The Impact of Bilirubin on 7 α - and 7 β -Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

: 7 α - and 7 β -hydroxysteroid dehydrogenases (HSDHs) are enzymes that can catalyze the isomerization of hydroxyl groups at site seven of bile acids. In a previous study, we found that the activities of 7 α - and 7 β -HSDHs can be inhibited by bilirubin. In order to clarify the impact, the effects of bilirubin on enzymes were studied by kinetics, spectrum, and docking analysis. The relative activity of 7 α -HSDH remained less than 40% under 1 mM bilirubin, and only 18% activity of 7 β -HSDH kept in the same condition. Using taurochenodeoxycholic acid (TCDCA) as substrate, the K m of 7 α -HSDH was up to 0.63 mM from 0.24 mM after binding with bilirubin and the K m of 7 β -HSDH rose from 1.14 mM to 1.87 mM for the catalysis of tauroursodeoxycholic acid (TUDCA). The afﬁnity of 7 α - and 7 β -HSDHs to substrates decreased with the effect of bilirubin. The binding of bilirubin with 7 α - or 7 β -HSDHs was analyzed by UV–vis, ﬂuorescence, and circular dichroism (CD) spectroscopy. The results reﬂected that bilirubin caused a slight change in the secondary structure of 7 α - or 7 β -HSDHs, and the changes were correlated with the ratio of bilirubin to enzymes. Ten candidate molecular docking results were presented to reﬂect the binding of bilirubin with 7 α - or 7 β -HSDHs and to explore the inhibition mechanism. This research provides a more in-depth understanding of the effect of bilirubin on 7 α - and 7 β -HSDHs.


Introduction
Bile acids are mainly synthesized in the liver from cholesterol, stored in the gallbladder, and metabolized in the intestine by the gut microbiota [1,2]. The specific bile acids can activate important nuclear receptors, the farnesoid X receptor (FXR), and one G protein-coupled receptor (TGR5). Microbial modifications of bile acids influence the host metabolism via the bile acid receptors FXR and TGR5, which suggest that such agonists may prove useful in the treatment of many diseases [2]. Among the bile acids, ursodeoxycholic acid (UDCA) and its conjugated form tauroursodeoxycholic acid (TUDCA) have the highest medicinal value. UDCA is the only drug for the treatment of primary biliary cirrhosis (PBC) approved by the FDA so far. UDCA has been effectively used to treat hepatobiliary disease by relieving hepatic steatosis and reducing liver fibrosis [3][4][5]. UDCA and TUDCA can prevent vision and hearing loss and can slow retinal degeneration [6,7]. With the blood-brain barrier crossing function, UDCA and TUDCA exert effects on the central nervous system, especially in Alzheimer's disease (AD) and Parkinson's disease therapies [8,9]. Furthermore, TUDCA can promote vascular repair [10], inhibit obesity [11,12], and enhance islet function [13]. A paper published in November 2022 reported that the down regulation of ACE2 mediated by UDCA can reduce susceptibility to SARS-CoV-2 [14].
According to previous studies, chenodeoxycholic acid (CDCA), and its conjugated form taurochenodeoxycholic acid (TCDCA), could be directly transformed into UDCA or TUDCA in vitro with the catalysis of 7α-and 7β-hydroxysteroid dehydrogenases cleotide phosphate (NADP + and NADPH), the two steps coupling reaction is highly efficient in preparation of UDCA and TUDCA. 7α-and 7β-HSDHs belong to the short chain dehydrogenase/reductase (SDR) family, which was discovered from a series of gut microbes such as Clostridium absonum [17], Bacteroides fragilis [18], Xanthomonas maltophilia [19], and Candidatus Ligilactobacillus [20]. Furthermore, 7α-and 7β-HSDHs have been successfully expressed in E. coli and purified using specific tags [17,21]. Furthermore, both free enzymes and immobilized enzymes are suitable for two-step catalytic reactions. The waste chicken bile is rich in TCDCA and a sufficient source of substrate can be ensured [22]. Bilirubin is one of the main components of waste chicken bile, from which substrate TCDCA is obtained. In summary, this strategy affords an efficient and clean process for production of TUDCA.
Bilirubin is a yellow compound that occurs in the normal catabolic pathway known for its antioxidant properties. Bilirubin is synthesized by the activity of biliverdin reductase on biliverdin. Both bilirubin and biliverdin are the products of heme catabolism. Bilirubin, when oxidized, reverts back to biliverdin [23,24]. Recently, bilirubin has been recognized as a hormone with endocrine actions [25]. Current studies also show that bilirubin can decrease adiposity and prevent metabolic and cardiovascular diseases [26]. Bilirubin can act as an agonist activate the melastatin-like transient receptor potential 2 TRPM2 channel to exacerbate neurotoxicity [27].The structure of bilirubin is presented in Figure 1b. The combination of substances can be achieved in a variety of ways, such as self-assembly [28,29], co-precipitation [30,31] and immobilization [32,33]. The combination of bilirubin and albumin can proceed spontaneously, and the binding of bilirubin and albumin has been explored by scientists for a long time as it controls a variety of physiological functions in human body [34][35][36]. Serum unbound bilirubin concentration can act as an ideal marker that reflects changes in bilirubin binding to albumin. Disorders of bilirubin binding to albumin may be associated with neurologic dysfunction [37]. The Bilirubin is a yellow compound that occurs in the normal catabolic pathway known for its antioxidant properties. Bilirubin is synthesized by the activity of biliverdin reductase on biliverdin. Both bilirubin and biliverdin are the products of heme catabolism. Bilirubin, when oxidized, reverts back to biliverdin [23,24]. Recently, bilirubin has been recognized as a hormone with endocrine actions [25]. Current studies also show that bilirubin can decrease adiposity and prevent metabolic and cardiovascular diseases [26]. Bilirubin can act as an agonist activate the melastatin-like transient receptor potential 2 TRPM2 channel to exacerbate neurotoxicity [27].The structure of bilirubin is presented in Figure 1b.
The combination of substances can be achieved in a variety of ways, such as selfassembly [28,29], co-precipitation [30,31] and immobilization [32,33]. The combination of bilirubin and albumin can proceed spontaneously, and the binding of bilirubin and albumin has been explored by scientists for a long time as it controls a variety of physiological functions in human body [34][35][36]. Serum unbound bilirubin concentration can act as an ideal marker that reflects changes in bilirubin binding to albumin. Disorders of bilirubin binding to albumin may be associated with neurologic dysfunction [37]. The research also found that bilirubin binding capacity and affinity to albumin is low and variable in premature infants. Specific exogenous drugs, intravenous lipids, and a series of clinical factors may adversely affect bilirubin-albumin binding [38]. Arriaga et al. found that C1 esterase activity was inhibited by unconjugated bilirubin [39]. Bilirubin showed a higher affinity for collagen (1 mg) at a concentration of approximately 25 nM and the affinity rate for bilirubin to collagen has been found to be 8.89 × 10 −3 s −1 [40]. The interaction between fibrinogen and bilirubin and the influence of bilirubin on the formation of fibrin and protection against oxidation are described by Gligorijević et al. [41].
In previous study, 7α-and 7β-HSDHs were co-immobilized on modified chitosan microspheres and used as recyclable biocatalyst for the catalysis of chicken bile [22]. We found that the activities of 7α-and 7β-HSDHs were affected by bilirubin and Cu 2+ . In particular, the TUDCA yield decreased rapidly in the presence of bilirubin. However, the previous research did not offer an in-depth analysis and explanation. It is meaningful to explore the effects of bilirubin on 7α-and 7β-HSDHs including the relative activity of enzymes, transformation rate of substrates, spectral changes in enzymes, and possible inhibition mechanisms. This research was focused on the impact of bilirubin on 7α-and 7β-HSDHs. Firstly, the properties of 7α-and 7β-HSDHs were studied after purification. Then, the effects of bilirubin on enzyme activity, kinetics, and substrate transformation were carefully measured by UV absorption and high-performance liquid chromatography (HPLC), respectively. In addition, the binding of bilirubin to 7α-or 7β-HSDHs was analyzed by UV-vis, fluorescence, and CD spectroscopy. Finally, molecular docking was used to present the binding conformation and explain the possible inhibition mechanism of bilirubin to enzymes.

Properties of 7α-and 7β-HSDHs
The target proteins, 7α-and 7β-HSDHs, were successfully expressed in recombination E. coli and purified by a GST gene fusion system in a mild condition without the process of denaturation by urea or guanidine hydrochloride. After purification and concentration, two enzymes were verified by SDS-PAGE as shown in Figure 2a. According to standard protein markers, the molecular mass of 7α-HSDH is approximately 27 kDa and 7β-HSDH is approximately 29 kDa. The results match the previously sequenced 7α-and 7β-HSDHs [17,18]. research also found that bilirubin binding capacity and affinity to albumin is low and variable in premature infants. Specific exogenous drugs, intravenous lipids, and a series of clinical factors may adversely affect bilirubin-albumin binding [38]. Arriaga et al. found that C1 esterase activity was inhibited by unconjugated bilirubin [39]. Bilirubin showed a higher affinity for collagen (1 mg) at a concentration of approximately 25 nM and the affinity rate for bilirubin to collagen has been found to be 8.89 × 10 −3 s −1 [40]. The interaction between fibrinogen and bilirubin and the influence of bilirubin on the formation of fibrin and protection against oxidation are described by Gligorijević et al. [41].
In previous study, 7α-and 7β-HSDHs were co-immobilized on modified chitosan microspheres and used as recyclable biocatalyst for the catalysis of chicken bile [22]. We found that the activities of 7α-and 7β-HSDHs were affected by bilirubin and Cu 2+ . In particular, the TUDCA yield decreased rapidly in the presence of bilirubin. However, the previous research did not offer an in-depth analysis and explanation. It is meaningful to explore the effects of bilirubin on 7α-and 7β-HSDHs including the relative activity of enzymes, transformation rate of substrates, spectral changes in enzymes, and possible inhibition mechanisms. This research was focused on the impact of bilirubin on 7α-and 7β-HSDHs. Firstly, the properties of 7α-and 7β-HSDHs were studied after purification. Then, the effects of bilirubin on enzyme activity, kinetics, and substrate transformation were carefully measured by UV absorption and high-performance liquid chromatography (HPLC), respectively. In addition, the binding of bilirubin to 7α-or 7β-HSDHs was analyzed by UV-vis, fluorescence, and CD spectroscopy. Finally, molecular docking was used to present the binding conformation and explain the possible inhibition mechanism of bilirubin to enzymes.

Properties of 7α-and 7β-HSDHs
The target proteins, 7α-and 7β-HSDHs, were successfully expressed in recombination E. coli and purified by a GST gene fusion system in a mild condition without the process of denaturation by urea or guanidine hydrochloride. After purification and concentration, two enzymes were verified by SDS-PAGE as shown in Figure 2a. According to standard protein markers, the molecular mass of 7α-HSDH is approximately 27 kDa and 7β-HSDH is approximately 29 kDa. The results match the previously sequenced 7αand 7β-HSDHs [17,18]. In addition, a peptide calculator (Qiangyao, Shanghai) was also used to explore the properties of 7α-and 7β-HSDHs [42]. 7α-and 7β-HSDHs were cloned from Clostridium absonum ATCC27555, Genbank number JN191345. The results are shown in Table 1. The variation in net charge with pH for 7α-and 7β-HSDHs is described in Figure 2b, c. Both 7α-and 7β-HSDHs are negatively charged at pH 7.0, −2.8 net charges for 7α-HSDH and In addition, a peptide calculator (Qiangyao, Shanghai, China) was also used to explore the properties of 7α-and 7β-HSDHs [42]. 7α-and 7β-HSDHs were cloned from Clostridium absonum ATCC27555, Genbank number JN191345. The results are shown in Table 1. The variation in net charge with pH for 7α-and 7β-HSDHs is described in Figure 2b,c. Both 7α-and 7β-HSDHs are negatively charged at pH 7.0, −2.8 net charges for 7α-HSDH and −4.6 net charges for 7β-HSDH. The isoelectric points of 7α-and 7β-HSDHs are 5.7 and 5.5, respectively. Two enzymes have the same ratio of hydrophilic residues (37%). The

The Effects of Bilirubin on Enzymatic Activity and Kinetics
The effects of bilirubin on the activities of 7α-and 7β-HSDHs were studied by testing various bilirubin concentrations ranging from 0 to 1 mM in 50 mM Tris-HCl buffer (pH 8.5). Enzymatic activity without the effect of bilirubin was regarded as 100%, and the results are shown in Figure 3. With the increase in bilirubin, the relative activity of two enzymes decreased sharply. The relative activity of 7α-HSDH remained less than 40% in 1 mM bilirubin. Furthermore, the activity 7β-HSDH was more seriously affected by bilirubin compared with 7α-HSDH. For 7β-HSDH, only 18% activity remained as the concentration of bilirubin was in the 1 mM in buffer. The different activity of 7α-and 7β-HSDHs at the same bilirubin concentration may be related to different inhibition mechanisms [16,17].
Catalysts 2023, 13, x FOR PEER REVIEW 4 of 18 −4.6 net charges for 7β-HSDH. The isoelectric points of 7α-and 7β-HSDHs are 5.7 and 5.5, respectively. Two enzymes have the same ratio of hydrophilic residues (37%). The molecular weights estimated by the peptide calculator (28,289.16 g/mol for 7α-HSDH, 29,382.85 g/mol for 7β-HSDH) were close to that determined by SDS-PAGE.

The Effects of Bilirubin on Enzymatic Activity and Kinetics
The effects of bilirubin on the activities of 7α-and 7β-HSDHs were studied by testing various bilirubin concentrations ranging from 0 to 1 mM in 50 mM Tris-HCl buffer (pH 8.5). Enzymatic activity without the effect of bilirubin was regarded as 100%, and the results are shown in Figure 3. With the increase in bilirubin, the relative activity of two enzymes decreased sharply. The relative activity of 7α-HSDH remained less than 40% in 1 mM bilirubin. Furthermore, the activity 7β-HSDH was more seriously affected by bilirubin compared with 7α-HSDH. For 7β-HSDH, only 18% activity remained as the concentration of bilirubin was in the 1 mM in buffer. The different activity of 7α-and 7β-HSDHs at the same bilirubin concentration may be related to different inhibition mechanisms [16,17]. Kinetic constants of 7α-HSDH and 7β-HSDH are presented in Table 2. By measuring the reduction of NADP + , we got a series of kinetic constants for 7α-and 7β-HSDHs, such as Vmax, Km, kcat, and kcat/Km. Km and Vmax of free 7α-HSDH were 0.24 mM and 22.47 µmol/min using TCDCA as substrate. After binding with bilirubin, the Km became 0.63 mM and Vmax was as low as 6.78 µmol/min. kcat changed from 6.25 × 10 3 s −1 to 1.02 × 10 3 s −1 . kcat/Km decreased from 2.60 × 10 4 s −1 mM −1 to 1.62 × 10 3 s −1 mM −1 . For cofactor NADP + , the Vmax of 7α-HSDH did not vary much. The Km of 7α-HSDH rose to 0.59 mM from 0.28 mM with the effect of bilirubin. Similarly, after binding with bilirubin, Vmax of 7β-HSDH for TUDCA decreased from 22.47 µmol/min to 4.32 µmol/min and Km of 7β-HSDH for TUDCA rose from 1.14 mM to 1.87 mM. kcat changed from 7.28 × 10 4 s −1 to 2.64 × 10 4 s −1 .  Kinetic constants of 7α-HSDH and 7β-HSDH are presented in Table 2. By measuring the reduction of NADP + , we got a series of kinetic constants for 7α-and 7β-HSDHs, such as V max , K m , k cat , and k cat /K m . K m and V max of free 7α-HSDH were 0.24 mM and 22.47 µmol/min using TCDCA as substrate. After binding with bilirubin, the K m became 0.63 mM and V max was as low as 6.78 µmol/min. k cat changed from 6.25 × 10 3 s −1 to 1.02 × 10 3 s −1 . k cat /K m decreased from 2.60 × 10 4 s −1 mM −1 to 1.62 × 10 3 s −1 mM −1 . For cofactor NADP + , the V max of 7α-HSDH did not vary much. The K m of 7α-HSDH rose to 0.59 mM from 0.28 mM with the effect of bilirubin. Similarly, after binding with bilirubin, V max of 7β-HSDH for TUDCA decreased from 22.47 µmol/min to 4.32 µmol/min and K m of 7β-HSDH for TUDCA rose from 1.14 mM to 1.87 mM. k cat changed from 7.28 × 10 4 s −1 to 2.64 × 10 4 s −1 . k cat /K m decreased from 6.39 × 10 4 s −1 mM −1 to 1.41 × 10 4 s −1 mM −1 . For cofactor NADP + , the V max of 7β-HSDH varied from 53.61 µmol/min to 19.65 µmol/min. The K m of 7β-HSDH only had a slight change from 1.37 mM to 1.32 mM with the effect of bilirubin. After binding with bilirubin, the V max of 7α-HSDH to NADP + only had a minor change and the K m of 7α-HSDH to NADP + had a significant increase. It was determined that the inhibition of bilirubin to 7α-HSDH attributed to competitive inhibition. Both the V max and K m of 7β-HSDH to NADP + decreased. It was determined that the inhibition of bilirubin to 7β-HSDH may be due to uncompetitive inhibition. After binding with bilirubin, the k cat for both 7α-HSDH and 7β-HSDH reduced, and the catalysis rate of enzymes slowed down with the inhibition of bilirubin. The results indicated that the catalytic efficiency and the transformation ability seriously decreased after the enzymes bound with bilirubin. Furthermore, the affinity of 7α-and 7β-HSDHs to substrates decreased after binding with bilirubin, which may be due to the change in their configuration. After immobilization on the modified chitosan microsphere, the kinetic parameters of 7α-and 7β-HSDHs changed similarly [16].

The Effect of Bilirubin on Enzymatic Reaction
TCDCA can be transformed into TUDCA by the isomerization of -OH at site seven with the catalysis of 7α-and 7β-HSDHs in two steps, as shown in Figure 1. The couplingreaction has been reported previously [18,19]. In order to obtain an accurate quantification of bile acids, the composition of reaction products was measured by high-performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD). The peak positions of TCDCA (approximately 4.0 min), T-7-KLCA (approximately 4.6 min), and TUDCA (approximately 6.2 min) are presented in Figure 4.
Due to the inhibition of bilirubin on enzyme activity, the rate of catalysis would slow down, and the substrate transformation and product yield would be less in a certain period of time. According to the literature, acetone-derived extracts were inhibitors for cellulase and decreased glucose yield for enzyme hydrolysis of Solka-Floc (a lignin-free cellulose) by 42% at 15 FPU/g glucan in 72 h. The probable inhibitors include phenolic compounds, monomeric and oligomeric sugars, furan derivatives, and acetic acid [43]. Baksi et al. reported that the conversion of biomass to biofuel is substantiated with specific kinetics controlling enzymatic hydrolysis in presence of unavoidable inhibitors, and reviewed that the addition of monosaccharides (excluding the end products) such as glucose, galactose, mannose, or fructose of similar concentration lowers the glucose yield to different degrees [44]. It was observed that for a particular cellulase-substrate (40 g/L)-time (eight hours) combination, cellobiose (20 g/L) posed maximum inhibition (72.3% reduction in reaction rate) whereas the reaction rate was reduced by 32%, 39%, and 49.7% with galactose, mannose, and glucose, respectively [45]. The results of the TCDCA transformation in the absence or presence of bilirubin are shown in Figures 4 and 5. With the increase in bilirubin, both TCDCA conversion and TUDCA yield were gradually decreased. Without the inhibition of bilirubin on two enzymes, TCDCA conversion was more than 90%, and the TUDCA yield was close to 60%. However, as the concentration of bilirubin was 0.8 mM in the reaction buffer, TCDCA conversion remained at 15% and the TUDCA yield was only 5% in four hours. Furthermore, from the change in peak area, we can see that there was almost no conversion of TCDCA when the concentration of bilirubin in the buffer was 0.8 mM. Except for bilirubin, the experiments showed that Cu 2+ and Ca 2+ have some inhibition to 7α-and 7β-HSDHs [22]. Due to the inhibition of bilirubin on enzyme activity, the rate of catalysis would slow down, and the substrate transformation and product yield would be less in a certain period of time. According to the literature, acetone-derived extracts were inhibitors for cellulase and decreased glucose yield for enzyme hydrolysis of Solka-Floc (a lignin-free cellulose) by 42% at 15 FPU/g glucan in 72 h. The probable inhibitors include phenolic compounds, monomeric and oligomeric sugars, furan derivatives, and acetic acid [43]. Baksi et al. reported that the conversion of biomass to biofuel is substantiated with specific kinetics controlling enzymatic hydrolysis in presence of unavoidable inhibitors, and reviewed that the addition of monosaccharides (excluding the end products) such as glucose, galactose, mannose, or fructose of similar concentration lowers the glucose yield to different degrees [44]. It was observed that for a particular cellulase-substrate (40 g/L)-time (eight hours) combination, cellobiose (20 g/L) posed maximum inhibition (72.3% reduction in reaction rate) whereas the reaction rate was reduced by 32%, 39%, and 49.7% with galactose, mannose, and glucose, respectively [45]. The results of the TCDCA transformation in the absence or presence of bilirubin are shown in Figure 4 and Figure 5. With the increase in bilirubin, both TCDCA conversion and TUDCA yield were gradually decreased. Without the inhibition of bilirubin on two enzymes, TCDCA conversion was more than 90%, and the TUDCA yield was close to 60%. However, as the concentration of bilirubin was 0.8 mM in the reaction buffer, TCDCA conversion remained at 15% and the TUDCA yield was only 5% in four hours. Furthermore, from the change in peak area, we can see that there was almost no conversion of TCDCA when the concentration of bilirubin in the buffer was 0.8 mM. Except for biliru- bin, the experiments showed that Cu 2+ and Ca 2+ have some inhibition to 7α-and 7β-HSDHs [22].

Analysis of the Binding of Enzymes and Bilirubin
To investigate the reactions of bilirubin and enzymes, samples of 7α-or 7β-HSDHs in the absence or presence of bilirubin were analyzed by UV-vis, fluorescence, and CD spectroscopy, respectively.
At first, the combinations of bilirubin to 7α-or 7β-HSDHs were analyzed by UV-vis

Analysis of the Binding of Enzymes and Bilirubin
To investigate the reactions of bilirubin and enzymes, samples of 7α-or 7β-HSDHs in the absence or presence of bilirubin were analyzed by UV-vis, fluorescence, and CD spectroscopy, respectively.
At first, the combinations of bilirubin to 7α-or 7β-HSDHs were analyzed by UV-vis spectrum from 250 nm to 600 nm. The results are presented in Figure 6. There were no absorption peaks from 300 nm to 600 nm for free 7α-and 7β-HSDHs. Bilirubin had an obvious absorption peak at approximately 438 nm, which was consistent with reference [46]. After binding to 7α-and 7β-HSDHs, the main absorption became higher. Upon binding to 7β-HSDH, the main absorption band of bilirubin shifted from 438 nm (bilirubin alone) to 442 nm. This result was similar to those previously reported. The main absorption band of bilirubin shifted from 438 nm to 460 nm after binding to human serum albumin [46]. The concentration of the substance TCDCA was 8mM with NADP + (2mM) as cofactor. The reaction time was four hours. The analyses were performed at 25 °C in 50 mM Tris-HCl buffer (pH 8.5).

Analysis of the Binding of Enzymes and Bilirubin
To investigate the reactions of bilirubin and enzymes, samples of 7α-or 7β-HSDHs in the absence or presence of bilirubin were analyzed by UV-vis, fluorescence, and CD spectroscopy, respectively.
At first, the combinations of bilirubin to 7α-or 7β-HSDHs were analyzed by UV-vis spectrum from 250 nm to 600 nm. The results are presented in Figure 6. There were no absorption peaks from 300 nm to 600 nm for free 7α-and 7β-HSDHs. Bilirubin had an obvious absorption peak at approximately 438 nm, which was consistent with reference [46]. After binding to 7α-and 7β-HSDHs, the main absorption became higher. Upon binding to 7β-HSDH, the main absorption band of bilirubin shifted from 438 nm (bilirubin alone) to 442 nm. This result was similar to those previously reported. The main absorption band of bilirubin shifted from 438 nm to 460 nm after binding to human serum albumin [46]. After the molecular interaction, the fluorescence intensity of samples decreased, and fluorescence quenching occurred. By studying this phenomenon, the binding mechanism of two molecules would be easily obtained. Quenching can be divided into After the molecular interaction, the fluorescence intensity of samples decreased, and fluorescence quenching occurred. By studying this phenomenon, the binding mechanism of two molecules would be easily obtained. Quenching can be divided into two forms: dynamic or static. In dynamic quenching, a collision happens between the fluorophore and quencher. In static quenching, a ground state complex forms between the fluorophore and quencher [47].
The interactions between bilirubin and enzymes were investigated by florescence quenching from 350 nm to 600 nm using 7α-and 7β-HSDHs as the fluorophore and bilirubin as the quencher. The results are presented in Figure 7. Figure 7a shows the fluorescence spectra emission of 7α-HSDH in the presence of bilirubin at 25 • C. 7α-HSDH exhibited a broadband emission with a maximum at 448 nm when it was excited at 280 nm. According to the literature, the shift in position of the emission maximum corresponded to the changes in the polarity of the chromospheres molecule [48,49]. The maximum emission wavelength of 7α-HSDH shifted from 448 to 511 nm, suggesting that the surroundings of the fluorophore are less polar when bilirubin interacts with the protein [50]. Figure 7b showed the fluorescence spectra emission of 7β-HSDH in the presence of bilirubin at 25 • C. Compared with 7α-HSDH, the fluorescence spectra emission of 7β-HSDH was more obvious after binding with bilirubin. The maximum emission of 7β-HSDH was at 440 nm when it was excited at 280 nm. The different results may be attributed to different compositions of tryptophan and tyrosine for 7α-and 7β-HSDHs. Previous research demonstrated that bilirubin was most possibly binding with arginine residue from human serum albumin. Bilirubin possessed an electron-rich surface that created van der Waals, stacking, and charge transfer interactions with tryptophan [46]. Bisphenols affected the activity of 11β-hydroxysteroid tein [50]. Figure 7b showed the fluorescence spectra emission of 7β-HSDH in the presence of bilirubin at 25 °C. Compared with 7α-HSDH, the fluorescence spectra emission of 7β-HSDH was more obvious after binding with bilirubin. The maximum emission of 7β-HSDH was at 440 nm when it was excited at 280 nm. The different results may be attributed to different compositions of tryptophan and tyrosine for 7α-and 7β-HSDHs. Previous research demonstrated that bilirubin was most possibly binding with arginine residue from human serum albumin. Bilirubin possessed an electron-rich surface that created van der Waals, stacking, and charge transfer interactions with tryptophan [46]. Bisphenols affected the activity of 11β-hydroxysteroid dehydrogenase 2 by binding to the steroid-binding site and interacting with the catalytic residue tyrosine232 [51]. The CD spectrum is widely used to evaluate the secondary structure, folding, and binding properties of proteins since different structural elements have unique CD spectra at a particular wavelength [52]. The reactions of bilirubin to enzymes were analyzed by CD spectra from 250 nm to 550 nm. Figure 8 shows the typical induced CD spectra of bilirubin bound to 7α-or 7β-HSDHs at molar ratios of 1:1. Bilirubin, 7α-, and 7β-HSDHs did not exhibit any dichroism. The solution of 7β-HSDH binding with 0.2 mM bilirubin exhibited similar CD spectra with 7α-HSDH binding with 0.2 mM bilirubin. 7α-HSDH induced a positive CD spectrum with a peak and a trough at 381 nm and 486 nm, respectively, and a cross-over at 415 nm. 7β-HSDH induces a positive bisignate CD spectrum with the peak, cross-over, and trough at 329, 375, and 475 nm, respectively. Probably, after binding with bilirubin, the entropy of 7α-and 7β-HSDHs were changed, which led to the change in secondary structure. The changes are reflected and recorded by the CD spectrum. The CD spectrum is widely used to evaluate the secondary structure, folding, and binding properties of proteins since different structural elements have unique CD spectra at a particular wavelength [52]. The reactions of bilirubin to enzymes were analyzed by CD spectra from 250 nm to 550 nm. Figure 8 shows the typical induced CD spectra of bilirubin bound to 7α-or 7β-HSDHs at molar ratios of 1:1. Bilirubin, 7α-, and 7β-HSDHs did not exhibit any dichroism. The solution of 7β-HSDH binding with 0.2 mM bilirubin exhibited similar CD spectra with 7α-HSDH binding with 0.2 mM bilirubin. 7α-HSDH induced a positive CD spectrum with a peak and a trough at 381 nm and 486 nm, respectively, and a cross-over at 415 nm. 7β-HSDH induces a positive bisignate CD spectrum with the peak, cross-over, and trough at 329, 375, and 475 nm, respectively. Probably, after binding with bilirubin, the entropy of 7α-and 7β-HSDHs were changed, which led to the change in secondary structure. The changes are reflected and recorded by the CD spectrum.  The CD spectra of bilirubin bound to 7α-or 7β-HSDHs with different proportions are shown in Figure 9. The molar ratios of 7α-or 7β-HSDHs to bilirubin were 1:1, 1:3, and 1:5, which are represented by black, red, and blue lines. The addition of bilirubin led to a gradual increase in the CD signal. Similar results are presented in both 7α-HSDH binding with bilirubin and 7β-HSDH binding with bilirubin. As Figure 9a shows, compared with the 7α-HSDH/bilirubin = 1/1 molar ratio, the original value increase in  The CD spectra of bilirubin bound to 7α-or 7β-HSDHs with different proportions are shown in Figure 9. The molar ratios of 7α-or 7β-HSDHs to bilirubin were 1:1, 1:3, and 1:5, which are represented by black, red, and blue lines. The addition of bilirubin led to a gradual increase in the CD signal. Similar results are presented in both 7α-HSDH binding with bilirubin and 7β-HSDH binding with bilirubin. As Figure 9a shows, compared with the 7α-HSDH/bilirubin = 1/1 molar ratio, the original value increase in 7α-HSDH/bilirubin = 1/5 reached 44% for positive components and 38% for negative components. As Figure 9b shows, compared with the 7β-HSDH/bilirubin = 1/1 molar ratio, the original value increase in 7β-HSDH/bilirubin = 1/5 reached 54% positive components and 46% negative components, respectively. We assume bilirubin can be bound to different sites of 7α-or 7β-HSDHs. More than one bilirubin molecule can be combined with enzymes at the same time. With the increase in the binding amount of bilirubin, the structure changes of 7α-or 7β-HSDHs may be more obvious. Figure 8. The analysis for the combination of bilirubin with 7α-or 7β-HSDHs by CD spectroscopy. Bilirubin bound to 7α-or 7β-HSDH at molar ratios of 1:1. (a) For 7α-HSDH; (b) for 7β-HSDH. The PBS buffer (10 mM, pH 7.4) was used as a background. 7α-HSDH + bilirubin and 7β-HSDH + bilirubin represented 7α-HSDH binding with 0.2 mM bilirubin and 7β-HSDH binding with 0.2 mM bilirubin, respectively.
The CD spectra of bilirubin bound to 7α-or 7β-HSDHs with different proportions are shown in Figure 9. The molar ratios of 7α-or 7β-HSDHs to bilirubin were 1:1, 1:3, and 1:5, which are represented by black, red, and blue lines. The addition of bilirubin led to a gradual increase in the CD signal. Similar results are presented in both 7α-HSDH binding with bilirubin and 7β-HSDH binding with bilirubin. As Figure 9a shows, compared with the 7α-HSDH/bilirubin = 1/1 molar ratio, the original value increase in 7α-HSDH/bilirubin = 1/5 reached 44% for positive components and 38% for negative components. As Figure 9b shows, compared with the 7β-HSDH/bilirubin = 1/1 molar ratio, the original value increase in 7β-HSDH/bilirubin = 1/5 reached 54% positive components and 46% negative components, respectively. We assume bilirubin can be bound to different sites of 7α-or 7β-HSDHs. More than one bilirubin molecule can be combined with enzymes at the same time. With the increase in the binding amount of bilirubin, the structure changes of 7α-or 7β-HSDHs may be more obvious.

Exploration of Inhibition Mechanism
Molecular docking is a computational simulation of a candidate ligand binding to a receptor, which is one of the most frequently used methods in structure-based binding of small molecules to functional proteins. In order to further clarify the inhibition

Exploration of Inhibition Mechanism
Molecular docking is a computational simulation of a candidate ligand binding to a receptor, which is one of the most frequently used methods in structure-based binding of small molecules to functional proteins. In order to further clarify the inhibition mechanisms of bilirubin on 7α-or 7β-HSDHs, random docking was conducted by AutoDock 4.2, and 10 candidate docking conformations were chosen for analysis as Table 3. mechanisms of bilirubin on 7α-or 7β-HSDHs, random docking was conducted by Au-toDock 4.2, and 10 candidate docking conformations were chosen for analysis as Table 3.
The probable structures of 7α-HSDH after binding with bilirubin are shown in Table 3 according to the binding energy. Structure one had the lowest energy value. The binding sites of bilirubin for structures one, three, five, and seven are in the adjacent area. This region is the most probable binding area of bilirubin and 7α-HSDH. From Figure S1, we can see the most probable binding site of bilirubin was close to NADP + , and bilirubin may be bound with Arg (16), Arg (20), Arg (194), and Glu (221) of 7α-HSDH. The previous study demonstrated that bilirubin was also most likely binding with the arginine residue from human serum albumin [46]. Li et al. reported that triclosan, triflumizole, and dichlone can bind to the NAD + -binding site of human placental 3β-hydroxysteroid dehydrogenase [53]. According to reference [54], this region was exactly located at the cofactor-binding site. Three arginines at positions 16, 38, and 194 of 7α-HSDH form a positive environment surrounded the 2′-phosphate adenine ribose of NADP + . Perhaps the hydrogen bonds were broken, and the transfer of H + was hindered after binding with bilirubin. It is unsurprising, then, that the activity of 7α-HSDH was inhibited. In summary, structure one was the most probable position for the binding of bilirubin to 7α-HSDH. After binding with bilirubin, the Vmax of 7α-HSDH to NADP + only had a minor change and the Km of 7α-HSDH to NADP + had a significant increase, as described in Table 2. It was therefore determined that the inhibition of bilirubin to 7α-HSDH is due to competitive inhibition. This was consistent with the results of the molecular docking simulation.
The probable structures of 7β-HSDH after binding with bilirubin are also shown in Table 3 according to binding energy. Structure one had the lowest energy value. For 7β-HSDH, the binding sites of bilirubin are located at the channel entrance for structures one, three, four, and nine. Five docking structures, 2, 5, 7, 8 and 10, reflect that bilirubin can enter the inner space of 7β-HSDH (as Table 3). Based on the analysis of optimum docking conformation, the most probable binding residues of bilirubin were Arg (40), Met (97), and Lys (107) of 7β-HSDH, as Figure S2 shows, which were located at the channel entrance for substrates and products. Access for substrates and products was restricted, which may lead to the activity decrease in 7β-HSDH. Structure one was the most probable position for the binding of bilirubin to 7β-HSDH. Furthermore, after binding with bilirubin, both the Vmax and the Km of 7β-HSDH to NADP + decreased. Therefore, it was determined that the inhibition of bilirubin to 7β-HSDH was due to uncompetitive inhibition and the inhibition mechanism of bilirubin to 7β-HSDH was different from 7α-HSDH. mechanisms of bilirubin on 7α-or 7β-HSDHs, random docking was conducted by Au-toDock 4.2, and 10 candidate docking conformations were chosen for analysis as Table 3.
The probable structures of 7α-HSDH after binding with bilirubin are shown in Table 3 according to the binding energy. Structure one had the lowest energy value. The binding sites of bilirubin for structures one, three, five, and seven are in the adjacent area. This region is the most probable binding area of bilirubin and 7α-HSDH. From Figure S1, we can see the most probable binding site of bilirubin was close to NADP + , and bilirubin may be bound with Arg (16), Arg (20), Arg (194), and Glu (221) of 7α-HSDH. The previous study demonstrated that bilirubin was also most likely binding with the arginine residue from human serum albumin [46]. Li et al. reported that triclosan, triflumizole, and dichlone can bind to the NAD + -binding site of human placental 3β-hydroxysteroid dehydrogenase [53]. According to reference [54], this region was exactly located at the cofactor-binding site. Three arginines at positions 16, 38, and 194 of 7α-HSDH form a positive environment surrounded the 2′-phosphate adenine ribose of NADP + . Perhaps the hydrogen bonds were broken, and the transfer of H + was hindered after binding with bilirubin. It is unsurprising, then, that the activity of 7α-HSDH was inhibited. In summary, structure one was the most probable position for the binding of bilirubin to 7α-HSDH. After binding with bilirubin, the Vmax of 7α-HSDH to NADP + only had a minor change and the Km of 7α-HSDH to NADP + had a significant increase, as described in Table 2. It was therefore determined that the inhibition of bilirubin to 7α-HSDH is due to competitive inhibition. This was consistent with the results of the molecular docking simulation.
The probable structures of 7β-HSDH after binding with bilirubin are also shown in Table 3 according to binding energy. Structure one had the lowest energy value. For 7β-HSDH, the binding sites of bilirubin are located at the channel entrance for structures one, three, four, and nine. Five docking structures, 2, 5, 7, 8 and 10, reflect that bilirubin can enter the inner space of 7β-HSDH (as Table 3). Based on the analysis of optimum docking conformation, the most probable binding residues of bilirubin were Arg (40), Met (97), and Lys (107) of 7β-HSDH, as Figure S2 shows, which were located at the channel entrance for substrates and products. Access for substrates and products was restricted, which may lead to the activity decrease in 7β-HSDH. Structure one was the most probable position for the binding of bilirubin to 7β-HSDH. Furthermore, after binding with bilirubin, both the Vmax and the Km of 7β-HSDH to NADP + decreased. Therefore, it was determined that the inhibition of bilirubin to 7β-HSDH was due to uncompetitive inhibition and the inhibition mechanism of bilirubin to 7β-HSDH was different from 7α-HSDH.   The bilirubin molecule is labeled green. The docking results are ranked from small to large by the binding free energy, and the axes are located in the upper left corner of each picture. Binding energy includes van der Waals forces, hydrogen bonds, desolvation energy, and electrostatic interactions.
The bilirubin used in this study was purchased from Sigma-Aldrich (Product code: 14370) and the purity of bilirubin was 95%. The protein marker was obtained from Sangon Biotech Co., Ltd., Shanghai, China (Product code: C610011-0250). The BCA protein assay reagent was a product of Beyotime (P0010, Shanghai, China). The standards of TCDCA and TUDCA were purchased from the National Institutes for Food and Drug Control (Beijing, China). Sodium taurine-7-ketolithocholic acid (T-7-KLCA) was synthesized by our laboratory. The reaction cofactors, NADP-Na2 and NADPH-Na4 (Purity ≥ 97%), were acquired from Roche (Basel, Switzerland). All the other chemicals used in this study were of analytical grade and used without further purification. Double-distilled water was used in all experiments.

Properties of 7α-and 7β-HSDH
After expression and purification, 7α-and 7β-HSDHs were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After the separation gel (12%) solidified, 5% spacer gel was prepared, and the appropriate comb was selected. The sample (10 µL) of 7α-or 7β-HSDHs was transferred to fresh tube and 2 µL of 6 × SDS loading buffer was added. The samples were heated for 5 min at 100 °C after a brief vortex. The gel was stained with Coomassie blue for approximately 30 min after running it for the appropriate length of time. It was decolorized with de-staining solution until the bands were clear. The concentration of 7α-or 7β-HSDHs was measured by BCA protein assay using bovine serum albumin as a standard. Purified enzymes were stored at −80 °C with 20% glycerol for further use. The properties of 7α-and 7β-HSDHs were estimated by a peptide calculator (Qiangyao, Shanghai, China) according to references
The bilirubin used in this study was purchased from Sigma-Aldrich (Product code: 14370) and the purity of bilirubin was 95%. The protein marker was obtained from Sangon Biotech Co., Ltd., Shanghai, China (Product code: C610011-0250). The BCA protein assay reagent was a product of Beyotime (P0010, Shanghai, China). The standards of TCDCA and TUDCA were purchased from the National Institutes for Food and Drug Control (Beijing, China). Sodium taurine-7-ketolithocholic acid (T-7-KLCA) was synthesized by our laboratory. The reaction cofactors, NADP-Na2 and NADPH-Na4 (Purity ≥ 97%), were acquired from Roche (Basel, Switzerland). All the other chemicals used in this study were of analytical grade and used without further purification. Double-distilled water was used in all experiments.

Properties of 7α-and 7β-HSDH
After expression and purification, 7α-and 7β-HSDHs were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After the separation gel (12%) solidified, 5% spacer gel was prepared, and the appropriate comb was selected. The sample (10 µL) of 7α-or 7β-HSDHs was transferred to fresh tube and 2 µL of 6 × SDS loading buffer was added. The samples were heated for 5 min at 100 °C after a brief vortex. The gel was stained with Coomassie blue for approximately 30 min after running it for the appropriate length of time. It was decolorized with de-staining solution until the bands were clear. The concentration of 7α-or 7β-HSDHs was measured by BCA protein assay using bovine serum albumin as a standard. Purified enzymes were stored at −80 °C with 20% glycerol for further use. The properties of 7α-and 7β-HSDHs were estimated by a peptide calculator (Qiangyao, Shanghai, China) according to references The probable structures of 7α-HSDH after binding with bilirubin are shown in Table 3 according to the binding energy. Structure one had the lowest energy value. The binding sites of bilirubin for structures one, three, five, and seven are in the adjacent area. This region is the most probable binding area of bilirubin and 7α-HSDH. From Figure S1, we can see the most probable binding site of bilirubin was close to NADP + , and bilirubin may be bound with Arg (16), Arg (20), Arg (194), and Glu (221) of 7α-HSDH. The previous study demonstrated that bilirubin was also most likely binding with the arginine residue from human serum albumin [46]. Li et al. reported that triclosan, triflumizole, and dichlone can bind to the NAD + -binding site of human placental 3β-hydroxysteroid dehydrogenase [53]. According to reference [54], this region was exactly located at the cofactor-binding site. Three arginines at positions 16, 38, and 194 of 7α-HSDH form a positive environment surrounded the 2 -phosphate adenine ribose of NADP + . Perhaps the hydrogen bonds were broken, and the transfer of H + was hindered after binding with bilirubin. It is unsurprising, then, that the activity of 7α-HSDH was inhibited. In summary, structure one was the most probable position for the binding of bilirubin to 7α-HSDH. After binding with bilirubin, the V max of 7α-HSDH to NADP + only had a minor change and the K m of 7α-HSDH to NADP + had a significant increase, as described in Table 2. It was therefore determined that the inhibition of bilirubin to 7α-HSDH is due to competitive inhibition. This was consistent with the results of the molecular docking simulation.
The probable structures of 7β-HSDH after binding with bilirubin are also shown in Table 3 according to binding energy. Structure one had the lowest energy value. For 7β-HSDH, the binding sites of bilirubin are located at the channel entrance for structures one, three, four, and nine. Five docking structures, 2, 5, 7, 8 and 10, reflect that bilirubin can enter the inner space of 7β-HSDH (as Table 3). Based on the analysis of optimum docking conformation, the most probable binding residues of bilirubin were Arg (40), Met (97), and Lys (107) of 7β-HSDH, as Figure S2 shows, which were located at the channel entrance for substrates and products. Access for substrates and products was restricted, which may lead to the activity decrease in 7β-HSDH. Structure one was the most probable position for the binding of bilirubin to 7β-HSDH. Furthermore, after binding with bilirubin, both the V max and the K m of 7β-HSDH to NADP + decreased. Therefore, it was determined that the inhibition of bilirubin to 7β-HSDH was due to uncompetitive inhibition and the inhibition mechanism of bilirubin to 7β-HSDH was different from 7α-HSDH.
The bilirubin used in this study was purchased from Sigma-Aldrich (Product code: 14370) and the purity of bilirubin was 95%. The protein marker was obtained from Sangon Biotech Co., Ltd., Shanghai, China (Product code: C610011-0250). The BCA protein assay reagent was a product of Beyotime (P0010, Shanghai, China). The standards of TCDCA and TUDCA were purchased from the National Institutes for Food and Drug Control (Beijing, China). Sodium taurine-7-ketolithocholic acid (T-7-KLCA) was synthesized by our laboratory. The reaction cofactors, NADP-Na 2 and NADPH-Na 4 (Purity ≥ 97%), were acquired from Roche (Basel, Switzerland). All the other chemicals used in this study were of analytical grade and used without further purification. Double-distilled water was used in all experiments.

Properties of 7α-and 7β-HSDH
After expression and purification, 7α-and 7β-HSDHs were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After the separation gel (12%) solidified, 5% spacer gel was prepared, and the appropriate comb was selected. The sample (10 µL) of 7α-or 7β-HSDHs was transferred to fresh tube and 2 µL of 6 × SDS loading buffer was added. The samples were heated for 5 min at 100 • C after a brief vortex. The gel was stained with Coomassie blue for approximately 30 min after running it for the appropriate length of time. It was decolorized with de-staining solution until the bands were clear. The concentration of 7α-or 7β-HSDHs was measured by BCA protein assay using bovine serum albumin as a standard. Purified enzymes were stored at −80 • C with 20% glycerol for further use. The properties of 7α-and 7β-HSDHs were estimated by a peptide calculator (Qiangyao, Shanghai, China) according to references [42], such as molecular weight, isoelectric point, charge condition, average hydrophilicity, and ratio of hydrophilic residues.

Enzymatic Activity and Kinetic Characterization
The activities of 7α-and 7β-HSDHa were assayed according to references [16,17] with slight modifications. For the determination of 7α-HSDH activity, the enzyme assay mixture in a total volume of 1 mL was: 2 mM TCDCA in 50 mM Tris-HCl, pH 8.5, and 0.2 mM NADP + . Determination of 7β-HSDH activity was performed in a total volume of 1 mL assay mixture containing: 2 mM TUDCA in 50 mM Tris-HCl, pH 8.5, and 0.2 mM NADP + . The amount of NADPH was proportional to the ultraviolet absorption at 340 nm. The change in absorbance at 340 nm was recorded by a spectrophotometer (UV 1800, Shimadzu, Japan) to evaluate enzyme activity. One unit of enzyme activity was defined as the amount of enzyme needed to convert 1 µM substrate (TCDCA/TUDCA) within 1 min at 25 • C in a buffer solution containing 50 mM Tris-HCl (pH 8.5).
The kinetic studies of 7α-HSDH and 7β-HSDH were performed by measuring the reduction of NADP + at 340 nm using UV-vis spectrophotometer (UV 1800, Shimadzu, Japan) according to the previously reported methods [16,17]. [S] is the substrate concentration. v and V max represent the initial and maximum rate of reaction, respectively. K m is the Michaelis constant.

Enzymatic Reaction
The reaction was carried out at 25 • C and pH 8.5 Tris-HCl (50 mM) using TCDCA (8 mM) as substrate. The final concentration of both 7α-HSDH and 7β-HSDH was 0.5 mg/L, and the concentration of NADP + was 2 mM. The reaction was continued for 4 h with the catalysis of free 7α-and 7β-HSDHs. All tests were performed in triplicate and expressed as the means ± SD (n = 3). TCDCA conversion and TUDCA yield were analyzed by HPLC-ELSD and evaluated as follows: TCDCA Conversion = Total amount of TCDCA added (g) − Total amount of TCDCA remaining (g) Total amount of TCDCA added (g) × 100% (1) TUDCA Yield = Total amount of TUDCA generated (g) Total amount of TCDCA added (g) × 100% (2)

The Effect of Bilirubin on Enzyme Activity and TCDCA Transformation
The effects of bilirubin on the activity of enzymes and TCDCA transformation were studied according to the following methods. For enzyme activity, bilirubin was added to the solution of TCDCA or TUDCA at 25 • C (50 mM Tris-HCl, pH 8.5), and the final concentration of bilirubin varied from 0 to 1.0 mM. Enzyme activity in the absence of bilirubin was set to 100% so that the relative activity in the presence of bilirubin could be obtained. Furthermore, the effects of bilirubin (0.2 mM) on the kinetic parameters of 7α-and 7β-HSDHs were investigated. The analyses of enzyme activity and kinetics were according to the protocol 3.3. For enzymatic reactions, the effect of bilirubin on the coupling reaction was determined by testing various bilirubin concentrations ranging from 0 to 0.8 mM with a gradient per 0.2 mM in 50 mM Tris-HCl buffer (pH 8.5). Furthermore, the composition of products was analyzed by HPLC-ELSD. TCDCA conversion and TUDCA yield were calculated based on the equations of protocol 3.4. Other reaction conditions were consistent. All tests were performed in triplicate.

UV-Vis
The binding of bilirubin and enzymes was analyzed by UV-vis. The enzyme solution (7α-HSDH or 7β-HSDH) was diluted to a concentration 1.2 × 10 −5 mol/L. Bilirubin solution with the same concentration was used for analysis. A mixture solution was obtained by mixing the equivalent volume of bilirubin stock solution and the equivalent volume of enzyme stock solution in a phosphate-buffered saline (PBS) of pH = 7.4. The samples were scanned against a PBS buffer (10 mM, pH 7.4) background by a wave number ranging from 250 nm to 600 nm with a 1 cm path length.

Fluorescence
The binding of bilirubin and enzymes was analyzed by fluorescence. Fluorescence measurements were performed on a spectrofluorimeter (LS55, PerkinElmer, Waltham, MA, USA) equipped with thermostatiation systems and a temperature controller using 5 nm excitation and 5 nm emission slit widths. The 3 samples (enzyme solution, bilirubin solution, and the mixture of 2 solutions with molar ratio 1:1 for enzyme and bilirubin) were excited at 280 nm and scanned in the range of 350-600 nm with 100 nm min −1 scanning speed. The PBS buffer (10 mM, pH 7.4) was used as a background.

CD Spectroscopy
The binding of bilirubin and enzymes was analyzed by CD spectroscopy. The CD spectra of 3 solutions (enzyme solution, bilirubin solution, the mixture solutions of enzyme and bilirubin with molar ratio 1:1, 1:3 and 1:5) were recorded on a CD spectrometer (MOS450, BioLogic, Seyssinet-Pariset, France) under a nitrogen atmosphere using a quartz cell with a path length of 1 cm. All experiments were run at 25 • C. The CD spectra were recorded at 250-550 nm wavelength region as an average of 3 scans measured with a 1 nm bandwidth, a 1 s response, and 100 nm min −1 scanning speed. All CD spectra of samples used a baseline PBS buffer (10 mM, pH 7.4) by using a spectrum of the solvent obtained under the same experimental conditions.

High-Performance Liquid Chromatography (HPLC)
HPLC analysis was performed using Agilent 1260 Infinity (Santa Clara, CA, USA) with an evaporative light-scattering detector, and chromatographic separations were conducted on a Welch ultimate XB-C18 column (4.6 × 250 mm, 5 µm). The analysis was carried out in a nitrogen environment. The speed of nitrogen was 1.2 L·min −1 and the temperature of the ELSD detector was set to 80 • C. Methanol was used as solvent and the injection volume was set to 10 µL. The mobile phase consisted of 2 solvents, solution A: 100% acetonitrile; solution B: 30 mM aqueous ammonium acetate solution (pH 4.5). The analysis was conducted with 42% solution A and 58% solution B. Furthermore, the analysis time was 10 min and a constant flow rate of 1.0 mL·min −1 was adopted.

Molecular Docking of Bilirubin and Enzymes
The goal of bilirubin protein docking is to predict the predominant binding modes of bilirubin with 7α-HSDH or 7β-HSDH, which have a known 3D structure. Furthermore, we can analyze the inhibition mechanisms of bilirubin to 7α-HSDH or 7β-HSDH based on the dockings. The 3D crystal structures of 7α-HSDH (PDB code: 5EPO) and 7β-HSDH (PDB code: 5GT9) were obtained from the Protein Data Bank [54,55]. The ionizable residues were set to protonation states (pH 7.4). In this state, all His, Arg, and Lys were protonated while Asp and Glu were deprotonated. The AutoDock 4.2 plugin was used for all dockings in this study. The docking parameters for AutoDock 4.2 were kept at their default values. The docking results were ranked by the binding free energy, and 10 candidate dockings were selected for analysis. The optimum docking conformation was chosen based on the lowest binding free energy and the most cluster members. Visual Molecular Dynamics 1.8.3 (VMD 1.8.3) was used to illustrate the binding results.

Conclusions
In conclusion, we systematically investigated the impact of bilirubin on 7α-and 7β-HSDHs, including the effect of bilirubin on enzymes activities and enzymatic reactions. After binding with bilirubin, the K m of 7α-HSDH for TCDCA rose from 0.24 mM to 0.63 mM and the K m of 7β-HSDH for TUDCA changed from 1.14 mM to 1.87 mM. The affinity of 7αand 7β-HSDHs to substrates decreased after binding with bilirubin. With the increase in bilirubin, both TCDCA conversion and TUDCA yield were gradually decreased. As the concentration of bilirubin was 0.8 mM in the reaction buffer, TCDCA conversion remained at 15% and the TUDCA yield was only 5%. The binding of bilirubin to 7α-or 7β-HSDHs will lead to significant changes in the enzyme spectrum, such as the UV-vis spectrum, fluorescence spectrum, and CD spectrum. The enzymatic activity loss and response of spectrums may be related to the structure changes in enzymes after binding with bilirubin. Molecular docking was conducted to explore the possible inhibition mechanism of bilirubin to enzymes. The most probable binding site of bilirubin to 7α-HSDH was close to NADP + , and the most probable binding residues of bilirubin to 7β-HSDH were located at the channel entrance for substrates and products. Regarding the kinetic parameters, we conclude that the inhibition of bilirubin to 7α-HSDH is due to competitive inhibition and the inhibition of bilirubin to 7β-HSDH us due to uncompetitive inhibition. Further research regarding the effects of bilirubin on other hydroxysteroid dehydrogenases is required.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/catal13060965/s1, Figure S1. The docking simulation of bilirubin and 7α-HSDH. (a) The overview of combination between bilirubin and 7α-HSDH; (b) The relative positions of cofactor NADP + and bilirubin; (c) The specific amino acid residues of 7α-HSDH combined with bilirubin. Molecular docking was performed on autodock 4.2 and the results were analyzed by VMD1.8.3; Figure S2. The docking simulation of bilirubin and 7β-HSDH. (a) The overview of combination between bilirubin and 7β-HSDH; (b) The relative positions of cofactor NADPH and bilirubin; (c) the specific amino acid residues of 7β-HSDH combined with bilirubin. Molecular docking was performed on autodock 4.2 and the results were analyzed by VMD1.8.3; Table S1 The fitting formulas for TCDCA, T-7-KLCA and TUDCA; Table S2 The low energy comformation of combination between bilirubin and 7α-HSDH; Table S3 The low energy comformation of combination between bilirubin and 7β-HSDH.
Author Contributions: Investigation, supervision, original draft preparation, and funding acquisition, Q.J.; methodology and data curation, J.C. and L.Z.; software and formal analysis, R.W.; writing-review and editing, supervision, and project administration, B.W. All authors discussed the results and contributed to the final manuscript. All authors have read and agreed to the published version of the manuscript.