BACKGROUND
the aldo-keto reductase  superfamily is mostly comprised of monomeric oxidoreductases that catalyze nadh-dependent reductions of a wide range of aldehydes and ketones including steroids, carbohydrates, bile acids, and prostaglandins  <cit> .
the akrs have been classified into  <dig> families  and akr <dig> family have been further divided into  <dig> subfamilies   <cit> .
among the subfamilies, akr1c enzymes are known as hydroxysteroid dehydrogenases  which play a pivotal role in metabolism and regulation of steroid hormones such as progesterone, 5α-dihydrotestosterone , and testosterone.
pig aldo-keto reductase family  <dig> member c <dig>  shows both 3α- and 20α-hsd activities and also plays a crucial role in progesterone metabolism, maintenance of pregnancy, and hormone regulation during the estrous cycle  <cit> .
it officially named as akrlcl <dig> , consists of  <dig> amino acid residues longer than that of general akr1c <dig>  <cit> .
the longer amino acid residues have been reported to alter enzymatic activities of several steroid hormones  <cit> .
the structures of akrs have the 8-barrel or tim-barrel motif and three conserved loop regions, loop a, b, and c, which are related with steroid hormone specificity  <cit> .
the enzymes catalyze an ordered bisequential kenetic process in which binding of cofactor is obligatory for the reaction  <cit> .
the nicotinamide group of nadph cofactor lies in anti-conformation with respect to the ribose group, so that 4-pro-r-hydride is transferred from the cofactor to the 3-ketosteroid substrate  <cit> .
the hydride transfer is mediated by a highly conserved catalytic tetrad consisting of asp <dig>  tyr <dig>  lys <dig>  and his <dig>  where tyr  <dig> acts as the general acid/base  <cit> .
recently we have identified two paralogous pig akr1c1s with or without c-terminal region  which was truncated by a non-synonymous variation  <cit> .
also, the c-terminal region significantly affects the nadph-dependent reductase activity for 5α-dht reduction  <cit> .
in this study, we performed molecular modeling studies combined with kinetic experiments to examine structural difference between wild-type akr1c <dig>  and c-terminal-truncated akr1c <dig>  for 5α-dht.
since there was no available experimental structure of pig akr1c <dig>  we have carried out homology modeling to build 3d structure models of the both types, which were used for molecular dynamics  simulation study.
our findings provide structural insights into important role of c-terminal region of the enzyme.
it can be helpful for understanding different enzymatic properties for 5α-dht between c-type and t-type.
methods
materials
the following chemicals were used in the experiments; 5α-dihydrotestosterone , methylglyoxal,  <dig> -phenanthrenequinone and hydrindantin were purchased from sigma , and ni-nta chelating agarose cl-6b was purchased from peptron company .
bio-rad bradford protein assay kit was purchased from .
the others, including na2hpo <dig>  nah2po <dig>  nacl, bovine serum albumin , and imidazole, were purchased from sigma .
recombinant protein purification
in previous study  <cit> , two types of pproex htb-akr1cl <dig> clones were constructed for the production of his-tagged fusion proteins for c-type and t-type.
they were used for the iptg-induced expression of each of the clones in e. coli bl <dig>  in this study, the iptg-induced proteins were subjected to the affinity chromatography using ni-nta agarose, according to manufacturer's manual .
briefly, basal buffer for protein purification was prepared by 50 mm sodium phosphate buffer, ph <dig> , and 500 mm nacl.
imidazole  was added to required concentration according to purification manual with nta chelating agarose cl-6b .
the overexpressed cells were precipitated by centrifugation, and suspended by binding buffer including 5 mm imidazole.
the collected cells were lyzed by sonics vibracell vcx <dig> ultrasonic cell disruptor, which was done twice by conditions as following; 5 min by 2 sec interval of on/off and 35% amplitude during ice cooling.
the supernatant to obtain water-soluble protein was collected from the cells treated by centrifugation for 30 min at  <dig>  rpm, 4°c.
purification of the protein was done by nta chelating agarose cl-6b  according to manufacturer’s directions.
the purified recombinant proteins were concentrated by ultrafree- <dig>  centrifugal filter device .
the concentrated proteins were quantified by bio-rad bradford protein assay kit  by od <dig> nm in wavelength.
the purified proteins were added with 50% glycerol and 50 mm sodium phosphate buffer  for long-term storage at −20°c.
measurement of nadph-dependent carbonyl reductase activity
the reductase activity was measured under conditions described previously  <cit> .
reaction mixtures included 60 mm sodium phosphate , purified recombinant proteins such as c-type and t-type,  <dig>  mm nadph and  <dig>  mm substrates  and were incubated in a total volume of  <dig>  ml at 37°c.
the assay of reductase activity was spectrophotometrically carried out by monitoring the decrease in absorbance at 340 nm with time.
statistical analysis
to determine kinetic parameters with a michaelis–menten plot, the data were analyzed by nonlinear regression using graphpad prism  <dig> software .
enzyme concentrations of  <dig> and  <dig>  nmol/mg were used for the calculation of turnover rates  for c-type and t-type, respectively.
the significant differences were analyzed by student’s t-test  using the above software.
the results are expressed as means ± standard errors  of at least  <dig> independent experiments.
homology modeling
the sequence of pig akr1c <dig> consisting of  <dig> amino acids, was obtained from uniprotkb  .
in order to build a structure model of pig akr1c <dig> homology modeling was conducted using phyre <dig> server  with intensive modeling mode  <cit> , which the server utilizes multiple templates and ab initio techniques to predict 3d structure model.
the generated homology model was subjected to energy minimization to refine the model as well as to reduce steric clashes.
the minimization with the steepest descent algorithm for  <dig>  steps was carried out by gromacs  <dig> .
<dig> package  <cit>  with charmm <dig> force field.
the stereochemical quality of the model was assessed by procheck  <cit> , prosa  <cit> , and errat  <cit> .
all other analyses including multiple sequence alignment were done by discovery studio v <dig>  .
molecular docking calculation
akr1c <dig> c-type and t-type in complex with nadph were subjected to molecular docking calculation.
the structure of t-type was prepared by deleting the c-terminal region  from c-type.
the coordinates of the cofactor were taken from the structure of human akr1c <dig> .
the substrate, 5α-dht, was downloaded from pubchem compound database   <cit> .
then 5α-dht was subjected to energy minimization with charmm force field and implicit solvent model using ds.
the binding pose of 5α-dht was predicted using gold v  <dig> .
<dig>   <cit>  which uses genetic algorithm  for docking flexible ligands in the binding site of the protein.
the binding site was assigned through define and edit binding site tool in ds.
all residues within the radius of 5 Å of the center of binding sphere were included in the calculation and the number of ga runs was set to  <dig>  all other parameters were used as their default values.
the docking poses were ranked based on gold fitness score and top solution was selected as initial conformation for md simulation.
molecular dynamics simulation
md simulations for c-type and t-type in complex with nadph and 5α-dht were performed using gromacs  <dig> .
<dig> with charmm <dig> force field.
topology files for the ligands were obtained from swissparam server  <cit> .
at the beginning, protonation states of the ionizable residues were set at ph <dig>  a water box with the size of  <dig>  nm from the protein surface was created to make an aqueous environment, and immersed using explicit tip3p water model  <cit> .
the size of the system was  <dig>  ×  <dig>  ×  <dig>  nm for c-type and  <dig>  ×  <dig>  ×  <dig>  nm for t-type, respectively.
several water molecules were replaced with sodium ions to neutralize the system.
energy minimization for  <dig>  steps was executed using steepest descent algorithm until the maximum force lower than 1000 kj/mol.
after minimization, the systems were subjected to 100 ps nvt equilibration at 300 k and then 100 ps npt equilibration at 300 k and 1 bar of pressure.
the equilibrated systems were used in 20 ns production runs under npt ensemble.
a constant temperature and pressure were kept using v-rescale thermostat  <cit>  and parrinello-rahman barostat  <cit> .
during the simulation, lincs  <cit>  and settle  <cit>  algorithms were used to constrain all bond lengths and the geometry of water molecules, respectively.
short-range interactions were treated with the cut-off value of  <dig>  nm and long-range electrostatic interactions were calculated by applying particle mesh ewald  method  <cit> .
the periodic boundary conditions were adopted to avoid edge effects.
a grid spacing of  <dig>  nm was applied for fast fourier transform calculations.
we repeated the simulations two times under the same conditions except that the simulation time was 10 ns.
all simulations were performed with the time step of 2 fs and the coordinates were saved every 1 ps for analyses.
RESULTS
the c-terminal region in akr1c <dig> alters significantly the enzymatic properties to 5α-dht
akr1c <dig> exhibits broadly enzymatic activities to various steroid hormones  <cit> .
among steroid hormones, akr1c <dig> originated from human, previously named as 20α-hydroxysteroid dehydrogenase, detects specifically to progesterone with high activity  <cit> .
a variant truncated at the c-terminus of pig akr1c <dig> was employed in this study.
in our previous study, we found a new novel single nucleotide variant  truncated in c-terminus, where the snv is a nonsense mutant lacking  <dig> amino acid residues  in c-terminus  <cit> .
during the evaluation of enzymatic activities with different steroid hormones, differential activities between akr1c <dig> c- and t-types were shown, 5α-dht being anyway the preferred substrate for both of them  <cit> .
therefore, 5α-dht was employed for enzymatic kinetics in this study.
in order to analyze enzymatic activity of akr1c1s, the enzymes were cloned into overexpression vector and then purified to homogeneity by affinity chromatography.
the purified akr1c1s were applied for enzymatic kinetics with substrate 5α-dht and cofactor nadph.
the vmax value of akr1c <dig> t-type was  <dig>  fold higher than that of c-type, but km value was lower  <dig>  fold .
furthermore, the values of kcat and catalytic efficiency of the t-type were  <dig>  and  <dig>  folds higher than those of c-type.
these results suggest that c-terminal truncated akr1c <dig> improves the values of vmax,kcat and catalytic efficiency.figure  <dig>
a michaelis-menten plot from measurement of the nadph-dependent reduction of 5α-dht by recombinant akr1c <dig>  or akr1c <dig> .
the data, obtained from measurement of the nadph-dependent reduction of 5α-dht by recombinant akr1c <dig>  and akr1c <dig> , were analyzed by nonlinear regression using graphpad prism  <dig> software .
each spot represents the mean ± s.e.
.
kinetic parameters for 5α-dht reduction measured by spectrophotometer
enzyme
v
max
k
m
k
cat
k
cat
/k
m
each value indicates mean ± sem .
kinetic parameters were determined using data in figure  <dig> through graphpad prism  <dig> software .
the structure prediction of pig akr1c <dig> using homology modeling
since crystal structure of pig akr1c <dig> has not been determined yet, we have constructed the 3d structure model using four structures as templates; rat akr1c <dig> , human akr1c <dig> , human akr1c <dig> , and rabbit akr1c <dig> .
the multiple sequence alignment with the four templates revealed that catalytic tetrad of asp <dig>  tyr <dig>  lys <dig>  and his <dig> were conserved and they have high sequence identity and similarity with each template;  <dig> % and  <dig> % between pig akr1c <dig> and rat akr1c <dig>   <dig> % and  <dig> % between pig akr1c <dig> and human akr1c <dig>   <dig> % and  <dig> % between pig akr1c <dig> and human akr1c <dig>   <dig> % and  <dig> % between pig akr1c <dig> and rabbit akr1c <dig>  respectively .
since there was no proper structural information for  <dig> residues at the end of the c-terminal region of akr1c <dig>  the region was modeled by ab initio method.
the homology model for pig akr1c <dig> was refined through the energy minimization and it showed conserved loop regions which are structural features of akr superfamily .
the stereochemical quality of the generated model was evaluated using three programs.
ramachandran plot obtained from procheck showed that  <dig> % of residues were in most favored regions and only one residue was in disallowed region .
overall quality factor scores calculated from errat and prosa were  <dig>  and − <dig> , respectively.figure  <dig>
the result of homology modeling.
a multiple sequence alignment of pig akr1c <dig> with rat akr1c <dig> , human akr1c <dig> , human akr1c <dig> , and rabbit akr1c <dig> .
b the 3d structure model of pig akr1c <dig>  protein is represented as cartoon model and colored by secondary structure.
loop a, loop b, and loop c are displayed as yellow.
c ramachandran plot of pig akr1c <dig> structure.
binding mode of 5α-dht in the active site of akr1c <dig> c-type and t-type
a molecular docking study was performed to discover proper binding conformations for 5α-dht at the active site of the c-type and t-type.
the docking conformations were clustered and ranked according to their gold fitness scores.
a conformation having high fitness score in the most populated cluster was selected as putative binding pose of each system.
the docking results revealed that 5α-dht bound to the both types of akr1c <dig> in a similar manner forming hydrophobic interactions with tyr <dig>  leu <dig>  trp <dig>  phe <dig>  leu <dig>  trp <dig>  phe <dig>  and tyr <dig> which have been reported as key residues for steroid binding  <cit> .
we found that 5α-dht formed two hydrogen bonds with tyr <dig> and his <dig> in the c-type, whereas, in the t-type, there was additional hydrogen bond with leu <dig> as well as two hydrogen bonds.
the final docking poses in c-type and t-type were used as initial structures in md simulation study to understand the effect of c-terminal region on the enzymatic activity in atomic level.
we evaluated the overall stability of md simulations by calculating cα root-mean-square deviation , potential energy, and the number of intra-hydrogen bonds .
the rmsd values for each system were converged to around  <dig>  nm in c-type and  <dig>  nm in t-type .
during the whole simulation time, the rmsd value of c-type was relatively higher than that of t-type with the average value of  <dig>  nm and  <dig>  nm, respectively.
the rmsd plot for only 5α-dht also revealed that the substrates in both c- and t-types achieved stabilization and their average values were  <dig>  nm .
moreover, potential energy and the number of intra-hydrogen bonds for the systems remained constant for the simulation time .
these results indicate that the md simulations for both systems were successfully completed and there were no abnormal behaviors in the structures throughout the simulation time.
a structural comparison between the c-type and t-type was performed using their representative structures which were the closest snapshot to the average of all snapshots obtained from the last 5 ns.
although there were no significant conformational changes in both systems, they showed a difference in 5α-dht binding in terms of hydrogen bond interactions .
the c-type showed only one hydrogen bond interaction between oxygen atom of 5α-dht and hydrogen atom of his <dig> with the distance of  <dig>  nm .
on the other hand, oxygen atoms of 5α-dht formed hydrogen bonds with hydrogen atoms of tyr <dig> and his <dig> in t-type and the distances of bonds were within  <dig>  nm .
the residues tyr <dig>  leu <dig>  trp <dig>  leu <dig>  leu <dig>  trp <dig>  phe <dig>  and tyr <dig> in both structures were participated in hydrophobic interactions which were similar to that observed in the initial docked structures.
these further stabilized 5α-dht binding in both active sites.figure  <dig>
the overall stability of md simulation.
a rmsd for protein cα atoms and 5α-dht , b potential energy, and c the number of intra-hydrogen bonds were calculated during 20 ns simulation time.
akr1c <dig> c-type and t-type are represented as blue and red lines, respectively.
superposition of c-type and t-type.
the c-type was superimposed into t-type using cα atoms of the proteins.
the c-type  and t-type  are shown as cartoon models and loop regions are drawn as more dark colors.
nadph and 5α-dht are indicated as stick models.
only polar hydrogens are shown.
binding mode of 5α-dht in the active site of akr1c <dig>  a 5α-dht binding in c-type, b in t-type.
the c-type  and t-type  are depicted as cartoon models while nadph, 5α-dht, and the residues involving molecular interactions with 5α-dht are shown as stick model and labeled.
the hydrogen bonds are shown as black dash lines.
difference in 5α-dht binding between c- type and t-type
a major difference in 5α-dht binding for c-type and t-type of akr1c <dig> was the relative distance from tyr <dig> which is important to initiate the catalytic reaction of the enzyme.
the c <dig> ketone of 5α-dht in the t-type was positioned much closer to the catalytic tetrad and the 4-pro-r hydride of the nadph than in the c-type .
the distance between c <dig> position of 5α-dht and c <dig> position in nicotinamide ring of nadph were  <dig>  nm in c-type and  <dig>  nm in t-type.
in contrast, the distance between the c <dig> of the 5α-dht and hydroxyl group of tyr <dig> in t-type was  <dig>  nm which is much shorter than the value of  <dig>  nm in c-type.
the monitoring these distances during 20 ns simulation time revealed that the both distances were relatively short in t-type compared to c-type .
superimposition of the two structures showed that 5α-dht in t-type was sandwiched between leu <dig> and trp <dig> and its β-face was oriented toward trp <dig>  whereas in the case of c-type, the flipping of the side chain of trp <dig> hindered the interaction with β-face of 5α-dht .
the side chain of tyr <dig> also showed different conformation in the both types and that was probably due to displacement of trp <dig>  from the structural comparison, it appears that binding conformation of 5α-dht in t-type was more favorable for catalytic reaction than that of c-type.
in root mean square fluctuation  plot, it was observed that the residues 226– <dig> in t-type exhibited higher flexibility than in c-type, while flexibilities of other residues were quite similar in the both structures except for highly flexible regions such as n- or c-terminal part of the protein .
these differences might be explained by flipping of trp <dig> in c-type.
from rmsd plot calculated using all atoms of trp <dig>  the rmsd value in c-type showed relatively high with the average of  <dig>  nm and it started to increase from 5 ns .
in the simulation for c-type, the flipping of trp <dig> side chain was observed and 5α-dht was gradually alienated from tyr <dig> during that time.
compared to c-type, rmsd value of trp <dig> in t-type was very stable, less than  <dig>  nm, throughout 20 ns simulation time and the average value was  <dig>  nm.
this might be related to the observation that flipping of trp <dig> hardly ever happened in t-type.
these analyses demonstrated that the instability of trp <dig> caused by flipping of the side chain might be correlated with the distance from 5α-dht to the hydroxyl group of tyr <dig>  additionally, the interaction energy between 5α-dht and the protein was − <dig>  kcal/mol in c-type and − <dig>  kcal/mol in t-type.
it also indicated that 5α-dht in t-type had energetically favorable conformation.figure  <dig>
the measurement of key distance for reduction reaction.
a the crucial distance to initiate catalytic reaction and relative position of the catalytic tetrad and cofactor in the active site of c-type and t-type.
the catalytic tetrad and 5α-dht in c-type  and t-type  are displayed as stick models.
the key distances are given in nm.
b the distance between c <dig> of 5α-dht and c4n of nadph.
c the distance between c <dig> of 5α-dht and oh of tyr <dig>  the both distances in c- and t-types were measured during 20 ns simulation time.
blue and red lines indicate c-type and t-type, respectively.
comparison of the 5α-dht binding in the active site of c-type and t-type.
interacting residues and 5α-dht in c-type  and t-type  are drawn as stick models.
rmsf plot showing the atomic fluctuations by residues of c-type and t-type.
rmsf values for cα atoms of the proteins are drawn as blue and red lines, respectively.
rmsd plot for trp <dig> of c-type and t-type.
rmsd values for all atoms of trp <dig> are displayed as blue and red lines, respectively.
CONCLUSIONS
the study of enzyme kinetics revealed that the c-terminal region in akr1c <dig> contributed significantly the enzymatic properties for 5α-dht reduction.
to gain structural insights into the difference between c-type and t-type of akr1c <dig> for 5α-dht reduction, md simulations for both structures were carried out.
prior to the simulation, we generated homology model structure for akr1c <dig> due to lack of experimentally determined structures.
then c-type and t-type in complex with 5α-dht obtained from molecular docking study were used as initial conformations for md simulation.
although there were no significant conformational changes in both systems during 20 ns simulation time, binding conformations of 5α-dht were different in the active site of c-type and t-type.
the structural comparisons showed that t-type formed strong hydrogen bonds with tyr <dig> and his <dig>  while only his <dig> was found in c-type.
to initiate catalytic reaction, the c <dig> ketone group of 5α-dht should be close to tyr <dig> and the nicotinamide ring of nadph which are involved in hydride transfer.
the distances between these groups were monitored during 20 ns simulation time.
as a result, 5α-dht was close to the cofactor in the both structures, whereas the distance between 5α-dht and tyr <dig> in t-type was relatively much shorter than c-type.
on the contrary, the flipping of the side chain of trp <dig> in c-type might disrupt the interaction with β-face of 5α-dht.
the interaction energies between 5α-dht and the proteins also indicated that t-type was energetically stable compared to c-type.
taken together, our simulation results demonstrated that binding conformation of 5α-dht in t-type was more favorable for catalytic reaction than that of c-type.
these structural explanations were also in agreement with kinetic experimental results.
our findings will be useful to understand molecular mechanism for the enzyme kinetics of akr1c <dig> protein.
minky son, chanin park and seul gi kwon contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
ms and cp performed structural modeling and analyzed data.
sgk, wyb, and swk designed the study and carried out the experiments and statistical analysis.
ms, cp, sgk, wyb, and swk wrote the manuscript.
cwk and kwl interpreted the data and correct the manuscript.
all authors read and approved the final manuscript.
this work was supported by grants from priority research centers program  and management of climate change program  through the national research foundation of korea  funded by the ministry of education of republic of korea, the project, “search & discovery of utility value from biological resources ”, from the national institute of biological resources of korean government, the export promotion technology development program  of ministry of food, agriculture, forestry and fisheries, republic of korea.
and this work was also supported by the next-generation biogreen  <dig> program  from rural development administration  of republic of korea.

 the aldo-keto reductase superfamily is mostly comprised of monomeric oxidoreductases that catalyze nadh-dependent reductions of a wide range of aldehydes and ketones including steroids, carbohydrates, bile acids, and prostaglandins. the akrs have been classified into families and akr family have been further divided into subfamilies. among the subfamilies, akr1c enzymes are known as hydroxysteroid dehydrogenases which play a pivotal role in metabolism and regulation of steroid hormones such as progesterone, 5α-dihydrotestosterone, and testosterone. pig aldo-keto reductase family member c shows both 3α- and 20α-hsd activities and also plays a crucial role in progesterone metabolism, maintenance of pregnancy, and hormone regulation during the estrous cycle. it officially named as akrlcl, consists of amino acid residues longer than that of general akr1c. the longer amino acid residues have been reported to alter enzymatic activities of several steroid hormones. the structures of akrs have the 8-barrel or tim-barrel motif and three conserved loop regions, loop a, b, and c, which are related with steroid hormone specificity. the enzymes catalyze an ordered bisequential kenetic process in which binding of cofactor is obligatory for the reaction. the nicotinamide group of nadph cofactor lies in anti-conformation with respect to the ribose group, so that 4-pro-r-hydride is transferred from the cofactor to the 3-ketosteroid substrate. the hydride transfer is mediated by a highly conserved catalytic tetrad consisting of asp tyr lys and his where tyr acts as the general acid/base. recently we have identified two paralogous pig akr1c1s with or without c-terminal region which was truncated by a non-synonymous variation. also, the c-terminal region significantly affects the nadph-dependent reductase activity for 5α-dht reduction. in this study, we performed molecular modeling studies combined with kinetic experiments to examine structural difference between wild-type akr1c and c-terminal-truncated akr1c for 5α-dht. since there was no available experimental structure of pig akr1c we have carried out homology modeling to build 3d structure models of the both types, which were used for molecular dynamics simulation study. our findings provide structural insights into important role of c-terminal region of the enzyme. it can be helpful for understanding different enzymatic properties for 5α-dht between c-type and t-type. methods materials the following chemicals were used in the experiments; 5α-dihydrotestosterone, methylglyoxal, -phenanthrenequinone and hydrindantin were purchased from sigma, and ni-nta chelating agarose cl-6b was purchased from peptron company. bio-rad bradford protein assay kit was purchased from. the others, including na2hpo nah2po nacl, bovine serum albumin, and imidazole, were purchased from sigma. recombinant protein purification in previous study, two types of pproex htb-akr1cl clones were constructed for the production of his-tagged fusion proteins for c-type and t-type. they were used for the iptg-induced expression of each of the clones in e. coli bl in this study, the iptg-induced proteins were subjected to the affinity chromatography using ni-nta agarose, according to manufacturer's manual. briefly, basal buffer for protein purification was prepared by 50 mm sodium phosphate buffer, ph, and 500 mm nacl. imidazole was added to required concentration according to purification manual with nta chelating agarose cl-6b. the overexpressed cells were precipitated by centrifugation, and suspended by binding buffer including 5 mm imidazole. the collected cells were lyzed by sonics vibracell vcx ultrasonic cell disruptor, which was done twice by conditions as following; 5 min by 2 sec interval of on/off and 35% amplitude during ice cooling. the supernatant to obtain water-soluble protein was collected from the cells treated by centrifugation for 30 min at  rpm, 4°c. purification of the protein was done by nta chelating agarose cl-6b according to manufacturer’s directions. the purified recombinant proteins were concentrated by ultrafree- centrifugal filter device. the concentrated proteins were quantified by bio-rad bradford protein assay kit by od nm in wavelength. the purified proteins were added with 50% glycerol and 50 mm sodium phosphate buffer for long-term storage at −20°c. measurement of nadph-dependent carbonyl reductase activity the reductase activity was measured under conditions described previously. reaction mixtures included 60 mm sodium phosphate, purified recombinant proteins such as c-type and t-type,  mm nadph and  mm substrates and were incubated in a total volume of  ml at 37°c. the assay of reductase activity was spectrophotometrically carried out by monitoring the decrease in absorbance at 340 nm with time. statistical analysis to determine kinetic parameters with a michaelis–menten plot, the data were analyzed by nonlinear regression using graphpad prism software. enzyme concentrations of and  nmol/mg were used for the calculation of turnover rates for c-type and t-type, respectively. the significant differences were analyzed by student’s t-test using the above software. the results are expressed as means ± standard errors of at least independent experiments. homology modeling the sequence of pig akr1c consisting of amino acids, was obtained from uniprotkb. in order to build a structure model of pig akr1c homology modeling was conducted using phyre server with intensive modeling mode, which the server utilizes multiple templates and ab initio techniques to predict 3d structure model. the generated homology model was subjected to energy minimization to refine the model as well as to reduce steric clashes. the minimization with the steepest descent algorithm for steps was carried out by gromacs. package with charmm force field. the stereochemical quality of the model was assessed by procheck, prosa, and errat. all other analyses including multiple sequence alignment were done by discovery studio v. molecular docking calculation akr1c c-type and t-type in complex with nadph were subjected to molecular docking calculation. the structure of t-type was prepared by deleting the c-terminal region from c-type. the coordinates of the cofactor were taken from the structure of human akr1c. the substrate, 5α-dht, was downloaded from pubchem compound database. then 5α-dht was subjected to energy minimization with charmm force field and implicit solvent model using ds. the binding pose of 5α-dht was predicted using gold v. which uses genetic algorithm for docking flexible ligands in the binding site of the protein. the binding site was assigned through define and edit binding site tool in ds. all residues within the radius of 5 Å of the center of binding sphere were included in the calculation and the number of ga runs was set to all other parameters were used as their default values. the docking poses were ranked based on gold fitness score and top solution was selected as initial conformation for md simulation. molecular dynamics simulation md simulations for c-type and t-type in complex with nadph and 5α-dht were performed using gromacs. with charmm force field. topology files for the ligands were obtained from swissparam server. at the beginning, protonation states of the ionizable residues were set at ph a water box with the size of  nm from the protein surface was created to make an aqueous environment, and immersed using explicit tip3p water model. the size of the system was × ×  nm for c-type and × ×  nm for t-type, respectively. several water molecules were replaced with sodium ions to neutralize the system. energy minimization for steps was executed using steepest descent algorithm until the maximum force lower than 1000 kj/mol. after minimization, the systems were subjected to 100 ps nvt equilibration at 300 k and then 100 ps npt equilibration at 300 k and 1 bar of pressure. the equilibrated systems were used in 20 ns production runs under npt ensemble. a constant temperature and pressure were kept using v-rescale thermostat and parrinello-rahman barostat. during the simulation, lincs and settle algorithms were used to constrain all bond lengths and the geometry of water molecules, respectively. short-range interactions were treated with the cut-off value of  nm and long-range electrostatic interactions were calculated by applying particle mesh ewald method. the periodic boundary conditions were adopted to avoid edge effects. a grid spacing of  nm was applied for fast fourier transform calculations. we repeated the simulations two times under the same conditions except that the simulation time was 10 ns. all simulations were performed with the time step of 2 fs and the coordinates were saved every 1 ps for analyses.  the c-terminal region in akr1c alters significantly the enzymatic properties to 5α-dht akr1c exhibits broadly enzymatic activities to various steroid hormones. among steroid hormones, akr1c originated from human, previously named as 20α-hydroxysteroid dehydrogenase, detects specifically to progesterone with high activity. a variant truncated at the c-terminus of pig akr1c was employed in this study. in our previous study, we found a new novel single nucleotide variant truncated in c-terminus, where the snv is a nonsense mutant lacking amino acid residues in c-terminus. during the evaluation of enzymatic activities with different steroid hormones, differential activities between akr1c c- and t-types were shown, 5α-dht being anyway the preferred substrate for both of them. therefore, 5α-dht was employed for enzymatic kinetics in this study. in order to analyze enzymatic activity of akr1c1s, the enzymes were cloned into overexpression vector and then purified to homogeneity by affinity chromatography. the purified akr1c1s were applied for enzymatic kinetics with substrate 5α-dht and cofactor nadph. the vmax value of akr1c t-type was fold higher than that of c-type, but km value was lower fold. furthermore, the values of kcat and catalytic efficiency of the t-type were and folds higher than those of c-type. these results suggest that c-terminal truncated akr1c improves the values of vmax,kcat and catalytic efficiency.figure a michaelis-menten plot from measurement of the nadph-dependent reduction of 5α-dht by recombinant akr1c or akr1c. the data, obtained from measurement of the nadph-dependent reduction of 5α-dht by recombinant akr1c and akr1c, were analyzed by nonlinear regression using graphpad prism software. each spot represents the mean ± s.e.. kinetic parameters for 5α-dht reduction measured by spectrophotometer enzyme v max k m k cat k cat /k m each value indicates mean ± sem. kinetic parameters were determined using data in figure  through graphpad prism software. the structure prediction of pig akr1c using homology modeling since crystal structure of pig akr1c has not been determined yet, we have constructed the 3d structure model using four structures as templates; rat akr1c, human akr1c, human akr1c, and rabbit akr1c. the multiple sequence alignment with the four templates revealed that catalytic tetrad of asp tyr lys and his were conserved and they have high sequence identity and similarity with each template; % and % between pig akr1c and rat akr1c % and % between pig akr1c and human akr1c % and % between pig akr1c and human akr1c % and % between pig akr1c and rabbit akr1c respectively. since there was no proper structural information for residues at the end of the c-terminal region of akr1c the region was modeled by ab initio method. the homology model for pig akr1c was refined through the energy minimization and it showed conserved loop regions which are structural features of akr superfamily. the stereochemical quality of the generated model was evaluated using three programs. ramachandran plot obtained from procheck showed that % of residues were in most favored regions and only one residue was in disallowed region. overall quality factor scores calculated from errat and prosa were and −, respectively.figure the result of homology modeling. a multiple sequence alignment of pig akr1c with rat akr1c, human akr1c, human akr1c, and rabbit akr1c. b the 3d structure model of pig akr1c protein is represented as cartoon model and colored by secondary structure. loop a, loop b, and loop c are displayed as yellow. c ramachandran plot of pig akr1c structure. binding mode of 5α-dht in the active site of akr1c c-type and t-type a molecular docking study was performed to discover proper binding conformations for 5α-dht at the active site of the c-type and t-type. the docking conformations were clustered and ranked according to their gold fitness scores. a conformation having high fitness score in the most populated cluster was selected as putative binding pose of each system. the docking results revealed that 5α-dht bound to the both types of akr1c in a similar manner forming hydrophobic interactions with tyr leu trp phe leu trp phe and tyr which have been reported as key residues for steroid binding. we found that 5α-dht formed two hydrogen bonds with tyr and his in the c-type, whereas, in the t-type, there was additional hydrogen bond with leu as well as two hydrogen bonds. the final docking poses in c-type and t-type were used as initial structures in md simulation study to understand the effect of c-terminal region on the enzymatic activity in atomic level. we evaluated the overall stability of md simulations by calculating cα root-mean-square deviation, potential energy, and the number of intra-hydrogen bonds. the rmsd values for each system were converged to around  nm in c-type and  nm in t-type. during the whole simulation time, the rmsd value of c-type was relatively higher than that of t-type with the average value of  nm and  nm, respectively. the rmsd plot for only 5α-dht also revealed that the substrates in both c- and t-types achieved stabilization and their average values were  nm. moreover, potential energy and the number of intra-hydrogen bonds for the systems remained constant for the simulation time. these results indicate that the md simulations for both systems were successfully completed and there were no abnormal behaviors in the structures throughout the simulation time. a structural comparison between the c-type and t-type was performed using their representative structures which were the closest snapshot to the average of all snapshots obtained from the last 5 ns. although there were no significant conformational changes in both systems, they showed a difference in 5α-dht binding in terms of hydrogen bond interactions. the c-type showed only one hydrogen bond interaction between oxygen atom of 5α-dht and hydrogen atom of his with the distance of  nm. on the other hand, oxygen atoms of 5α-dht formed hydrogen bonds with hydrogen atoms of tyr and his in t-type and the distances of bonds were within  nm. the residues tyr leu trp leu leu trp phe and tyr in both structures were participated in hydrophobic interactions which were similar to that observed in the initial docked structures. these further stabilized 5α-dht binding in both active sites.figure the overall stability of md simulation. a rmsd for protein cα atoms and 5α-dht, b potential energy, and c the number of intra-hydrogen bonds were calculated during 20 ns simulation time. akr1c c-type and t-type are represented as blue and red lines, respectively. superposition of c-type and t-type. the c-type was superimposed into t-type using cα atoms of the proteins. the c-type and t-type are shown as cartoon models and loop regions are drawn as more dark colors. nadph and 5α-dht are indicated as stick models. only polar hydrogens are shown. binding mode of 5α-dht in the active site of akr1c a 5α-dht binding in c-type, b in t-type. the c-type and t-type are depicted as cartoon models while nadph, 5α-dht, and the residues involving molecular interactions with 5α-dht are shown as stick model and labeled. the hydrogen bonds are shown as black dash lines. difference in 5α-dht binding between c- type and t-type a major difference in 5α-dht binding for c-type and t-type of akr1c was the relative distance from tyr which is important to initiate the catalytic reaction of the enzyme. the c ketone of 5α-dht in the t-type was positioned much closer to the catalytic tetrad and the 4-pro-r hydride of the nadph than in the c-type. the distance between c position of 5α-dht and c position in nicotinamide ring of nadph were  nm in c-type and  nm in t-type. in contrast, the distance between the c of the 5α-dht and hydroxyl group of tyr in t-type was  nm which is much shorter than the value of  nm in c-type. the monitoring these distances during 20 ns simulation time revealed that the both distances were relatively short in t-type compared to c-type. superimposition of the two structures showed that 5α-dht in t-type was sandwiched between leu and trp and its β-face was oriented toward trp whereas in the case of c-type, the flipping of the side chain of trp hindered the interaction with β-face of 5α-dht. the side chain of tyr also showed different conformation in the both types and that was probably due to displacement of trp from the structural comparison, it appears that binding conformation of 5α-dht in t-type was more favorable for catalytic reaction than that of c-type. in root mean square fluctuation plot, it was observed that the residues 226– in t-type exhibited higher flexibility than in c-type, while flexibilities of other residues were quite similar in the both structures except for highly flexible regions such as n- or c-terminal part of the protein. these differences might be explained by flipping of trp in c-type. from rmsd plot calculated using all atoms of trp the rmsd value in c-type showed relatively high with the average of  nm and it started to increase from 5 ns. in the simulation for c-type, the flipping of trp side chain was observed and 5α-dht was gradually alienated from tyr during that time. compared to c-type, rmsd value of trp in t-type was very stable, less than  nm, throughout 20 ns simulation time and the average value was  nm. this might be related to the observation that flipping of trp hardly ever happened in t-type. these analyses demonstrated that the instability of trp caused by flipping of the side chain might be correlated with the distance from 5α-dht to the hydroxyl group of tyr additionally, the interaction energy between 5α-dht and the protein was −  kcal/mol in c-type and −  kcal/mol in t-type. it also indicated that 5α-dht in t-type had energetically favorable conformation.figure the measurement of key distance for reduction reaction. a the crucial distance to initiate catalytic reaction and relative position of the catalytic tetrad and cofactor in the active site of c-type and t-type. the catalytic tetrad and 5α-dht in c-type and t-type are displayed as stick models. the key distances are given in nm. b the distance between c of 5α-dht and c4n of nadph. c the distance between c of 5α-dht and oh of tyr the both distances in c- and t-types were measured during 20 ns simulation time. blue and red lines indicate c-type and t-type, respectively. comparison of the 5α-dht binding in the active site of c-type and t-type. interacting residues and 5α-dht in c-type and t-type are drawn as stick models. rmsf plot showing the atomic fluctuations by residues of c-type and t-type. rmsf values for cα atoms of the proteins are drawn as blue and red lines, respectively. rmsd plot for trp of c-type and t-type. rmsd values for all atoms of trp are displayed as blue and red lines, respectively.  the study of enzyme kinetics revealed that the c-terminal region in akr1c contributed significantly the enzymatic properties for 5α-dht reduction. to gain structural insights into the difference between c-type and t-type of akr1c for 5α-dht reduction, md simulations for both structures were carried out. prior to the simulation, we generated homology model structure for akr1c due to lack of experimentally determined structures. then c-type and t-type in complex with 5α-dht obtained from molecular docking study were used as initial conformations for md simulation. although there were no significant conformational changes in both systems during 20 ns simulation time, binding conformations of 5α-dht were different in the active site of c-type and t-type. the structural comparisons showed that t-type formed strong hydrogen bonds with tyr and his while only his was found in c-type. to initiate catalytic reaction, the c ketone group of 5α-dht should be close to tyr and the nicotinamide ring of nadph which are involved in hydride transfer. the distances between these groups were monitored during 20 ns simulation time. as a result, 5α-dht was close to the cofactor in the both structures, whereas the distance between 5α-dht and tyr in t-type was relatively much shorter than c-type. on the contrary, the flipping of the side chain of trp in c-type might disrupt the interaction with β-face of 5α-dht. the interaction energies between 5α-dht and the proteins also indicated that t-type was energetically stable compared to c-type. taken together, our simulation results demonstrated that binding conformation of 5α-dht in t-type was more favorable for catalytic reaction than that of c-type. these structural explanations were also in agreement with kinetic experimental results. our findings will be useful to understand molecular mechanism for the enzyme kinetics of akr1c protein. minky son, chanin park and seul gi kwon contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions ms and cp performed structural modeling and analyzed data. sgk, wyb, and swk designed the study and carried out the experiments and statistical analysis. ms, cp, sgk, wyb, and swk wrote the manuscript. cwk and kwl interpreted the data and correct the manuscript. all authors read and approved the final manuscript. this work was supported by grants from priority research centers program and management of climate change program through the national research foundation of korea funded by the ministry of education of republic of korea, the project, “search & discovery of utility value from biological resources ”, from the national institute of biological resources of korean government, the export promotion technology development program of ministry of food, agriculture, forestry and fisheries, republic of korea. and this work was also supported by the next-generation biogreen program from rural development administration of republic of korea.
we believe that our study provides valuable information to understand important role of c-terminal region that affects enzymatic properties for 5α-dht, and further molecular mechanism for the enzyme kinetics of akr1c proteins. the results of the enzyme kinetics revealed that vmax and kcat values of the t-type were and folds higher than those of the c-type. also, the c-terminal region significantly contributes to the nadph-dependent reductase activity for 5α-dht reduction. our results showed that 5α-dht binding in t-type was more favorable for catalytic reaction to facilitate hydride transfer from the cofactor, and were consistent with experimental results. the structural comparisons between c-type and t-type showed that 5α-dht formed strong hydrogen bonds with catalytic residues such as tyr and his in t-type. moreover, catalytic efficiency was also fold higher in t-type compared to c-type. molecular modeling studies combined with kinetic experiments were performed to investigate structural and enzymatic differences between wild-type akr1c c-type and t-type. previously we have reported two paralogous pig akr1c1s, wild-type akr1c and c-terminal-truncated akr1c. pig aldo-keto reductase family member c belongs to akr superfamily which catalyzes the nadh-dependent reduction of various substrates including steroid hormones.
 pig aldo-keto reductase family member c belongs to akr superfamily which catalyzes the nadh-dependent reduction of various substrates including steroid hormones. previously we have reported two paralogous pig akr1c1s, wild-type akr1c and c-terminal-truncated akr1c. also, the c-terminal region significantly contributes to the nadph-dependent reductase activity for 5α-dht reduction. molecular modeling studies combined with kinetic experiments were performed to investigate structural and enzymatic differences between wild-type akr1c c-type and t-type.  the results of the enzyme kinetics revealed that vmax and kcat values of the t-type were and folds higher than those of the c-type. moreover, catalytic efficiency was also fold higher in t-type compared to c-type. since x-ray crystal structures of pig akr1c were not available, three dimensional structures of the both types of the protein were predicted using homology modeling methodology and they were used for molecular dynamics simulations. the structural comparisons between c-type and t-type showed that 5α-dht formed strong hydrogen bonds with catalytic residues such as tyr and his in t-type. in particular, c ketone group of the substrate was close to tyr and nadph in t-type.  our results showed that 5α-dht binding in t-type was more favorable for catalytic reaction to facilitate hydride transfer from the cofactor, and were consistent with experimental results. we believe that our study provides valuable information to understand important role of c-terminal region that affects enzymatic properties for 5α-dht, and further molecular mechanism for the enzyme kinetics of akr1c proteins. keywords aldo-keto reductasehomology modelingmolecular dynamic simulationnadph-dependent reductionsteroid hormoneissue-copyright-statement© the author 2015 