492, 193–198. [CrossRef] 99. Banfield, M.J.; Salvucci, M.E.; Baker, E.N.; Smith, C.A. Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 Å resolution. J. Mol. Biol. 2001, 306, 239–250. [CrossRef] 100. Jin, Y.; Penning, T.M. Molecular docking simulations of steroid substrates into human cytosolic hydroxysteroid dehydrogenases (AKR1C1 and AKR1C2): Insights into positional and stereochemical preferences. Steroids 2006, 71, 380–391. [CrossRef][PubMed] 101. Penning, T.M.; Bennett, M.J.; Smith-Hoog, S.; Schlegel, B.P.; Jez, J.M.; Lewis, M. Structure and function of 3α-hydroxysteroid dehydrogenase. Steroids 1997, 62, 101–111. [CrossRef] 102. Khan, M.S.; Qais, F.A.; Rehman, T.; Ismail, M.H.; Alokail, M.S.; Altwaijry, N.; Alafaleq, N.O.; Alajmi, M.F.; Gaber, N.S.; Alqhatani, R. Mechanistic inhibition of non-enzymatic glycation and aldose reductase activity by naringenin: Binding, enzyme kinetics and molecular docking analysis. Int. J. Biol. Macromol. 2020, 159, 87–97. [CrossRef][PubMed] 103. Cooper, W.C.; Jin, Y.; Penning, T.M. Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydroge- nase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate. J. Biol. Chem. 2007, 282, 33484–33493. [CrossRef] [PubMed] 104. Borhani, D.; Harter, T.; Petrash, J. The crystal structure of the aldose reductase NADPH binary complex. J. Biol. Chem. 1992, 267, 24841–24847. [CrossRef] 105. Persson, B.; Kallberg, Y.; Oppermann, U.; Jörnvall, H. Coenzyme-based functional assignments of short-chain dehydroge- nases/reductases (SDRs). Chem. Interact. 2003, 144, 271–278. [CrossRef] 106. Ghosh, D.; Wawrzak, Z.; Weeks, C.M.; Duax, W.L.; Erman, M. The refined three-dimensional structure of 3α,20β-hydroxysteroid dehydrogenase and possible roles of the residues conserved in short-chain dehydrogenases. Structure 1994, 2, 629–640. [CrossRef] 107. Gani, O.A.B.S.M.; Adekoya, O.A.; Giurato, L.; Spyrakis, F.; Cozzini, P.; Guccione, S.; Winberg, J.-O.; Sylte, I. Theoretical Calculations of the Catalytic Triad in Short-Chain Alcohol Dehydrogenases/Reductases. Biophys. J. 2008, 94, 1412–1427. [CrossRef] 108. Koumanov, A.; Benach, J.; Atrian, S.; Gonzàlez-Duarte, R.; Karshikoff, A.; Ladenstein, R. The catalytic mechanism of Drosophi- laalcohol dehydrogenase: Evidence for a proton relay modulated by the coupled ionization of the active site Lysine/Tyrosine pair and a NAD+ ribose OH switch. Proteins Struct. Funct. Bioinform. 2003, 51, 289–298. [CrossRef][PubMed] 109. Winberg, J.-O.; Brendskag, M.K.; Sylte, I.; Lindstad, R.I.; McKinley-Mckee, J.S. The catalytic triad in Drosophila alcohol dehydro- genase: pH, temperature and molecular modelling studies. J. Mol. Biol. 1999, 294, 601–616. [CrossRef] 110. Hovik, R.; Winberg, J.-O.; McKinley-Mckee, J.S. Drosophila melanogaster alcohol dehydrogenase. Insect Biochem. 1984, 14, 345–351. [CrossRef] 111. Wuxiuer, Y.; Morgunova, E.; Cols, N.; Popov, A.; Karshikoff, A.; Sylte, I.; Gonzàlez-Duarte, R.; Ladenstein, R.; Winberg, J.-O. An intact eight-membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity. FEBS J. 2012, 279, 2940–2956. [CrossRef] Crystals 2021, 11, 224 24 of 24

112. Tanabe, T.; Tanaka, N.; Uchikawa, K.; Kabashima, T.; Ito, K.; Nonaka, T.; Mitsui, Y.; Tsuru, M.; Yoshimoto, T. Roles of the Ser146, Tyr159, and Lys163 Residues in the Catalytic Action of 7-Hydroxysteroid Dehydrogenase from Escherichia coli. J. Biochem. 1998, 124, 634–641. [CrossRef] 113. Penning, T.M.; Drury, J.E. Human aldo–keto reductases: Function, gene regulation, and single nucleotide polymorphisms. Arch. Biochem. Biophys. 2007, 464, 241–250. [CrossRef][PubMed] 114. Flynn, T.G.; Green, N.C.; Bhatia, M.B.; El-Kabbani, O. Structure and Mechanism of Aldehyde Reductase. Adv. Exp. Med. Biol. 1995, 372, 193–201. [CrossRef][PubMed] 115. Bohren, K.M.; Grimshaw, C.E.; Lai, C.J.; Harrison, D.H.; Ringe, D.; Petsko, G.A.; Gabbay, K.H. Tyrosine-48 Is the Proton Donor and Histidine-110 Directs Substrate Stereochemical Selectivity in the Reduction Reaction of Human Aldose Reductase: Enzyme Kinetics and Crystal Structure of the Y48H Mutant Enzyme. Biochemistry 1994, 33, 2021–2032. [CrossRef] 116. Tarle, I.; Borhani, D.W.; Wilson, D.K.; Quiocho, F.A.; Petrash, J.M. Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110. J. Biol. Chem. 1993, 268, 25687–25693. [CrossRef] 117. Bohren, K.; Grimshaw, C.; Gabbay, K. Catalytic effectiveness of human aldose reductase. Critical role of C-terminal domain. J. Biol. Chem. 1992, 267, 20965–20970. [CrossRef] 118. Grimshaw, C.E.; Bohren, K.M.; Lai, C.-J.; Gabbay, K.H. Human Aldose Reductase: Rate Constants for a Mechanism Including Interconversion of Ternary Complexes by Recombinant Wild-Type Enzyme. Biochemistry 1995, 34, 14356–14365. [CrossRef] 119. Schlegel, B.P.; Jez, J.M.; Penning, T.M. Mutagenesis of 3α-Hydroxysteroid Dehydrogenase Reveals a “Push−Pull” Mechanism for Proton Transfer in Aldo−Keto Reductases. Biochemistry 1998, 37, 3538–3548. [CrossRef] 120. Penning, T. Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3α-hydroxysteroid dehydrogenase. J. Steroid Biochem. Mol. Biol. 1999, 69, 211–225. [CrossRef] 121. Eggert, T.; Bakonyi, D.; Hummel, W. Enzymatic routes for the synthesis of ursodeoxycholic acid. J. Biotechnol. 2014, 191, 11–21. [CrossRef][PubMed] 122. Lee, J.-Y.; Arai, H.; Nakamura, Y.; Fukiya, S.; Wada, M.; Yokota, A. Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon. J. Lipid Res. 2013, 54, 3062–3069. [CrossRef] 123. Zheng, M.-M.; Chen, F.-F.; Li, H.; Li, C.-X.; Xu, J.-H. Continuous Production of Ursodeoxycholic Acid by Using Two Cascade Reactors with Co-immobilized Enzymes. ChemBioChem 2018, 19, 347–353. [CrossRef] 124. Savino, S.; Ferrandi, E.E.; Forneris, F.; Rovida, S.; Riva, S.; Monti, D.; Mattevi, A. Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity. Proteins Struct. Funct. Bioinform. 2016, 84, 859–865. [CrossRef] 125. Nakajima, K.; Hashimoto, T.; Yamada, Y. Two tropinone reductases with different stereospecificities are short-chain dehydroge- nases evolved from a common ancestor. Proc. Natl. Acad. Sci. USA 1993, 90, 9591–9595. [CrossRef][PubMed] 126. Nakajima, K.; Yamashita, A.; Akama, H.; Nakatsu, T.; Kato, H.; Hashimoto, T.; Oda, J.; Yamada, Y. Crystal structures of two tropinone reductases: Different reaction stereospecificities in the same protein fold. Proc. Natl. Acad. Sci. USA 1998, 95, 4876–4881. [CrossRef][PubMed] 127. Krishnakumar, A.M.; Nocek, B.P.; Clark, D.D.; Ensign, S.A.; Peters, J.W. Structural Basis for Stereoselectivity in the (R)- and (S)-Hydroxypropylthioethanesulfonate Dehydrogenases. Biochemistry 2006, 45, 8831–8840. [CrossRef][PubMed] 128. Sliwa, D.A.; Krishnakumar, A.M.; Peters, J.W.; Ensign, S.A. Molecular Basis for Enantioselectivity in the (R)- and (S)- Hydroxypropylthioethanesulfonate Dehydrogenases, a Unique Pair of Stereoselective Short-Chain Dehydrogenases/Reductases Involved in Aliphatic Epoxide Carboxylation. Biochemistry 2010, 49, 3487–3498. [CrossRef] 129. Chen, D.; Wu, S.; Xue, H.; Jiang, J. Stereoselective catabolism of compounds by microorganisms: Catabolic pathway, molecular mechanism and potential application. Int. Biodeterior. Biodegrad. 2020, 146, 104822. [CrossRef] 130. Parales, R.E.; Lee, K.; Resnick, S.M.; Jiang, H.; Lessner, D.J.; Gibson, D.T. Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme. J. Bacteriol. 2000, 182, 1641–1649. [CrossRef] 131. Parales, R.E.; Resnick, S.M.; Yu, C.-L.; Boyd, D.R.; Sharma, N.D.; Gibson, D.T. Regioselectivity and Enantioselectivity of Naphthalene Dioxygenase during Arene cis-Dihydroxylation: Control by Phenylalanine 352 in the α Subunit. J. Bacteriol. 2000, 182, 5495–5504. [CrossRef] 132. Cassetta, A.; Stojan, J.; Krastanova, I.; Kristan, K.; Švegelj, M.B.; Lamba, D.; Rižner, T.L. Structural basis for inhibition of 17β- hydroxysteroid dehydrogenases by phytoestrogens: The case of fungal 17β-HSDcl. J. Steroid Biochem. Mol. Biol. 2017, 171, 80–93. [CrossRef][PubMed] 133. Steckelbroeck, S.; Jin, Y.; Oyesanmi, B.; Kloosterboer, H.J.; Penning, T.M. Tibolone Is Metabolized by the 3α/3β-Hydroxysteroid Dehydrogenase Activities of the Four Human Isozymes of the Aldo-Keto Reductase 1C Subfamily: Inversion of Stereospecificity with a ∆5(10)-3-Ketosteroid. Mol. Pharmacol. 2004, 66, 1702–1711. [CrossRef][PubMed] 134. Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014, 42, W320–W324. [CrossRef][PubMed] 135. Dhagat, U.; Carbone, V.; Chung, R.P.-T.; Schulze-Briese, C.; Endo, S.; Hara, A.; El-Kabbani, O. Structure of 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: An insight into the bifunctionality of the enzyme. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2007, 63, 825–830. [CrossRef]