Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: Example of perfect enzyme engineering

Article abstract of DOI:10.1074/jbc.M111.338780

Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ⁴-3- ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His¹²⁰ (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ⁴-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ⁴-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.

Full text of DOI:10.1074/jbc.M111.338780

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2012/03/20/M111.338780.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 20, pp. 16609–16622, May 11, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Conversion of Human Steroid 5␤-Reductase (AKR1D1) into
3␤-Hydroxysteroid Dehydrogenase by Single Point Mutation
E120H
□
S
EXAMPLE OF PERFECT ENZYME ENGINEERING*
Received for publication, December 30, 2011, and in revised form, March 10, 2012 Published, JBC Papers in Press, March 20, 2012, DOI 10.1074/jbc.M111.338780
Mo Chen‡, Jason E. Drury‡, David W. Christianson§, and Trevor M. Penning‡1
From the ‡Department of Pharmacology and Center of Excellence in Environmental Toxicology, Perelman School of Medicine, and
the §Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Background: The aldo-keto reductase (AKR) subfamily AKR1D comprises steroid 5␤-reductases, whereas the AKR1C
subfamily comprises hydroxysteroid dehydrogenases.
Results: A single E120H mutation in AKR1D1 converts human steroid 5␤-reductase into a 3␤-hydroxysteroid
dehydrogenase.
Conclusion: Glu120 controls the positional specificity of hydride transfer and facilitates double bond reduction.
Significance: This is an example of a perfect change-of-function that can be achieved by a single point mutation.
Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C can invert stereochemistry when the steroid swings across the
enzymes are essential for bile acid biosynthesis and steroid hor- binding pocket. These studies show how a single point mutation
mone metabolism. AKR1D1 catalyzes the 5␤-reduction of ⌬4-3- in AKR1D1 can introduce HSD activity with unexpected config-
ketosteroids, whereas AKR1C enzymes are hydroxysteroid urational and stereochemical preference.
dehydrogenases (HSDs). These enzymes share high sequence
identity and catalyze 4-pro-(R)-hydride transfer from NADPH
to an electrophilic carbon but differ in that one residue in the
conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is
substituted by a glutamate in AKR1D1. We find that the
AKR1D1 E120H mutant abolishes 5␤-reductase activity and
introduces HSD activity. However, the E120H mutant unex-
pectedly favors dihydrosteroids with the 5␣-configuration and,
unlike most of the AKR1C enzymes, shows a dominant stereo-
chemical preference to act as a 3␤-HSD as opposed to a 3␣-HSD.
The catalytic efficiency achieved for 3␤-HSD activity is higher
than that observed for any AKR to date. High resolution crystal
structures of the E120H mutant in complex with epiandros-
terone, 5␤-dihydrotestosterone, and ⌬4-androstene-3,17-dione
elucidated the structural basis for this functional change. The
glutamate-histidine substitution prevents a 3-ketosteroid from
penetrating the active site so that hydride transfer is directed
toward the C3 carbonyl group rather than the ⌬4-double bond
and confers 3␤-HSD activity on the 5␤-reductase. Structures
indicate that stereospecificity of HSD activity is achieved
because the steroid flips over to present its ␣-face to the A-face
of NADPH. This is in contrast to the AKR1C enzymes, which
Two subfamilies of NAD(P)(H)-dependent steroid trans-
forming aldo-keto reductase (AKR)2 exist in humans: 5␤-re-
ductase (AKR1D1) and hydroxysteroid dehydrogenases (HSDs;
also known as AKR1C1–1C4). AKR1D1 catalyzes the irrevers-
ible reduction of ⌬4-3-ketosteroids to 5␤-dihydrosteroids to
introduce a 90° bend at the steroid A/B ring junction (1). This
cis-configuration is critical for bile acids to properly emulsify
dietary fats and cholesterol (2). 5␤-Reduced steroid hormones
may be involved in parturition (3), porphyrinogenesis (4, 5), and
xenoprotection (6, 7). By contrast, AKR1C enzymes catalyze
the interconversion between 3-, 17-, and 20-ketosteroids and
3␣/␤-, 17␤-, and 20␣-hydroxysteroids (Scheme 1) (8). By func-
tioning as HSDs, AKR1C enzymes regulate the ratio of active
and inactive androgens, estrogens, and progestogens that can
bind to nuclear receptors in target tissues (9).
AKR1D1 and AKR1C enzymes are highly homologous
enzymes, sharing over 50% sequence identity and a common
(␣/␤)8 barrel fold (10–12). Both subfamilies of enzymes bind
the steroid perpendicularly to the NAD(P)(H) cofactor in the
active site so that either the steroid A or D ring faces the nico-
tinamide ring. During reduction, a hydride from the 4-pro-(R)-
position on the nicotinamide ring is stereospecifically trans-
ferred to the recipient double bond/carbonyl group on the
steroid substrate (13). AKR1C enzymes perform the hydride
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
(NIH) Grants R01-DK40715 and P30-ES013508 (to T. M. P.), NIH Grant
GM-056838 (to D. W. C.), and NIH, NIDDK, Grant F32DK089827 (to M. C.).
□S
This article contains supplemental Figs. S1–S7.
The atomic coordinates and structure factors (codes 3UZW, 3UZX, 3UZY, and
3UZZ) have been deposited in the Protein Data Bank, Research Collaboratory 2 The abbreviations used are: AKR, aldo-keto reductase; ⌬4-adione, ⌬4-andro-
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
stene-3,17-dione; AKR1C1 to AKR1C4, human hydroxysteroid dehydroge-
nases; AKR1C9, rat 3␣-hydroxysteroid dehydrogenase; AKR1D1, human
steroid 5␤-reductase; AKR1D2, rat steroid 5␤-reductase; DHT, dihydrotes-
tosterone; epiA, epiandrosterone (3␤-hydroxy-5␣-androstan-17-one);
HSD, hydroxysteroid dehydrogenase; 20␣-OHP, 20␣-hydroxyprogester-
one; PDB, Protein Data Bank.
1 To whom correspondence should be addressed: Dept. of Pharmacology,
Perelman School of Medicine, University of Pennsylvania, 130C John Mor-
ganBldg., 3620HamiltonWalk, Philadelphia, PA19104-6084. Tel.: 215-898-
9445; Fax: 215-573-2236; E-mail: penning@upenn.edu.
MAY 11, 2012•VOLUME 287•NUMBER 20
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Engineering 3␤-HSD into Steroid 5␤-Reductase
SCHEME 2. Proposed mechanism for AKR1C enzyme-catalyzed 3-keto-
steroid reduction (left) and AKR1D1-catalyzed 5␤-reduction (right).
acid group of Glu120 is presumed to adopt the fully protonated
anti-oriented conformation in order to donate a hydrogen
bond directly to the steroid carbonyl oxygen. This arrangement
together with the general acid Tyr58 creates a superacidic envi-
ronment that facilitates hydride transfer by activating the
⌬4-double bond and stabilizing the enolate intermediate
(Scheme 2, right) (10).
Despite the homology between AKR1D1 and the AKR1C
enzymes, no mixed activity has been observed between the two
subfamilies. The glutamate-histidine substitution appears to be
the key mutation that distinguishes 5␤-reduction from HSD
activity. To examine the role of Glu120 further, we introduced
the E120H mutation in AKR1D1. Our kinetic characterization
of the mutant demonstrates that the E120H mutation com-
pletely abolishes the 5␤-reductase activity and converts
AKR1D1 into a highly efficient 3␤-HSD that exhibits opposite
stereopreference to the majority of AKR1C enzymes, which
show preference for 3␣-HSD activity. Analysis of the crystal
structures of the E120H mutant in complex with epiandros-
terone (epiA; 3␤-hydroxy-5␣-androstan-17-one), 5␤-dihy-
drotestosterone (5␤-DHT), and ⌬4-androstene-3,17-dione
(⌬4-Adione) reveals that the bulky histidine imidazole ring
introduced by the E120H substitution prevents penetration of
the steroid into the active site so that hydride transfer is now
only possible to the C3-position for ketosteroid reduction. The
binding conformations of different steroid isomers also illus-
trate the mechanism by which HSD stereospecificity is
controlled.
SCHEME 1. Reactions catalyzed by AKR1D1 and human AKR1C enzymes.
Functions of the AKR1C enzymes are assigned based on their preferred sub-
strate in the reduction of 3-/17-/20-ketosteroids. Common names and sub-
strate preference of the AKR1C enzymes are as follows: AKR1C1 (20(3)␣-HSD),
20-ketosteroid Ͼ 3-ketosteroid ϳ 17-ketosteroid; AKR1C2 (3␣-HSD type 3),
3-ketosteroid Ͼ 20-ketosteroid; AKR1C3 (17␤-HSD type 5 or 3␣-HSD type 2),
17-ketosteroid Ͼ20-ketosteroid Ͼ3-ketosteroid; AKR1C4 (3␣-HSD type 1), 3-ke-
tosteroid Ͼ 20-ketosteroid (8, 23).
transfer utilizing a well characterized “push-pull” mechanism
(14). In the reduction direction, Tyr58 acts as a general acid, and
His120 facilitates proton donation by hydrogen-bonding to
Tyr58. In the oxidation direction, Tyr58 acts as a general base
assisted by Lys87 for proton removal, where Asp53 balances the
positive charge on Lys87 through a salt bridge (Scheme 2, left).
All residues in this report are numbered according to AKR1D1.
Corresponding positions in AKR1C enzymes are Asp50, Tyr55,
Lys84, and His117). These four residues are conserved across
nearly all the AKR subfamilies and are collectively known as the
catalytic tetrad.
EXPERIMENTAL PROCEDURES
ϩ
ϩ
Materials—NADPH, NADH, NADP , and NAD were pur-
chased from Roche Applied Science. Steroids were purchased
from Steraloids. Synthetic oligonucleotides were obtained from
Invitrogen. E. coli strain C41 (DE3) was provided by Dr. J.E.
Walker (Medical Research Council Laboratory of Molecular
Biology, Cambridge, UK). The QuikChange II site-directed
mutagenesis kit was purchased from Stratagene. DEAE-Sep-
harose Fast Flow resin and HisTrap Fast Flow column (5 ml)
were purchased from GE Healthcare. [4-14C]Testosterone (50
mCi/mmol), [4-14C]⌬4-androstene-3,17-dione (48 mCi/mmol),
[4-14C]17␤-estradiol (53 mCi/mmol), [1,2,4,5,6,7-3H]5␣-dihy-
drotestosterone (5␣-DHT) (110 Ci/mmol), and [9,11-3H]andros-
terone (54 Ci/mmol) were purchased from PerkinElmer Life Sci-
ences. [4-14C]Progesterone (55 mCi/mmol) was purchased from
American Radiolabeled Chemicals. All other reagents were of
The mechanism for 5␤-reduction has not been fully eluci-
dated. Even so, based on the sequence and structural similarity,
it is reasonable to assume that AKR1D1 employs features of the
hydride transfer mechanism displayed by the AKR1C enzymes.
However, in all known 5␤-reductases, one of the tetrad resi-
dues, His120, is specifically substituted by a glutamate (15).
Compared with ketone reduction, the 5␤-reduction of a car-
bon-carbon double bond is much harder to achieve chemically.
It requires a strong reductant and stereo-controlled hydride
addition to achieve the A/B cis ring junction. Common chemi-
cal reductants like borohydride will reduce ⌬4-3-ketosteroids
to yield the 3␤-allylic alcohol, and when forced, the double
bond is reduced to the 5␣-configuration, but the 5␤-configura-
tion is rarely obtained (16, 17). Thus, Glu120 has been proposed
to play a unique role in steroid 5␤-reduction. According to the
recently solved crystal structure of AKR1D1, the carboxylic American Chemical Society quality or higher.
16610 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 20•MAY 11, 2012

Engineering 3␤-HSD into Steroid 5␤-Reductase
Mutagenesis, Expression, and Purification of Recombinant synthesized steroids was assessed by TLC as described below.
AKR1D1 E120H Mutant—The expression plasmid for the The specific radioactivity of the synthesized substrates was
AKR1D1 E120H mutant was made using the pET28a-AKR1D1 based on the value for the starting material.
construct (10) as a template by conducting site-directed
Product Identification by TLC—The 5␤-reductase activity of
mutagenesis using the QuikChange method (Stratagene) fol- the AKR1D1 E120H mutant was examined using testosterone,
lowing the manufacturer’s protocol. The forward primer was progesterone, and ⌬4-Adione as substrates with NADPH as
5Ј-dGGA TCT TTA CAT CAT TCA CGT ACC AAT GGC cofactor. The ketosteroid reductase activity of the E120H
CTT TAA GCC-3Ј, and the reverse primer was 5Ј-dGGC TTA mutant was examined using 5␣-DHT, 5␤-DHT, androsterone,
AAG GCC ATT GGT ACG TGA ATG ATG TAA AGA TCC-3Ј. ⌬4-Adione, and progesterone as substrates and NADPH as
The mutation was verified by dideoxysequencing. The mutant cofactor. The reactions were performed in 200-␮l systems con-
enzyme was overexpressed in Escherichia coli C41(DE3) cells in taining 100 mM potassium phosphate buffer (pH 6.0), 4% ace-
LB-Miller medium (BD Diagnostics) by the addition of isopro- tonitrile, 200 ␮M NADPH, 2.8 ␮g of AKR1D1 E120H, and one
pyl 1-thio-␤-D-galactopyranoside (1 mM final concentration) at of the following substrates: 5 ␮M [14C]testosterone (20 mCi/
inoculation. The cells were grown for 20 h at 37 °C, harvested by mmol), 5 ␮M [14C]progesterone (5 mCi/mmol), 2 ␮M [14C]⌬4-
centrifugation, and resuspended in buffer A (20 mM Tris (pH Adione (48 mCi/mmol), 1 ␮M [3H]5␣-DHT (495 mCi/mmol),
8.6)) supplemented with 1 mg/ml lysozyme and 1 ␮g/ml DNase 1.9 ␮M [14C]5␤-DHT (50 mCi/mmol), or 5 ␮M [3H]andros-
I (Roche Applied Science). The cells were lysed by sonication, terone (530 mCi/mmol).
and the protein in the soluble fraction was purified using a
The hydroxysteroid oxidase activity of the E120H mutant
DEAE-Sepharose column with a linear salt gradient using was evaluated using androsterone, testosterone, 5␣-DHT,
buffer A and buffer B (20 mM Tris (pH 8.6), 250 mM NaCl). The 5␤-DHT, 17␤-estradiol, and 20␣-OHP as substrates, using
ϩ
mutant enzyme eluted at around 60 mM NaCl. Fractions con- NAD(P) as cofactor. The reactions were performed in 200-␮l
taining the mutant were identified by SDS-PAGE analysis and systems containing 100 mM potassium phosphate buffer (pH
ϩ
ϩ
pooled, and the final imidazole concentration was adjusted to 7.0), 4% acetonitrile, 2.2 mM NAD (200 ␮M NADP for
20 mM using buffer C (20 mM Tris (pH 7.9), 400 mM imidazole, 5␣-DHT and androsterone oxidation), 2.8 ␮g of AKR1D1
500 mM NaCl). The pooled fractions were then applied to a E120H, and one of the following substrates: 5 ␮M [3H]andros-
HisTrap FF column (5 ml), and the enzyme was purified by terone (530 mCi/mmol), 5 ␮M [14C]testosterone (20 mCi/
stepwise elution using 50% buffer C and 50% buffer D (20 mM mmol), 5 ␮M [3H]5␣-DHT (580 mCi/mmol), 5 ␮M [14C]5␤-
Tris (pH 7.9), 20 mM imidazole, 500 mM NaCl). The purity of DHT (13 mCi/mmol), 2 ␮M [14C]17␤-estradiol (53 mCi/mmol),
the protein was assessed by SDS-PAGE, and the protein con- or 5 ␮M [14C]20␣-OHP (7.4 mCi/mmol).
centration was determined by a Bradford protein assay using
All reactions were incubated at 37 °C and quenched by 1 ml
bovine serum albumin as a standard. The reductase activity of of ethyl acetate at different time intervals. Control reactions
the pooled fractions after each chromatographic step was that contained the same components as the reaction mixture
measured by the fluorimetric assay described under “Kinetic less enzyme were performed for each substrate using a 60-min
Analysis” using 1 ␮M 5␣-DHT and 7.5 ␮M NADPH as sub- incubation. The aqueous phase was extracted with ethyl ace-
strates. The mutant was obtained in a 65% yield.
tate, and the extracts were applied to an LK6D Silica 60 TLC
Enzymatic Synthesis of Radiolabeled Steroids—[14C]5␤-DHT plate (Whatman), and the radioactive peaks were detected by a
was enzymatically synthesized from [14C]testosterone by wild TLC-linear analyzer (Bioscan Imaging Scanner system 200-
type (WT) AKR1D1. The reaction contained 100 mM potas- IBM with AutoChanger 3000; Bioscan). Products of the reac-
sium phosphate buffer (pH 6.0), 5% acetonitrile, 5% ethanol, tions were identified by co-chromatography with authentic
400 ␮M NADPH, 40 ␮M [14C]testosterone (50 mCi/mmol), and synthetic standards. The chromatograms for reactions contain-
57.6 ␮g AKR1D1 in a final volume of 1 ml. The reaction was ing 5␣-DHT, ⌬4-androstene-3,17-dione, androsterone, or 17␤-
incubated at 37 °C for 1 h to achieve complete consumption of estradiol were developed three times in CH2Cl2/ether (110:10,
testosterone. [3H]5␣-Androstane-3,17-dione was enzymati- v/v). The chromatograms for reactions containing testosterone
cally synthesized from [3H]androsterone by AKR1C9. The or 5␤-DHT were developed twice in toluene/acetone (80:20,
reaction contained 100 mM K2HPO4, 5% ethanol, 2.2 mM v/v). The chromatograms for reactions containing progester-
ϩ
NAD , 0.9 ␮M [3H]androsterone (54 Ci/mmol), and 15 ␮g of one or 20␣-OHP were developed once in CH2Cl2/ethyl acetate
AKR1C9 in a final volume of 1 ml. The reaction was incubated (80:20, v/v).
at 37 °C for 1 h to achieve complete consumption of androste-
Kinetic Analysis—Reductase activity was monitored fluori-
rone. [14C]20␣-Hydroxyprogesterone (20␣-OHP) was synthe- metrically by measuring the disappearance of the NADPH sig-
sized from [14C]progesterone by AKR1C1. The reaction con- nal (␭ex ϭ 340 nm, ␭em ϭ 460 nm) in 1-ml systems containing
tained 100 mM potassium phosphate buffer (pH 6.0), 4% 100 mM potassium phosphate buffer (pH 6.0), 4% acetonitrile,
ethanol, 950 ␮M NADPH, 76 ␮M [14C]progesterone (55 mCi/ 7.2 ␮M NADPH, and varied concentrations of steroid (0.2–60
mmol), and 180 ␮g of AKR1C1 in a final volume of 0.5 ml. The ␮M) at 37 °C. The oxidase activity was monitored fluorimetri-
reaction was incubated at 37 °C for 2 h with ϳ75% conversion of cally by measuring the appearance of NADH signal (␭ex ϭ 340
progesterone. [14C]20␣-OHP required further purification nm, ␭em ϭ 460 nm) in 1-ml systems containing 100 mM potas-
ϩ
from the unreacted progesterone by chromatography (silica 60, sium phosphate buffer (pH 7.0), 4% acetonitrile, 2.3 mM NAD ,
CH2Cl2/ether, 60:5 (v/v)). After incubation, all reactions were and varied concentrations of steroid (0.2–60 ␮M) at 37 °C.
quenched and extracted with ethyl acetate. The purity of the Reactions were initiated by the addition of enzyme and were
MAY 11, 2012•VOLUME 287•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 16611

Engineering 3␤-HSD into Steroid 5␤-Reductase
corrected for the non-enzymatic rate. Kinetic constants were The programs CNS (20), PHENIX (21), and COOT (22) were
ϩ
calculated by fitting the initial velocity data to either the used for refinement and model fitting. NADP and steroids
Michaelis-Menten equation or the modified Michaelis-Menten were built into the electron density map at the final stage of
equation with a term for substrate inhibition (Equation 1) using refinement. The quality of the models was verified with Mol-
GraFit (Erithacus Software Ltd.). The iterative fits gave esti- probity and PROCHECK. PROCHECK identified residue
mates of the steady-state kinetic parameters, mean Ϯ S.E.
max[S]
Km ϩ [S] ϩ [S]2/Ki
Crystallography—Four different complexes were obtained
Thr224 in both monomers in all of the AKR1D1 E120H com-
plexes in the disallowed region. The refinement statistics are
reported in Table 1.
V
v ϭ
(Eq. 1)
RESULTS
Loss of 5␤-Reductase Activity—The E120H mutation effec-
ϩ
ϩ
for the E120H mutant (E120HꢀNADP , E120HꢀNADP ꢀepiA, tively abolished the 5␤-reductase activity found in WT
ϩ
ϩ
E120HꢀNADP ꢀ⌬4-Adione, and E120HꢀNADP ꢀ5␤-DHT) AKR1D1. No 5␤-reduced product was observed with the
using the hanging drop vapor diffusion method at 4 °C. Besides AKR1D1 E120H mutant when [14C]testosterone, [14C]proges-
the steroid, drops typically contained 3.0 ␮l of protein solution terone, or [14C]⌬4-Adione was employed as the substrate in the
ϩ
(10 mg/ml AKR1D1 E120H mutant, 2.0 mM NADP , and 10 presence of NADPH after a 1-h-long incubation at 37 °C. The
mM Tris-HCl (pH 7.4)) and 3.0 ␮l of well solution (0.1 M limit of detection of the assay was 0.2 pmol/min, and we esti-
Tris-HCl (pH 7.0–7.7), 14–20% (w/v) polyethylene glycol 4000, mated that if any residual activity remained, it was less than
and 10% isopropyl alcohol). Steroid solutions of 100 mM were 0.8% of wild-type enzyme.
prepared in isopropyl alcohol and added to the protein solution
3␤-Hydroxysteroid Dehydrogenase Activity of AKR1D1
in co-crystallization trials or to the soaking solution to obtain E120H Mutant—The ability of the E120H mutant to act as an
the desired steroid concentration without adjustment of the HSD was examined for the reduction of 3-, 17-, and 20-ke-
final isopropyl alcohol concentration to a constant value. tosteroids and the oxidation of the corresponding hydroxy-
Crystals appeared and grew to a suitable size for diffraction steroids (Scheme 3). Enzyme activity was monitored by
in approximately
a week. The ternary complex of measuring the change of fluorescence emission of the cofac-
ϩ
E120HꢀNADP ꢀepiA was obtained by co-crystallization in the tor to generate steady-state kinetic parameters. Concur-
presence of 2 mM 5␣-DHT, and the E120HꢀNADP ꢀ⌬4-Adione rently, reactions were performed with radiolabeled steroid
ϩ
complex was obtained in the presence of 2 mM testosterone. substrates so that product profiles of the reactions could be
The steroids in the structures were different from those used in ascertained. Product identity was achieved by TLC by meas-
co-crystallization due to substrate turnover (see “Results”). The uring co-migration of the radiolabeled peaks with authentic
ϩ
E120HꢀNADP ꢀ5␤-DHT complex was obtained by soaking the synthetic standards.
ϩ
binary complex E120HꢀNADP in the mother liquor aug-
The E120H mutant was expected to function as a 3␣-HSD
ϩ
mented with 5 mM NADP and 5 mM 5␤-DHT for 6 h. Prior to like the AKR1C enzymes but to maintain a preference for 3-ke-
flash-cooling, crystals were soaked for 5 min in a 16% (w/v) tosteroids with a 5␤-configuration because these are produced
Jeffamine ED-2001 cryoprotective solution containing 2 mM by WT AKR1D1. To our surprise, the E120H mutant acted
ϩ
NADP and 2 mM steroid to maintain full occupancy of the predominantly as a 3␤-HSD that preferred steroids with a
ϩ
steroid ligand. Diffraction data of the E120HꢀNADP , 5␣-configuration over those with a 5␤-configuration. In keto-
ϩ
ϩ
E120HꢀNADP ꢀ⌬4-Adione, and the E120HꢀNADP ꢀ5␤-DHT steroid reduction, the mutant stereospecifically reduced
complexes were collected at beamline 24-ID-C (␭ ϭ 0.979 Å) of 5␣-DHT to 5␣-androstane-3␤,17␤-diol (Fig. 1) with a catalytic
the Advanced Photon Source at Argonne National Laboratory efficiency of 2.1 ϫ 105 Ϫ1 sϪ1, which was 10-fold higher than
M
ϩ
(Argonne, IL). The data of the E120HꢀNADP ꢀepiA complex that for the reduction of 5␤-DHT, which has the opposite con-
were collected at beamline X29 (␭ ϭ 1.075 Å) of the National figuration (Table 2). For hydroxysteroid oxidation, the mutant
Synchrotron Light Source at Brookhaven National Laboratory oxidized the 3␤-hydroxysteroid, epiA, to 5␣-androstane-3,17-
(Upton, NY). All crystals are of the P212121 space group with dione with a catalytic efficiency of 1.3 ϫ 105 Ϫ1 sϪ1, which was
M
unit cell parameters a ϭ 50.03, b ϭ 109.71, and c ϭ 129.34 Å, at least 15-fold higher than the catalytic efficiency of the other
similar to those of the WT AKR1D1 (10). The asymmetric unit tested hydroxysteroids regardless of the configuration of the
of the unit cell contains two monomers of the E120H mutant. steroid A/B ring junction and the position of catalysis (3-, 17-,
Data were integrated and scaled with HKL2000 and Scalepack or 20-position) (Table 3).
(18). Data collection and reduction statistics are reported in
Comparison of AKR1D1 E120H Mutant with AKR1C
Table 1 for all complexes.
Enzymes—AKR1C enzymes perform ketosteroid reduction and
ϩ
The structure of the E120HꢀNADP complex was solved by exhibit similar catalytic efficiencies for the reduction of 5␣- and
molecular replacement performed with PHASER (19) from the 5␤-DHT and preferably form the 3␣-hydroxysteroid over the
ϩ
CCP4 suite using the coordinates of the WT AKR1D1ꢀNADP
3␤-hydroxysteroid (23, 24). The exception is AKR1C1, which
binary complex (PDB code 3BV7) (10) less ligand and solvent reduces 5␣-DHT to a mixture of 3␣- and 3␤-isomers in a 1:3
molecules as a search model. The structures of ternary com- ratio. The AKR1D1 E120H mutant exhibited superior specific-
plexes E120HꢀNADP ꢀepiA, E120HꢀNADP ꢀ⌬4-Adione, and ity and efficiency for the reduction of 5␣-DHT to yield 5␣-an-
ϩ
ϩ
ϩ
E120HꢀNADP ꢀ5␤-DHT were solved by the difference Fourier drostane-3␤,17␤-diol than observed with AKR1C1 (Table 2).
ϩ
method using the E120HꢀNADP complex as a starting model. The E120H mutant produced only the 3␤-isomer and had a
16612 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 20•MAY 11, 2012

Engineering 3␤-HSD into Steroid 5␤-Reductase
TABLE 1
Data collection and refinement statistics
؉
؉
E120HꢀNADP ꢀepiA
؉
E120HꢀNADP ꢀ5␤-DHT
Structure
E120HꢀNADP
E120HꢀNADP ꢀ ⌬4-Adione
؉
Data collection
Resolution range (Å)
Total reflections
50.0–1.89
50.0–1.64
50.0–1.82
50.0–1.83
286,303 (25,145)a
57,071 (5350)a
0.099 (0.43)a
13.5 (3.0)a
342,248 (32,768)a
86,051 (8402)a
0.055 (0.23)a
23.0 (5.5)a
282,951 (30,478)a
58,850 (6220)a
0.069 (0.46)a
20.7 (3.2)a
295,651 (28,129)a
58,578 (5985)a
0.087 (0.405)a
15.5 (3.8)a
Unique reflections measured
b
Rmerge
I/␴(I)
Completeness (%)
98.9 (94.3)a
97.4 (96.4)a
90.7 (96.9)a
92.0 (95.7)a
Refinement statistics
Reflections used in refinement/test set
54,659/2754
84,885/4299
56,182/2845
56,863/2892
c
R/Rfree
0.168/0.211
0.184/0.219
0.191/0.246
0.173/0.218
Protein atomsd
5256
622
2
5256
596
2
5256
504
2
5256
550
2
Water moleculesd
ϩ
NADP moleculesd
Chloride iond
3
4
1
2
Steroid moleculesd
Root mean square deviations
Bond lengths (Å)
2
2
2
0.006
1.0
0.004
1.0
21
0.006
1.1
22
0.007
1.1
21
Bond angles (degrees)
Overall B-factor (Å2)
Cofactor B-factor (Å2)
Steroid B-factor (Å2)
Ramachandran statisticse
Allowed (%)
21
12
13
14
13
28
41
43
90.4
9.3
90.0
9.6
89.0
10.7
0.0
90.2
9.4
Additionally allowed (%)
Generously allowed (%)
Disallowed (%)
0.0
0.0
0.2
0.3
0.3
0.3
0.2
a The number in parentheses refers to the outer 0.1-Å shell of data.
b
R
merge ϭ ⌺͉I Ϫ ͗I͉͘/⌺I, where I is the observed intensity and ͗I͘ is the average intensity calculated for replicate data.
c Crystallographic R-factor, R ϭ ⌺(͉Fo͉ Ϫ ͉Fc͉)/⌺͉Fo͉ for reflections contained in the working set. Free R-factor, Rfree ϭ ⌺(͉Fo͉ Ϫ ͉Fc͉)/⌺͉Fo͉ for reflections contained in the test
set excluded from refinement. ͉Fo͉ and ͉Fc͉ are the observed and calculated structure factor amplitudes, respectively.
d Per asymmetric unit.
e Ramachandran statistics were calculated with PROCHECK (42). Thr224 in both monomers of all the AKR1D1 E120H complexes was found in the disallowed region.
catalytic efficiency for 5␣-DHT that was 400-fold greater than (supplemental Fig. S3) or a 3␣,5␤- or 3␤,5␤-configuration to
that observed for the same reduction reaction catalyzed by the corresponding 3-ketosteroids with similar catalytic effi-
AKR1C1. In addition, the kcat/Km value for the reduction of ciencies. Their catalytic efficiencies were much lower than that
5␣-DHT catalyzed by the E120H mutant was about 15-fold of the oxidation of epiA, which contains the preferred 3␤,5␣-
greater than that observed for the reduction of this steroid configuration (Table 3). The E120H mutant also oxidized 17␤-
to the opposite stereoisomer, 5␣-androstane-3␣,17␤-diol, hydroxysteroids and converted 5␣-DHT to 5␣-androstane-
catalyzed by AKR1C2. The values obtained with the mutant 3,17-dione, 17␤-estradiol to estrone, testosterone to
were similar to that observed for the reduction of 5␣-DHT to ⌬4-Adione, and 5␤-DHT to 5␤-androstane-3,17-dione (sup-
5␣-androstane-3␣,17␤-diol catalyzed by AKR1C4, the most plemental Fig. S4 and Table 3). Oxidation of 5␣-DHT at the
efficient human AKR1C enzyme (23).
17␤-position exhibited slightly higher catalytic efficiency than
Functional Promiscuity of E120H Mutant as Hydroxysteroid that observed for the oxidation of the 3-hydroxysteroids except
Dehydrogenase—The E120H mutant also exhibited subsidiary for epiA. The oxidation of the 17␤-position of 5␣-DHT was also
functional promiscuity by catalyzing the reduction of select 3- and preferred over the oxidation of the 17␤-position of 5␤-DHT
17-ketosteroids and oxidation of 3␣/␤-, 17␤-, and 20␣-hydroxys- and gave a catalytic efficiency that was 50-fold higher. The
teroids with different stereochemical outcomes.
E120H mutant also catalyzed a very slow oxidation of 20␣-hy-
In the reduction direction, the AKR1D1 E120H mutant non- droxyprogesterone to progesterone, but accurate kinetic
stereospecifically reduced the 3-ketosteroid 5␤-DHT to a parameters were not obtained due to the slow reaction rate and
mixture of 5␤-androstane-3␣,17␤-diol and 5␤-androstane- substrate inhibition (supplemental Fig. S5 and Table 3). The
3␤,17␤-diol in a 4:1 ratio with a catalytic efficiency 10-fold catalytic efficiencies of these minor oxidative activities on the
lower than that observed with 5␣-DHT (supplemental Fig. S1 3␣/␤-, 17␤-, and 20␣-hydroxysteroids were 20–800-fold lower
and Table 2). Interestingly, the stereopreference for the than that obtained for the oxidation of epiA catalyzed by the
5␤-DHT reduction catalyzed by the E120H mutant is now con- 3␤-HSD activity of the E120H mutant.
sistent with the stereopreference of the AKR1C enzymes, which
Overview of Structure of E120H Mutant—We determined
greatly favors the production of the 3␣-isomer when the sub- the crystal structures of the E120H mutant in complex with
strate has the 5␤-configuration (24). The E120H mutant NADP , NADP and epiA, NADP and ⌬4-Adione, and
ϩ
ϩ
ϩ
ϩ
also reduced 17-ketosteroids and converted androsterone to NADP and 5␤-DHT to develop a structural explanation for
5␣-androstane-3␣,17␤-diol and ⌬4-Adione to testosterone, but the observed change of function. Each complex contained
the reaction rates were too slow to be captured by the fluori- two monomers of the E120H mutant in the asymmetric unit.
metric assay (supplemental Fig. S2).
Monomer B typically exhibited lower thermal B-factors
In the oxidation direction, the E120H mutant slowly compared with monomer A, especially in the loop regions
converted 3-hydroxysteroids with a 3␣,5␣-configuration (Loop A, Ile119–Leu147; Loop B, Tyr219–Leu238; Loop C,
MAY 11, 2012•VOLUME 287•NUMBER 20
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Engineering 3␤-HSD into Steroid 5␤-Reductase
SCHEME 3. Representative reactions monitored to determine substrate specificity of the AKR1D1 E120H mutant. The reactive centers in the steroid are
shown in red (3-position), blue (17-position), and green (20-position).
ϩ
Leu302–Tyr326), although no significant conformational dif- Glu120. NADP maintained the same hydrogen bond network
ferences were evident between the two monomers. This as well as the salt link between Arg279 and the 2Ј-phosphate of
finding is consistent with the reported structure of WT AMP moiety that is responsible for the tight binding of NADPH
AKR1D1 (10). The E120H mutation did not perturb the (10, 25). The positioning of the nicotinamide ring of the cofac-
overall structure of the protein. The root mean square devi- tor in the central active site cavity retained the same location as
ϩ
ations between the AKR1D1ꢀNADP ꢀcortisone structure found in WT AKR1D1.
ϩ
ϩ
(PDB code 3CMF) and the structures of the four complexes of
E120HꢀNADP Complex—The E120HꢀNADP complex was
ϩ
the E120H mutant were within 0.18 Å for the 325 residues in obtained by co-crystallization with NADP . In the binary com-
monomer B and were similar to the root mean square devia- plex, a water molecule occupied the oxyanion site through
tions when the individual mutant complexes were compared hydrogen-bonding to Tyr58 and His120 (Fig. 2B). In the absence
ϩ
(0.09–0.12 Å). When the AKR1D1ꢀNADP ꢀHEPES complex of a steroid ligand, Trp230 swung into the steroid binding chan-
ϩ
structure is compared with the AKR1D1 E120HꢀNADP com- nel, triggering a slight movement of Loop B.
ϩ
ϩ
plex structure, the similarity in the structures is apparent (Fig.
E120HꢀNADP ꢀepiA Complex—The E120HꢀNADP ꢀepiA
2A). The His120 residue in the E120H mutant adopts the same complex was obtained by co-crystallization with 5␣-DHT. Sim-
conformation as the Glu120 residue in WT AKR1D1. The two ulated annealing omit maps indicated that two androgens dis-
residues were essentially superimposed with each other at their tinct from 5␣-DHT were bound in the two monomers of the
backbones and side chains up to the C-␥ atom (Fig. 2B). Cofac- complex. EpiA agreed well with the electron density map in
tor binding was undisturbed by the mutation, as expected, due monomer B with lower average B-factors (Fig. 3A), whereas
to the lack of direct interaction between the cofactor and 5␣-androstane-3␤,17␤-diol best depicted the map in monomer
16614 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 20•MAY 11, 2012

Engineering 3␤-HSD into Steroid 5␤-Reductase
trated by the position of the 3-ketone group in testosterone in
ϩ
the AKR1C9ꢀNADP ꢀtestosterone complex (PDB code 1ASF)
(Fig. 3C).
ϩ
ϩ
E120HꢀNADP ꢀ⌬4-Adione Complex—The E120HꢀNADP ꢀ⌬4-
Adione was obtained by co-crystallization with testosterone.
Akin to the phenomenon observed with the E120Hꢀ
ϩ
NADP ꢀepiA complex, oxidation of the 17␤-hydroxy group of
testosterone was observed. ⌬4-Adione was found in monomer
B of the complex with lower average B-factors (Fig. 4A),
whereas testosterone was found in monomer A (supplemental
Fig. S7). Both ⌬4-3-ketosteroids occupied identical positions in
the steroid binding channel. ⌬4-Adione bound to the steroid
binding channel with its A-ring, rather than the D-ring, point-
ing toward the active site, placing the 3-ketone group at the
oxyanion site in a nonproductive orientation. Despite the struc-
tural resemblance between ⌬4-Adione and epiA, the binding
orientation of ⌬4-Adione resembles 5␤-DHT by orientating its
␤-face toward the cofactor (i.e. the steroid face has been
ϩ
flipped). In the WT AKR1D1ꢀNADP ꢀtestosterone structure,
testosterone was bound perpendicular to the steroid binding
channel in an alternative binding pocket, which is responsible
for the reported substrate inhibition of WT AKR1D1 (Fig. 4B)
(26). In the E120H mutant structure, testosterone was bound in
the steroid binding channel, although the mutation did not dis-
rupt the alternative binding pocket. Substrate inhibition
observed for testosterone oxidation catalyzed by the mutant
could be elicited either by steroid fitting into the alternative
binding pocket as in the wild type AKR1D1 or binding nonpro-
ductively in the steroid channel so that its A-ring is in proximity
with the cofactor.
FIGURE 1. Identification of the products obtained by the E120H mutant
catalyzed reduction of 5␣-DHT. The mutant stereospecifically reduced
5␣-DHT to 5␣-androstane-3␤,17␤-diol. 5␣A-3␣,17␤-diol, 5␣-androstane-
3␣,17␤-diol; 5␣A-3␤,17␤-diol, 5␣-androstane-3␤,17␤-diol. Distance, plate
length. The bottom of the plate is marked as zero.
ϩ
ϩ
E120HꢀNADP ꢀ5␤-DHT Complex—The E120HꢀNADP ꢀ
5␤-DHT complex was obtained by soaking 5␤-DHT into the
ϩ
A (supplemental Fig. S6). An assay mimicking the crystalliza- E120HꢀNADP complex. Although 5␤-DHT is a substrate for
tion condition was performed and showed that 5␣-DHT oxi- the E120H mutant, no turnover was observed due to its low
doreduction occurred during the crystallization process and catalytic efficiency and relatively short incubation time with the
produced a mixture of products that included epiA, 5␣-andro- enzyme. The steroid nucleus of 5␤-DHT occupied a similar
stane-3␤,17␤-diol, and 5␣-androstane-3,17-dione (supple- position in the steroid binding channel as epiA with the A-ring
mental Fig. S4). The electron density map of the ligand identi- approaching the cofactor (Fig. 5A). However, 5␤-DHT bound
fied the steroid that occupied the active site most frequently. distally from the catalytic site and instead a water molecule was
Despite the difference in functional groups at the C17-position, anchored to the oxyanion site. This represents a non-produc-
the two 3␤,5␣-tetrahydrosteroids occupied identical positions tive binding mode witnessed in monomer A of the AKR1D1ꢀ
ϩ
in the steroid binding channel. The structure of monomer B NADP ꢀ5␤-DHP complex (PDB code 3CAV) (25), which resem-
with epiA was selected to represent the complex. EpiA was bles a conformation that precedes steroid product release from the
oriented with the A-ring toward the active site center, placing WT enzyme. The ␤-face of 5␤-DHT was directed toward the
the 3␤-hydroxy group in the oxyanion site through interactions cofactor, retaining the same orientation utilized for steroid 5␤-re-
with Tyr58 and His120. Trp230 receded from the steroid channel, duction and coincides with the stereopreference of the mutant to
packing against the ␣-face of the steroid. The distance of 3.2 Å produce the 3␣,5␤-reduced steroid product from 5␤-DHT. Slight
between the C3-position of the steroid and the C4 atom on the movements of Trp230 and Tyr132 were observed to accommodate
nicotinamide ring was within a suitable distance for hydride the flip of the C18 and C19 angular methyl groups. In the WT
ϩ
transfer. The steroid ␣-face was directed toward the re-face of AKR1D1ꢀNADP ꢀ5␤-DHT structure (PDB code 3DOP) (27), the
the cofactor, which allowed oxidoreduction of the 3␤,5␣-re- steroid was also bound through a water molecule; however, the
duced hydroxysteroid, consistent with the observed stereopref- 17␤-hydroxy group of the steroid was directed toward the oxyan-
erence of the E120H mutant. Compared with WT AKR1D1, the ion site instead of the 3-ketone group (Fig. 5B).
E120H mutant bound steroids with an opposite facial orienta-
DISCUSSION
tion, and the oxyanion site in the mutant was shifted “upward”
by 1.5 Å (Fig. 3B). This movement placed the C3-position of the
Glutamate-Histidine Substitution Controls 5␤-Reductase
steroid in the E120H mutant at a position that could be super- and HSD Activity—Engineered enzymes usually exhibit crip-
imposed on the oxyanion site in the AKR1C enzymes, illus- pled catalytic efficiency and partially retain their original activ-
MAY 11, 2012•VOLUME 287•NUMBER 20
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Engineering 3␤-HSD into Steroid 5␤-Reductase
TABLE 2
Kinetic constants for 3-ketosteroid reduction catalyzed by the E120H mutant and AKR1C2
Substrate
Product specificity
kcat
Km
␮M
Ki
␮M
kcat/Km
Ϫ1
Ϫ1
ϫ 10Ϫ3 s
ϫ 102 Ϫ1 s
M
E120H
5␣-DHT
5␤-DHT
3␤
33.8 Ϯ 0.8
26.8 Ϯ 0.7
0.16 Ϯ 0.02
1.5 Ϯ 0.1
3.9 Ϯ 0.2
2100
180
3␣ (80%), 3␤ (20%)
47.1 Ϯ 3.6
AKR1C1
5␣-DHTa
3␣ (25%), 3␤ (75%)b
NAc
NA
4.8
AKR1C2
5␣-DHT
5␤-DHT
3␣ (95%), 3␤ (5%)b
3␣ (88%), 3␤ (11%)d
123 Ϯ 3
8.2 Ϯ 0.8
2.0 Ϯ 0.2
150
110
21.7 Ϯ 0.3
a AKR1C1 failed to display saturation kinetics due to the limit of substrate solubility. The kcat/Km values were thus estimated from the slope of the initial velocity-substrate
concentration curve.
b Data from Refs. 23 and 28. The ratio was determined at pH 7.0 using radiometric assays.
c NA, not available.
d Data from Ref. 24. The ratio was determined at pH 7.0 by LC-MS.
TABLE 3
Kinetic constants for hydroxysteroid oxidation catalyzed by the E120H mutant and AKR1C2
Substrate
Substrate stereospecificity
kcat
Km
␮M
Ki
␮M
kcat/Km
Ϫ1
Ϫ1
ϫ 10Ϫ3 s
ϫ 102 Ϫ1 s
M
E120H
Androsterone
epiA
3␣, 5␣
3␤, 5␣
3␣, 5␤
3␤, 5␤
17␤
47 Ϯ 2
27.5 Ϯ 3.1
1.9 Ϯ 0.3
36.4 Ϯ 2.3
15.3 Ϯ 2.2
0.8 Ϯ 0.1
2.8 Ϯ 0.1
1.8 Ϯ 0.3
5.7 Ϯ 1.2
Ͻ0.52
17
253 Ϯ 8
125 Ϯ 3
43 Ϯ 2
1300
34
3␣-Hydroxy-5␤-androstan-17-one
3␤-Hydroxy-5␤-androstan-17-one
5␣-DHT
28
5.8 Ϯ 0.3
36.7 Ϯ 7.2
40.4 Ϯ 7.9
Yes
73
17␤-Estradiol
17␤
5.87 Ϯ 0.07
0.77 Ϯ 0.05
0.85 Ϯ 0.05
Ͼ0.3
21
Testosterone
17␤
4.2
1.5
NAb
5␤-DHT
17␤
20␣-Hydroxyprogesteronea
20␣
AKR1C2
epiAa
Androsterone
3␣, 5␣
3␤, 5␣
3␣, 5␤
3␤, 5␤
17␤
21.7 Ϯ 0.8
ϳ0.07
9.3 Ϯ 0.9
Ͻ5.86
23
NA
4.9
0.2
0.19
2.6
0.2
NA
3.5
3␣-Hydroxy-5␤-androstan-17-one
3␤-Hydroxy-5␤-androstan-17-one
5␣-DHT
14 Ϯ 1
29.0 Ϯ 4.2
9.4 Ϯ 1.2
15.3 Ϯ 3.3
6.2 Ϯ 1.2
17.3 Ϯ 1.1
Ͻ1.89
0.183 Ϯ 0.008
0.28 Ϯ 0.02
1.6 Ϯ 0.1
17␤-Estradiol
17␤
Testosterone
17␤
0.348 Ϯ 0.008
0.112 Ϯ 0.005
2.12 Ϯ 0.05
5␤-DHTa
17␤
20␣-Hydroxyprogesterone
20␣
6.0 Ϯ 0.5
a Kinetic constants were not determined due to slow reaction rate.
b NA, not available.
FIGURE2. StructureoftheAKR1D1E120HꢀNADP؉ binarycomplex. Superpositionoftheoverallstructure(A)andactivesite(B)oftheAKR1D1E120HꢀNADPϩ
(green), AKR1D1 E120HꢀNADPϩꢀepiA (yellow), and WT AKR1D1ꢀNADPϩꢀHEPES complexes (red; PDB code 3BUV), showing that no significant conformational
change was induced by the E120H mutation. NADPϩ and epiA molecules in A are colored in black. The steroid A-ring is shown in a ball-and-stick representation.
In the binary complexes, a water molecule resides in the oxyanion site, and Trp230 swings 90° into the steroid binding channel. The green dashed lines indicate
hydrogen bond interaction in the AKR1D1 E120HꢀNADPϩ complex with the water molecule. epiA was omitted from B for clarity. All structural figures were
generated with PyMOL (Version 1.2r1 Schro¨dinger, LLC).
ity. Here we demonstrate that a single glutamate-to-histidine 3␤-HSD activity is much higher than observed with AKR1C1
mutation in AKR1D1 eliminates its 5␤-reductase activity and and is comparable with and in some cases even higher than
converts the enzyme into a proficient 3␤-HSD. The stereopref- native 3␤-HSDs in the short-chain dehydrogenase/reductase
erence of the E120H mutant is different from most of the family (29). We have previously engineered 5␤-reductase activ-
AKR1C enzymes except AKR1C1 (23, 28). The introduced ity into rat 3␣-HSD AKR1C9 by introducing the corresponding
16616 JOURNAL OF BIOLOGICAL CHEMISTRY
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Engineering 3␤-HSD into Steroid 5␤-Reductase
FIGURE 3. Structure of the AKR1D1 E120HꢀNADP؉ꢀepiA complex. A, stereoviews of the simulated annealing omit map (Fo Ϫ Fc) of epiA contoured at
2.8 ␴. Atoms are colored-coded as follows. Yellow, protein carbon; black, steroid carbon; blue, nitrogen; red, oxygen; orange, phosphorus. Hydrogen
bonds are indicated by red dashed lines. The steroid A-rings are shown in ball-and-stick representations. B, superposition of AKR1D1 E120HꢀNADPϩꢀepiA
(yellow) and WT AKR1D1ꢀNADPϩꢀcortisone complex (PDB code 3CMF; blue). The AKR1D1 E120H mutation allows the C3 carbonyl to reside ϳ1 Å closer
to the 4-pro-(R)-hydride on the nicotinamide ring. The trajectory of hydride transfer is indicated by a green dashed line. C, superposition of AKR1D1
E120HꢀNADPϩꢀ (yellow), AKR1C2ꢀNADPϩꢀursodeoxycholate (PDB code 1IHI; gray), and AKR1C9ꢀNADPϩꢀtestosterone complex (PDB code 1ASF; purple).
The testosterone 3-ketone group in AKR1C9 superimposed well with the 3-hydroxy group of epiA in the E120H mutant. Residues are numbered as in
AKR1D1.
H120E mutation (numbering according to AKR1D1) (30). AKR1D1 or the position of the cofactor (Fig. 2). The histi-
Characterization and kinetic analyses on the AKR1C9 H120E dine backbone in the E120H mutant is placed at essentially
mutant showed that it exhibited moderate kcat values ϳ0.005 the same position as the glutamate residue in WT AKR1D1
s
Ϫ1 and Km values of ϳ20 ␮M toward four different ⌬4-3-ketos- (Fig. 3B). However, compared with the glutamate, which
teroids, with catalytic efficiencies approximately one-thirtieth “swings” its side chain away from the steroid, the bulky imid-
of the corresponding values of purified rat liver 5␤-reductase azole ring of the histidine residue in the E120H mutant pro-
AKR1D2 (31). The two studies complement each other and trudes toward the catalytic center, adopting an orientation
prove that the functional difference between AKR1D1 and that closely resembles the conformation exhibited by His120
AKR1C enzymes originates from a single glutamate-histidine in the AKR1C enzymes (Fig. 3C). As a result, the steroid can
substitution in the catalytic tetrad residue at position 120. Crys- no longer penetrate deep into the active site, and the oxyan-
tal structures of the AKR1D1 E120H mutant were determined ion site in the mutant is shifted upward away from the cofac-
to elucidate the structural basis for the change-of-function. tor to a location almost identical to the one it occupies in the
Glu120 probably plays a dual role in determining the activity of AKR1C enzymes. This shift changes the recipient group for
AKR1D1 and the AKR1C enzymes; this residue controls the hydride transfer, from the C5-position to the C3-position of
positional specificity of hydride transfer and facilitates double the steroid, and results in the loss of 5␤-reductase activity
bond reduction.
and the gain of 3␤-HSD activity. This phenomenon is remi-
Glu120 Controls Positional Specificity of Hydride Transfer— niscent of what is seen in the short-chain dehydrogenase/
The E120H mutation does not disturb the overall structure of reductase family, in which plant Digitalis lanata progester-
MAY 11, 2012•VOLUME 287•NUMBER 20
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Engineering 3␤-HSD into Steroid 5␤-Reductase
FIGURE 4. Structure of the AKR1D1 E120HꢀNADP؉ꢀ⌬4-Adione complex. A, stereoviews of the simulated annealing omit map (Fo Ϫ Fc) of ⌬4-Adione
contoured at 2.6 ␴. Atoms are color-coded as described in the legend to Fig. 3, except the carbons of the AKR1D1 E120H mutant are colored in magenta.
Hydrogen bonds are indicated by red dashed lines. The steroid A-rings are shown in ball-and-stick representations. B, superposition of AKR1D1
E120HꢀNADPϩꢀ⌬4-Adione(magenta)andWTAKR1D1ꢀNADPϩꢀtestosteronecomplex(PDBcode3BUR;green). Thetwosteroidbindingmodesareperpendicular
to each other, although the residues involved in hydrogen binding to testosterone in WT AKR1D1 do not move in the mutant.
one 5␤-reductase corresponds to AKR1D1, and human 17␤- anism of 5␤-reduction. Studies on AKR1C9 H120E mutant
HSD type I corresponds to the AKR1C enzymes. In these two
short-chain dehydrogenase/reductases, the change from a
5␤-reductase to a 17␤-HSD is achieved by repositioning of a
catalytic tyrosine. The shift of this tyrosine alters the depth
of the steroid binding pocket so that either the steroid dou-
ble bond or ketone group is positioned for reduction (32).
However, in the short-chain dehydrogenase/reductase fam-
ily, relocation of the catalytic tyrosine is accompanied by
significant sequence and structural changes, whereas in the
AKR family, a single glutamate-histidine substitution easily
shifts the position of the steroid without rearrangement of
the catalytic site.
show that the 5␤-reductase activity is abolished by the Y58F/
H120E double mutation but not by the H120A mutation,
indicating that Glu120 greatly facilitates the reaction but is
not obligatory for catalysis (30). The pH-rate dependence of
these AKR1C9 mutants confirms that Tyr58 is the general
acid for 5␤-reduction and suggests that Glu120 lowers the
pKb of the general acid by hydrogen bonding to Tyr58, resem-
bling the role played by the histidine residue in AKR1C9
(Scheme 2, left) (30). However, the crystal structure of
AKR1D1 disagrees with this mechanism. The distance
between Glu120 and Tyr58 of 4.3 Å is too long for a hydrogen
bond to exist between these two residues. Instead, the struc-
ture suggests that Glu120 donates a superacidic hydrogen
bond to the steroid C3 carbonyl group, which activates the
␣,␤-unsaturated steroid double bond by stabilizing the eno-
lic intermediate during hydride transfer (Scheme 2, right)
(10). The common feature of the two mechanisms is that
Glu120 aids 5␤-reduction by promoting an acidic environ-
ment. Currently, there are no kinetic data to support either
mechanism directly. Characterization of the other Glu120
Catalytic Role of Glu120—The AKR1D1 E120A mutation
allows steroid penetration in the active site but eradicates
both 5␤-reductase and HSD activity.3 The H120A mutation
in AKR1C9 also exhibits diminished 5␤-reductase activity
compared with the H120E mutant (30). These findings imply
that the glutamate residue plays a role in the chemical mech-
3
J. E. Drury and T. M. Penning, unpublished data.
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Engineering 3␤-HSD into Steroid 5␤-Reductase
FIGURE 5. Structure of the AKR1D1 E120HꢀNADP؉ꢀ5␤-DHT complex. A, stereoviews of the simulated annealing omit map (Fo Ϫ Fo) contoured at 2.5 ␴.
5␤-DHT adopts a non-productive binding mode in the active site, allowing a water molecule to occupy the oxyanion site. Atoms are color-coded as described
in the legend to Fig. 3, except the carbons of the AKR1D1 E120H mutant are colored in blue. Hydrogen bonds are indicated by red dashed lines. The steroid
A-rings are shown in ball-and-stick representations. B, superposition of E120HꢀNADPϩꢀ5␤-DHT (blue) and WT AKR1D1ꢀNADPϩꢀ5␤-DHT complex (PDB code
3DOP; red). 5␤-DHT binds with its A-ring approaching the oxyanion site in the AKR1D1 E120H mutant and with its D-ring approaching the oxyanion site in the
WT AKR1D1. Water molecules are shown as spheres. Only monomer B is shown.
mutations, including E120D, E120Q, and E120N, will prob- and Trp230 are parallel to the steroid plane, and Tyr26 is
ably unveil the catalytic property of this residue. A similar perpendicular to the steroid plane (Fig. 3A). This shape
mechanistic problem existed for Pseudomonas ⌬5-3-ketos- allows easy accommodation of 5␣-DHT but not 5␤-DHT
teroid isomerase, in which Tyr14 functions as the general that has a 90° bend at the A/B ring junction. By contrast,
acid and Asp38 acts as the general base, but the role of a using AKR1C2 as an example, Tyr132 is replaced by a smaller
neighboring Asp99 was uncertain. This residue could be residue, and the indole ring of Trp230 is placed almost per-
either hydrogen-bonded directly to the steroid substrate (33, pendicular to its corresponding position in AKR1D1 (Fig.
34) or to Tyr14 to form a catalytic diad (35, 36). The role of 3C). These changes forge the steroid binding channel into a
Asp99 was only resolved via NMR studies using a series of curved shape that tolerates the cis-configuration better,
catalytic residue single/double mutants, which showed that resulting in a similar or even slightly favorable Km for
Asp99 was directly hydrogen-bonded to the steroid (37).
5␤-DHT over 5␣-DHT in 3-ketosteroid reduction (Table 2).
SubstratePreferenceforSteroidtrans-A/BRingFusion—AKR1C
Stereospecificity of 3-Ketosteroid/Hydroxysteroid Oxido-
enzymes reduce 5␣-DHT and 5␤-DHT with similar catalytic reduction—The majority of the AKR1C enzymes prefer the pro-
efficiencies (23, 24). The preference of the AKR1D1 E120H duction of 3␣-hydroxysteroids regardless of the steroid A/B-ring
mutant to reduce a 3-ketosteroid with an A/B trans-ring configuration. The AKR1D1 E120H mutant exhibits a different
configuration over a cis-configuration by more than 10-fold stereopreference. It acts as a 3␤-HSD in the presence of 5␣-DHT
was unexpected. This preference mainly results from the dif- but produces a mixture of 3␣- and 3␤-hydroxysteroids in a 4:1
ference in substrate binding affinity, as reflected by a 10-fold ratio when 5␤-DHT is the substrate. Two mechanisms have been
difference in Km values of 5␣-DHT and 5␤-DHT. The steroid proposed to control the stereopreference of these HSDs, each
binding channel of the E120H mutant has a cylindrical shape through a unique motion of the steroid involving either swinging
that packs the steroid tightly on three sides, where Tyr132 or flipping.
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Engineering 3␤-HSD into Steroid 5␤-Reductase
␤-face of the steroid is directed toward the cofactor for 3␣-re-
duction in AKR1C9.
Positional Specificity of Mutant—The E120H mutant exhib-
its residual activity at the 17- and 20-positions of the steroid.
AKR1C enzymes possess this functional plasticity as well
(Scheme 1) (8). The functional plasticity derives from the
symmetric nature of the steroid molecule and the plasticity
of the AKR1C active site. Among the ternary complex struc-
tures solved for the human AKR1C enzymes (12, 39–41), none of
the structures depict steroids in a productive binding mode. Only
ϩ
ϩ
in the AKR1C1ꢀNADP ꢀ20␣-OHP and AKR1C2ꢀNADP ꢀ
testosterone complexes do the steroids adopt orientations that
correspond to observed positional preferences of the enzymes.
The AKR1D1 E120H mutant catalyzes the 17␤-hydroxysteroid
oxidation of testosterone to yield ⌬4-Adione; however, the
FIGURE 6. Residues in the steroid binding channel in AKR1C9 (purple)
and the AKR1D1 E120H mutant (yellow). A, testosterone is tightly packed
by Leu57 and Trp230 parallel to the steroid plane and Tyr313 perpendicular to
the steroid plane. epiA is packed by Tyr132 and Trp230 parallel to the steroid
plane and Tyr26 perpendicular to the steroid plane. The steroid A-rings are
shown in ball-and-stick representations. B, a rotated view shows the residues
that may be involved in the determination of stereospecificity. Residues are
numbered as in AKR1D1.
ϩ
structure of the E120HꢀNADP ꢀ⌬4-Adione complex also fails
to display a productive binding mode (Fig. 4). Instead, a reverse
The swinging mechanism was proposed based on docking binding orientation with the 3-ketone group tethered to the
simulations of 5␣-DHT into AKR1C1 and AKR1C2, where oxyanion site is observed. The preference for the 3-ketone over
the two enzymes show opposite stereospecificity for the 17␤-hydroxyl group in the oxyanion site reflects the kinetic
5␣-DHT reduction (23, 38). The docking models suggest data, where the Km values for the 3-ketosteroid reduction for
that the steroid swings across the steroid binding pocket to 5␣- and 5␤-DHT are lower than the Km values for the 17␤-
present different faces for 4-pro-(R)-hydride transfer from hydroxysteroid oxidation of the same steroids. In addition, the
the cofactor to ensure different stereochemical outcomes. fact that the E120H mutant allows nonproductive binding of
This swinging mechanism is achieved by the size of the res- the substrate by reversing the A/D-ring orientation may be the
idue at position 57 (AKR1D1 numbering). The longer side cause of substrate inhibition observed with 5␣-DHT, testoster-
chain of Leu57 in AKR1C1 pushes the steroid toward Trp230 one, and 20␣-OHP in this mutant.
so that the steroid ␣-face is presented to the cofactor to
In conclusion, we have successfully engineered steroid
produce a 3␤-hydroxysteroid, whereas the shorter side chain 5␤-reductase into a highly efficient 3␤-HSD by a single glu-
of Val57 in AKR1C2 ensures that the steroid ␤-face is pre- tamate-to-histidine mutation. The bulky histidine alters the
sented to the cofactor to produce a 3␣-hydoxysteroid. How- shape of the active site, resulting in a shift of the relative
ever, this mechanism cannot be applied to AKR1D1 because position of the steroid to the cofactor, which changes the
Ile57 is no longer in close proximity to the steroid and is enzyme function. This E120H mutant elicits a change in ste-
unable to play a central role in steroid orientation. Also, this reospecificity of the HSD reaction by flipping the steroid,
swinging mechanism may not be generalized to the other and it shows an unexpected preference for C19 steroids with
AKR1C enzymes because both AKR1C3 and AKR1C4 con- a 5␣-configuration over C19 steroids with a 5␤-configura-
tain the Leu57 as in AKR1C1, yet they favor the production of tion due to the presence of a cylindrical binding channel.
the 3␣-hydroxysteroid as seen in AKR1C2.
Engineered enzymes rarely exhibit adequate catalytic effi-
Our structures of the E120H mutant support a mechanism in ciency for a new activity with complete eradication of the
which the faces of the steroid are flipped. The structures of the native activity, but here we demonstrate an example of a
ϩ
ϩ
E120HꢀNADP ꢀepiA and E120HꢀNADP ꢀ⌬4-Adione com- perfect change of function achieved by a single point
plexes show that the steroid may present either its ␣- or ␤-face mutation.
toward the cofactor by flipping 180° along its long molecular
axis, using the stationary oxyanion site as the center of rotation
Acknowledgments—We thank Dr. Julie A. Aaron for assistance in data
collecting and processing and Dr. Yi Jin for helpful discussions. Part of this
(Figs. 3A and 4A). The flipping mechanism complies with the
different stereopreference seen with the AKR1D1 E120H workwasbaseduponresearchconductedattheAdvancedPhotonSource
on the Northeastern Collaborative Access Team beamline 24_ID_C
(supported by National Center for Research Resources, National Insti-
tutesofHealth, GrantRR-15301). UseoftheAdvancedPhotonSource, an
Office of Science User Facility operated for the United States Department
of Energy Office of Science by Argonne National Laboratory, was sup-
ported by the United States Department of Energy under Contract
DE-AC02–06CH11357. Part of this work was based upon research con-
ducted at beamline X29 of the National Synchrotron Light Source.
Financial support comes principally from the Offices of Biological and
Environmental Research and of Basic Energy Sciences of the United
States Department of Energy and from National Center for Research
Resources, National Institutes of Health, Grant P41RR012408.
mutant and AKR1C9. The competitive inhibitor testosterone in
AKR1C9 binds to the active site at almost the same position as
epiA in the E120H mutant, except that the two steroids present
opposite faces to the cofactor (i.e. the two steroids are flipped)
(Fig. 6). Like the E120H mutant, AKR1C9 also packs testoster-
one on three sides, with Leu57 and Trp230 parallel to the steroid
plane and Tyr313 perpendicular to the steroid plane, but Tyr313
in AKR1C9 resides on the opposite side of the steroid against
Tyr26 in the E120H mutant. The tyrosine residues cause the
steroid B-ring to be flipped away from them to avoid clashing,
and, as a result, the ␣-face of the steroid is directed toward the
cofactor for 3␤-reduction in the E120H mutant, whereas the
16620 JOURNAL OF BIOLOGICAL CHEMISTRY
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Engineering 3␤-HSD into Steroid 5␤-Reductase
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