Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum

Accepted Manuscript Title: Functional contribution of coenzyme specificity-determining sites of 7␣-hydroxysteroid dehydrogenase from Clostridium absonum Authors: Deshuai Lou, Yue Wang, Jun Tan, Liancai Zhu, Shunlin Ji, Bochu Wang PII: DOI: Reference:

S1476-9271(16)30639-9 http://dx.doi.org/doi:10.1016/j.compbiolchem.2017.08.004 CBAC 6712

To appear in:

Computational Biology and Chemistry

Received date: Revised date: Accepted date:

26-11-2016 25-5-2017 6-8-2017

Please cite this article as: Lou, Deshuai, Wang, Yue, Tan, Jun, Zhu, Liancai, Ji, Shunlin, Wang, Bochu, Functional contribution of coenzyme specificity-determining sites of 7␣-hydroxysteroid dehydrogenase from Clostridium absonum.Computational Biology and Chemistry http://dx.doi.org/10.1016/j.compbiolchem.2017.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum

Authors: Deshuai Lou1,2, Yue Wang2, Jun Tan3,*, Liancai Zhu2, Shunlin Ji2, Bochu Wang2,*

1

Postdoctoral research station of biology, Chongqing University, Chongqing 400030, China

2

Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of

Education, College of Bioengineering, Chongqing University, Chongqing 400030, China 3

Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of

Biological & Chemical engineering, Chongqing University of Education, Chongqing 400067, China

*Corresponding authors: Bochu Wang and Jun Tan [email protected] (Wang B) and [email protected] (Tan J) Tel.: +86-023-65112840; Fax: +86-023-65112877. Postal addresses: College of Bioengineering, Chongqing University, No.174, Shapingba Main Street, Chongqing, P. R. China.

Graphical abstract

The natural coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum allowed the greatest NADP(H) binding affinity (the lowest Km), but not the best activity (kcat) toward coenzyme. 1

Highlights 

Functional contribution of CSDSs of CA 7α-HSDH was analysed.



Mutant, R194A, with increased catalytic efficiency toward NADP+ was probed.



We confirmed the function of R38 for coenzyme anchoring.

ABSTRACT Studies of the molecular determinants of coenzyme specificity help to reveal the structure-function relationship of enzymes, especially with regards to coenzyme specificity-determining sites (CSDSs) that usually mediate complex interactions. NADP(H)-dependent 7α-hydroxysteroid dehydrogenase from Clostridium absonum (CA 7α-HSDH), a member of the short-chain dehydrogenase/reductase superfamily (SDRs), possesses positively charged CSDSs that mainly contain T15, R16, R38, and R194, forming complicated polar interactions with the adenosine ribose C2 phosphate group of NADP(H). The R38 residue is crucial for coenzyme anchoring, but the influence of the other residues on coenzyme utilization is still not clear. Hence, we performed alanine scanning mutagenesis and molecular dynamic (MD) simulations. The results suggest that the natural CSDSs have the greatest NADP(H)-binding affinity, but not the best activity (kcat) toward NADP+. Compared with the wild type and other mutants, the mutant R194A showed the highest catalytic efficiency (kcat/Km), which was more than three-times that of the wild type. MD simulation and kinetics analysis suggested that the importance of the CSDSs of CA 7α-HSDH should be in accordance with the following order R38>T15>R16>R194, and S39 may have a supporting role in NADP(H) anchoring for mutants R16A/T194A and T15A/R16A/T194A.

Keywords: 7α-Hydroxysteroid dehydrogenase, Clostridium absonum, Coenzyme specificity, Enzyme engineering, Molecular dynamics simulation 1. Introduction 7α-Hydroxysteroid dehydrogenase (7α-HSDH) reversibly catalyzes the oxidation of the C7 hydroxyl of steroids that plays a critical role in the enterohepatic circulation of bile acids (BAs). Further, 7α-HSDH is promising for bioconversion due to the high stereoselectivity in asymmetric reactions and the broad

2

substrate spectrum. In addition to BAs and their conjugates with glycine or taurine, a series of aromatic and bulky aliphatic α-ketoesters (Zhu et al., 2006) and benzaldehyde analogues (Liu et al., 2011) can also be asymmetrically reduced by 7α-HSDH. To date, five 7α-HSDHs have been cloned and functionally verified from Bacteroides fragilis (Bennett et al., 2003), Clostridium scindens (Baron et al., 1991), Clostridium sordellii (Coleman et al., 1994), Clostridium absonum (Ferrandi et al., 2012) and Escherichia coli (Yoshimoto et al., 1991). The sequence alignment is shown in Fig. 1.

The multiple secondary structures of enzymes cooperate precisely with each other to accomplish substrate binding and product dissociation; for instance, the active centre is generally located in a hydrophobic cleft that contains more non-polar amino acids. On the other hand, polar interactions, especially hydrogen bonds and salt bridges are essential for the substrate to anchor at the right orientation. Complicated polar interactions between dehydrogenases and coenzyme NAD(H) or NADP(H), especially those involving coenzyme specificity-determining sites (CSDSs) have attracted a lot of attention. All of the 7α-HSDHs mentioned above belong to the short-chain dehydrogenase/reductase superfamily (SDRs) that have a highly similar α/β folding pattern (Rossmann-fold) (Oppermann et al., 2003). Generally, for SDRs, the CSDSs mainly involve sites that interact with the adenosine ribose C2 hydroxyl (NAD(H)) or the phosphate group (NADP(H)), and previous studies suggest that NAD(H)-dependent 7α-HSDHs generally possess a conserved negatively-charged residue (Asp or Glu) at the end of the second β strand (dashed-line box in Fig. 1) (Bellamacina, 1996). A conserved, positively-charged residue (Arg or Lys) is located at a position that is just behind the second β strand of NADP(H)-dependent 7α-HSDHs (solid lined box in Fig. 1) (Kallberg et al., 2002). Besides the key residues mentioned above, in most cases, some other amino acids are also important members of the CSDSs. Clostridium absonum was isolated from soil by Nakamura in 1973 (NAKAMURA et al., 1973) and was later experimentally shown to generate 7α-HSDH and 7β-HSDH (Macdonald et al., 1981). Both of these enzymes were cloned and functionally characterized in 2011 (Ferrandi et al., 2012). The crystal structure of Clostridium absonum 7α-HSDH (CA 7α-HSDH) complexed with taurochenodeoxycholic acid (TCDCA) and NADP+ was determined recently (Lou et al., 2016). The CSDSs of CA 7α-HSDH mainly consist of T15, R16, R38 and R194 that form a positively-charged hole, with three arginines anchoring the adenosine ribose C2 phosphate group of NADP(H) through complicated polar interactions 3

(Fig. 2). Previously, R38 was found to be crucial for anchoring NADP+ with not only the C2 phosphate group but also the adenine ring as a key residue for coenzyme specificity (Lou et al., 2016). Nonetheless, the influence of the other sites on coenzyme binding has not been probed; thus, in this study, we performed alanine scanning mutagenesis and molecular dynamics (MD) simulation analysis to clarify the functional contribution of CSDSs to CA 7α-HSDH.

2. Materials and Methods 2.1. Heterologous Expression and Purification of CA 7α-HSDH and the Mutants The gene codons for the wild-type CA 7α-HSDH were optimized according to the codon preferences of E. coli and a GST-fusion expression vector pGEX-6p-1 (GE Healthcare) was used for the expression of all enzymes (restriction sites: BamH I/Not I). Details of the experimental operations were determined by the previously reported methods (Lou et al., 2014; Lou et al., 2016).

2.2. Molecular Dynamic Simulations The Amber Molecular Dynamics Package (AMBER12) was used for all molecular dynamic (MD) simulations (Case et al.). The detailed operation of MD simulations and MM/PBSA analyses were in accordance with a previous report (Lou et al., 2016). Subunit A of wild-type (WT) CA 7α-HSDH complexed with TCDCA and NADP+ (PDB: 5EPO) and was extracted for the MD simulation. The initial coordinates of the mutants were prepared from the WT with the SYBYL program. All complexes were immersed in a periodic TIP3 water box with a minimum 10 Å water shell. Sodium ions were added to neutralize negative charges of the system. The LEAP module was then applied to generate topology and coordinate files. Three rounds of minimization were performed for: 1) solvent and ions (4000 steps); 2) solution and side chains (5000 steps); and 3) the whole system (10000 steps). A steepest descent and conjugate gradient combination method was carried out for the minimizations. The temperature of the system increased from 0 to 300K within 50 ps, and the NPT ensemble (50 ps) and MD simulation (5 ns) were performed at constant temperature. The last 1 ns trajectory was analysed for system stability (RMSD) and structural changes. Molecular structure superposition and illustrations were prepared with PyMOL.

4

2.3. Site-Directed Mutagenesis A total of seven mutants were prepared, as follows: two mutants (T15A and R16A) were prepared using long forward primers through traditional PCR; two mutants (R38A and R194A) were prepared using overlapping PCR, based on the R194A mutant; two-point mutants (R16A/R194A and R38A/R194A) were prepared through traditional and overlapping PCR, respectively. The gene for T15A/R16A/R194A mutants was synthesized and verified at Sangon Biotech (Sangon, Shanghai, China). All other genes were verified by DNA sequencing (TaKaRa, Dalian, China). The primers involved in the PCR experiments are summarized in Table S1 (supplementary material).

2.4. Assay of Enzyme Activity The enzyme assays and kinetic analyses of wild-type CA 7α-HSDH and the mutants were carried out in 2 mL reaction mixture, containing 50 mM Tris-HCl (pH 8.5), 200 mM NaCl, 0.5 mM NADP+ and 0.5 mM TCDCA at 20 °C. Cuvette with path length of 0.5 cm was used to determine the absorbance changes at 340 nm to measure enzyme activity. The kinetic constants of enzymes were measured with NADP + concentrations from 0.5 to 8 mM and the values of Km and kcat were obtained by the Lineweaver-Burk plot.

3. Results and Discussion Table 1 summarizes the kinetic constants of WT CA 7α-HSDH and the mutants. Mutant R194A was the most compelling mutant, with a catalytic efficiency (kcat/Km) toward coenzyme (NADP+) that increased by more than two-times, compared to that of WT. This result suggests that the excessive positive charges decreased the efficiency of coenzyme utilization. In addition, the removal of positively-charged residues should be chosen carefully because of their different functional contributions. Here, the detecting activity of mutants R16A/R194A and T15A/R16A/R194A, and the non-detecting activity of mutants R38A and R38A/R194A confirm the conclusion that R38 is the crucial CSDS of CA 7α-HSDH (Lou et al., 2016). Serine or threonine seemed to be very important for NADP(H) anchoring, because the catalytic efficiency of mutant T15A decreased by more than 4.5-times, compared to the wild type and the affinity dropped dramatically (Km value increased by nearly 17-times). Judging from the changes in catalytic efficiency toward NADP+, the importance of CSDSs in CA 7α-HSDH for coenzyme recognition should be in accordance with the following order: R38>T15>R16>R194. 5

The effects of CSDSs on coenzyme utilization were further investigated in this study. Six enzymes with activity, WT, T15A, R16A, R194A, R16A/R194A, T15A/R16A/R194A, complexed with NADP+ and TCDCA were studied with MD simulations. The conformational stabilities were verified by the root mean square deviations (RMSD) of the main chain of proteins, compared to the initial structure (see supplementary Fig. S1). All of the structures quickly reached stability at the start of the 5 ns MD simulation. The calculated average amino acid backbone root mean square fluctuations (RMSF) indicated that all of the complexes possessed similar flexibility, and the most flexible parts were localized in the C-terminal region (see supplementary Fig. S2). The average structures of WT and the mutants mentioned above were calculated to evaluate the change of interacting pattern between enzyme and coenzyme. Fig. 3 shows the structural comparisons of coenzyme and CSDSs between the mutants and WT. For the three single-point mutants T15A, R16A and R194A, overall structures and interacting patterns did not change significantly except that the related polar interactions disappeared, which is in accordance with the decrease in NADP+-binding free energies (Table 2). For R16A/R194A and T15A/R16A/R194A, significant twist occurred in NADP+, including the pyrophosphate bridge, and the adenine and ribose 2'-phosphate (Fig. 3). Based on the results of the MD simulations, the average structures of the enzyme-substrate complexes were comprehensively analysed. Specifically, S39 formed hydrogen bonds with the 2'-phosphate of adenine ribose of NADP+ in the mutants R16A/R194A and T15A/R16A/R194A (Fig. 4).

NAD(H) and NADP(H), acting as helper molecules in transferring hydride, are required for enzymatic redox reactions. Although the structural differences between the two coenzymes are only in the groups (hydroxyl or phosphate) that covalently bind the C2 of adenine ribose, the market price of NADP(H) is about 10-times higher than that of NAD(H). Hence, functional studies of CSDSs are mainly focused on the site characteristics and how to invert the coenzyme specificity (Moon et al., 2012; Scrutton et al., 1990; Takase et al., 2014). Evidence suggests that CSDSs tend to contain an essential residue. Single point mutations of mouse lung carbonyl reductase (an SDR), T38D, switched the coenzyme preference from NADP(H) to NAD(H) (Nakanishi et al., 1997), and some residues, other that the essential one, have been useful for completely inverting the coenzyme specificity (Rosell et al., 2003). Nevertheless, such coenzyme specificity-inverting mutants have a strong tendency to decrease the enzyme activity (Dudek et al., 2010), suggesting that each related enzyme formed complicated CSDSs in 6

harmony with the whole structure during long-term evolution. Overall, the molecular modification of an enzyme (protein) should be considered on a case-by-case basis. According to the theory mentioned above, A37 should be the key residue for the NAD(H) reorganization of CA 7α-HSDH, but mutant A37D did not show any activity toward NAD(H) or NADP(H), even when R38 was simultaneously mutated to Ile, Tyr, or Val (Lou et al., 2016). These results indicate that CA 7α-HSDH may differ from other members of the SDR superfamily, possible because of the complicated interactions between CSDSs and NADP(H).

Nine NADP(H)-dependent dehydrogenases/reductases were selected at random from PDB (Protein Data Bank, http://www.rcsb.org/) and their CSDSs are shown in Fig. 5. Obviously, the CSDSs of all nine enzymes are comprised of no more than two positively-charged residues (Arg or Lys). Except for the five 7α-HSDHs, only CA 7α-HSDH has an extra arginine at position 194 (labelled with an arrow in Fig. 1). Alanine scanning mutagenesis was performed to probe the functional contributions of the CSDSs, and unexpectedly, mutants T15A, R16A, and R194A had increased kcat values, while R194A had an increased catalytic efficiency (kcat/Km). Therefore, the CSDSs of CA 7α-HSDH not only determine coenzyme specificity, but also significantly influence coenzyme binding affinity. A higher substrate binding affinity generates a lower kcat and kcat/Km. Conversely, without disrupting the key interactions of the catalytic residues and the anchoring of the coenzyme in the active pocket, a moderate reduction in the substrate-binding affinity could be a feasible approach for generating mutants with greater catalytic efficiency toward NADP(H).

4. Conclusion For NADP(H)-dependent SDRs, their CSDSs generally involve more than one arginine that interacts with adenine ribose 2'-phosphate. Our findings from alanine scanning mutagenesis and molecular dynamic (MD) simulations of CA 7α-HSDH suggest that the excessive arginine may decrease NADP(H) utilization. From another perspective, according to the relative importance of CSDSs, a moderate reduction in the affinity between the enzyme and NADP(H) may improve the activity.

7

Funding This work was supported by the Fundamental Research Funds for the Central Universities (grant numbers 106112016CDJCR231213 and 106112015CDJXY230001); National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (grant number 2014ZX09301306-007); the Natural Science Foundation Project of CQ CSTC (grant number cstc2015jcyjA10094); Three Gorges Natural Medicine Engineering Research Center of Chongqing University of Education (grant number 167011).

Conflict of Interest None declared.

8

References Baron, S.F., Franklund, C.V., Hylemon, P.B. 1991. Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708. J. Bacteriol. 173 (15), 4558-4569. Bellamacina, C.R. 1996. The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins. FASEB J. 10 (11), 1257-1269. Bennett, M.J., McKnight, S.L., Coleman, J.P. 2003. Cloning and characterization of the NAD-dependent 7 alpha-hydroxysteroid dehydrogenase from Bacteroides fragilis. Curr. Microbiol. 47 (6), 475-484. doi:10.1007/s00284-003-4079-4 Case, D., Darden, T., Cheatham III, T., Simmerling, C., Wang, J., Duke, R., et al., AMBER 12; University of California: San Francisco, 2012. There is no corresponding record for this reference, Coleman, J.P., Hudson, L.L., Adams, M.J. 1994. Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii. J. Bacteriol. 176 (16), 4865-4874. Dudek, H.M., Torres Pazmino, D.E., Rodriguez, C., de Gonzalo, G., Gotor, V., Fraaije, M.W. 2010. Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca. Appl. Microbiol. Biotechnol. 88 (5), 1135-1143. Ferrandi, E.E., Bertolesi, G.M., Polentini, F., Negri, A., Riva, S., Monti, D. 2012. In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7 alpha- and 7 beta-hydroxysteroid dehydrogenases from Clostridium absonum. Appl. Microbiol. Biotechnol. 95 (5), 1221-1233. Kallberg, Y., Oppermann, U., Jornvall, H., Persson, B. 2002. Short-chain dehydrogenases/reductases (SDRs) - Coenzyme-based functional assignments in completed genomes. Eur. J. Biochem. 269 (18), 4409-4417. Liu, Y., Lv, T., Ren, J., Wang, M., Wu, Q.Q., Zhu, D.M. 2011. The catalytic promiscuity of a microbial 7 alpha-hydroxysteroid dehydrogenase. Reduction of non-steroidal carbonyl compounds. Steroids 76 (10-11), 1136-1140. Lou, D., Wang, B., Tan, J., Zhu, L. 2014. Carboxyl-terminal and Arg38 are essential for activity of the 7alpha-hydroxysteroid dehydrogenase from Clostridium absonum. Protein Pept. Lett. 21 (9), 894-900. Lou, D.S., Wang, B.C., Tan, J., Zhu, L.C., Cen, X.X., Ji, Q.Z., et al., 2016. The three-dimensional structure of Clostridium absonum 7 alpha-hydroxysteroid dehydrogenase: new insights into the conserved arginines for NADP(H) recognition. Scientific Reports 6. Macdonald, I.A., Hutchison, D.M., Forrest, T.P. 1981. Formation of urso- and ursodeoxy-cholic acids from primary bile acids by Clostridium absonum. J. Lipid Res. 22 (3), 458-466. Moon, H.-J., Tiwari, M.K., Singh, R., Kang, Y.C., Lee, J.-K. 2012. Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase. Appl.

9

Environ. Microb. 78 (9), 3079-3086. Nakamura, S., Shimamura, T., Hayase, M., Nishida, S. 1973. Numerical taxonomy of saccharolytic clostridia, particularly Clostridium perfringens-like strains: descriptions of Clostridium absonum sp. n. and Clostridium paraperfringens. Int. J. Syst. Bacteriol. 23 (4), 419-429. Nakanishi, M., Matsuura, K., Kaibe, H., Tanaka, N., Nonaka, T., Mitsui, Y., et al., 1997. Switch of coenzyme specificity of mouse lung carbonyl reductase by substitution of threonine 38 with aspartic acid. J. Biol. Chem. 272 (4), 2218-2222. Oppermann, U., Filling, C., Hult, M., Shafqat, N., Wu, X., Lindh, M., et al., 2003. Short-chain dehydrogenases/reductases (SDR): the 2002 update. Chem. Biol. Interact. 143-144, 247-253. Rosell, A., Valencia, E., Ochoa, W.F., Fita, I., Parés, X., Farrés, J. 2003. Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase. J. Biol. Chem. 278 (42), 40573-40580. Scrutton, N.S., Berry, A., Perham, R.N. 1990. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343 (6253), 38-43. Takase, R., Mikami, B., Kawai, S., Murata, K., Hashimoto, W. 2014. Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism. J. Biol. Chem. 289 (48), 33198-33214. Yoshimoto, T., ., Higashi, H., ., Kanatani, A., ., Lin, X.S., Nagai, H., ., Oyama, H., ., et al., 1991. Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme. J. Bacteriol. 173 (7), 2173-2179. Zhu, D., Stearns, J.E., Ramirez, M., Hua, L. 2006. Enzymatic enantioselective reduction of α-ketoesters by a thermostable 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis. Tetrahedron 62 (18), 4535-4539.

10

Figure Legends: Fig. 1. Multiple sequence alignment of reported 7α-HSDHs. CLOabso: Clostridium absonum (GenBank: JN191345); CLOscin: Clostridium scindens (GenBank: M58473); CLOsord: Clostridium sordellii (GenBank: L12058); BACfrag: Bacteroides fragilis (GenBank: AF173833); ESCcoli: Escherichia coli (GenBank: HDHA_ECOLI). Arginines (solid-lined box) indicate NADP(H)-preferred enzymes and aspartic acids (dashed-line box) NAD(H)-preferred ones. Fig. 2. Polar interactions between CSDSs and NADP + in CA 7α-HSDH. Fig. 3. Structural alignments of coenzyme and residues at positions 15, 16, 38, and 194 from WT and the four mutants. The CSDSs of WT and the mutants are colored green and cyan, respectively, and NADP+ is colored green and red, respectively: A: T15A; B: R16A; C: R194A; D: R16A/R194A; E: T15A/R16A/R194A. Fig. 4. Interactions between NADP+ and the two mutants of CA 7α-HSDH: (A) R16A/R194A; (B) T15A/R16A/R194A. Fig. 5. Coenzyme specificity-related structure of 9 NADP(H)-dependent SDRs: (A) 17β-HSDH (Cochliobolus lunatus, PDB: 3QWF); (B) Estradiol 17β-dehydrogenase 1 (Homo sapiens, PDB: 1QYV); (C) Carbonyl reductase (Mus musculus, PDB: 1CYD); (D) Trihydroxynaphthalene reductase (Magnaporthe grisea, PDB: 1DOH); (E) Carbonyl reductase/20beta-hydroxysteroid dehydrogenase (Sus scrofa, PDB: 1N5D); (F) Sepiapterin reductase (Mus musculus, PDB: 1NAS); (G) Mannitol dehydrogenase (Agaricus bisporus, PDB: 1H5Q); (H) Beta-keto acyl carrier protein reductase (Brassica napus, PDB: 1EDO); (I) 1,3,6,8-tetrahydroxynaphthalene reductase (Magnaporthe grisea, PDB: 1JA9).

11

12

13

14

15

16

Table 1. Kinetic constants of WT CA 7α-HSDH and the mutants toward NADP+. Enzyme

Km (mM)

kcat (s-1)

kcat/K (s-1·mM-1)

WT

0.69

4.89

7.10

T15A

12.33

15.85

1.29

R16A

4.25

25.26

5.95

R194A

4.71

111.70

23.72

R16A/R194A

0.96

3.86

4.02

T15A/R16A/R194A

2.95

3.23

1.09

R38A

ND

/

/

R38A/R194A

ND

/

/

ND: not detected.

Table 2. Binding free energies between NADP+ and CA 7α-HSDH. Enzyme

∆G binding (kcal/mol)

WT

-105.18±8.57

T15A

-94.91±6.50

R16A

-86.06±9.55

R194A

-79.61±8.21

R16A/R194A

-67.82±5.62

T15A/R16A/R194A

-51.06±5.93

17