Structure and catalytic mechanism of 3-ketosteroid-Δ4-(5α)- dehydrogenase from Rhodococcus jostii RHA1 genome

Article abstract of DOI:10.1074/jbc.M112.374306

3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)- KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 A resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4β-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs.

Full text of DOI:10.1074/jbc.M112.374306

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2012/07/24/M112.374306.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 37, pp. 30975–30983, September 7, 2012
2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
©
Structure and Catalytic Mechanism of 3-Ketosteroid-⌬4-(5␣)-
□
S
*
dehydrogenase from Rhodococcus jostii RHA1 Genome
Received for publication, April 20, 2012, and in revised form, July 6, 2012 Published, JBC Papers in Press, July 24, 2012, DOI 10.1074/jbc.M112.374306
§1 ‡2
‡
1
§
§
Niels van Oosterwijk , Jan Knol , Lubbert Dijkhuizen , Robert van der Geize , and Bauke W. Dijkstra
‡
§
From the Laboratory of Biophysical Chemistry and the Department of Microbiology, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands
Background: Ketosteroid dehydrogenases are enzymes of biotechnological relevance that introduce a double bond into
steroids as a first step toward their degradation.
Results: First structures of a 3-ketosteroid-⌬4-(5␣)-dehydrogenase combined with mutational analysis allowed the identifica-
tion of residues essential for catalysis.
Conclusion: Tyr-319, Tyr-466, and Ser-468 have essential roles in catalysis.
Significance: These structures may facilitate the development of better catalysts for steroid conversion.
3
-Ketosteroid ⌬4-(5␣)-dehydrogenases (⌬4-(5␣)-KSTDs) are duction of bioactive steroids, making Rhodococcus a particu-
enzymes that introduce a double bond between the C4 and C5 larly interesting organism for steroid bioconversion (2).
atoms of 3-keto-(5␣)-steroids. Here we show that the ro05698
An important step in the microbial degradation of various
gene from Rhodococcus jostii RHA1 codes for a flavoprotein naturally occurring sterols is the desaturation of the steroid A
with ⌬4-(5␣)-KSTD activity. The 1.6 Å resolution crystal struc- ring (Fig. 1). Several different 3-ketosteroid dehydrogenases
3
ture of the enzyme revealed three conserved residues (Tyr-319, (KSTDs) are known to promote this desaturation, such as
Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring 3-ketosteroid ⌬1-dehydrogenase (⌬1-KSTD), 3-ketosteroid
system of the FAD co-factor. Site-directed mutagenesis of these ⌬4-(5␣)-dehydrogenase (⌬4-(5␣)-KSTD), and 3-ketosteroid
residues confirmed that they are absolutely essential for cata- ⌬4-(5␤)-dehydrogenase (⌬4-(5␤)-KSTD), which are all FAD-
lytic activity. A crystal structure with bound product 4-andro- containing enzymes. Although ⌬1-KSTDs desaturate the
stene-3,17-dione showed that Ser-468 is in a position in which it C1-C2 position, ⌬4-KSTDs introduce a double bond at the
can serve as the base abstracting the 4␤-proton from the C4 C4-C5 position (3, 4).
atom of the substrate. Ser-468 is assisted by Tyr-319, which pos-
Amino acid sequence analysis of (putative) KSTDs showed
sibly is involved in shuttling the proton to the solvent. Tyr-466 is that ⌬1-KSTDs and ⌬4-KSTDs are clearly distinct, belonging to
at hydrogen bonding distance to the C3 oxygen atom of the sub- different branches of the phylogenetic tree. They display
strate and can stabilize the keto-enol intermediate occurring 20–24% sequence similarity to fumarate reductases (5). The
during the reaction. Finally, the FAD N5 atom is in a position to highest homology is found in the FAD-binding domain; the
be able to abstract the 5␣-hydrogen of the substrate as a hydride substrate-binding domain shows lower sequence similarity.
ion. These features fully explain the reaction catalyzed by The residues involved in the dehydrogenation of the substrate
⌬
4-(5␣)-KSTDs.
by KSTDs are currently unknown, although chemical modifi-
cation and mutagenesis studies on ⌬1-KSTDs have implicated
histidine and tyrosine residues in catalysis (6, 7). Mutational
studies confirming the involvement of these residues in
Rhodococcus is a genus of aerobic Gram-positive bacteria
⌬4-(5␣)-KSTDs have not been reported so far.
closely related to Mycobacterium and Corynebacterium (1). Its
members show a broad catabolic diversity and a wide range of
unique enzymatic capabilities, among which is the ability to
degrade naturally occurring phytosterols. Intermediates of the
phytosterol degradation pathway find application in the pro-
KSTDs also have an important function in Mycobacterium
tuberculosis where they are part of the metabolic pathway for
cholesterol degradation. In M. tuberculosis this pathway is cen-
tral to the unusual ability of the bacterium to survive inside
macrophages (8). Interestingly, transposon mutagenesis has
suggested that the ⌬4-KSTD homologue Rv1817 is involved in
the pathogenicity of M. tuberculosis (9), and it has been pro-
*
This was project was supported in part by the Netherlands Ministry of Eco-
nomic Affairs and the B-Basic partner organizations through B-Basic, a posed that KSTDs could be valuable targets for the develop-
public-private Advanced Chemical Technologies for Sustainability pro-
ment of antituberculosis drugs (8).
gram. This work was also supported by Schering-Plough.
This article contains supplemental Table S1 and Figs. S1–S3.
□S
In the Rhodococcus jostii RHA1 genome (1), a single ⌬4kstD
The atomic coordinates and structure factors (codes 4AT0 and 4AT2) have been gene (ro05698) is present. It codes for a protein of 490 amino
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ(http://www.rcsb.org/).
acid residues, with an estimated molecular mass of 51.9 kDa.
1
Both authors contributed equally to this work.
2
3
To whom correspondence should be addressed: Laboratory of Biophysical
Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Nether-
lands. Tel.: 31503634381; Fax: 31503634800; E-mail: b.w.dijkstra@rug.nl.
The abbreviations used are: KSTD, ketosteroid dehydrogenase; 5␣-AD, 5␣-
androstane-3,17-dione; 1-(5␣)-AD, 1-(5␣)-androstene-3,17-dione; 4-AD, 4-
androstene-3,17-dione; ADD, 1,4-androstadiene-3,17-dione; RMSD, root
mean square deviation; DCPIP, dichlorophenolindophenol.
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3
-Ketosteroid-⌬4-(5␣)-dehydrogenase Reaction Mechanism
and without substrate were used to characterize the protein as a
flavoprotein and to detect the reduction of the FAD upon addi-
tion of substrate.
Noncovalent binding of the flavin co-factor was determined
as follows. Purified ⌬4-(5␣)-KSTD was applied to a SDS-PAGE
gel. Following electrophoresis, the gel was treated with 5% ace-
tic acid solution, and flavin was visualized by 254-nm UV irra-
diation. Lack of co-migration indicated noncovalent binding of
the flavin co-factor to the ⌬4-(5␣)-KSTD protein.
Activity Assays and Product Identification—Because E. coli
does not code for any KSTD enzymes, cell-free extracts were
used for product formation assays as described before (5).
Briefly, enzyme activities were determined spectrophotometri-
cally at 30 °C in 50 mM Tris-HCl buffer, pH 7.4, using 80 ␮M
dichlorophenolindophenol (DCPIP) as an artificial electron
acceptor and 0–200 ␮M 1-(5␣)-androstene-3,17-dione (1-(5␣)-
AD) as substrate (Steraloids). The initial reaction rates were
determined from the absorbance change at 600 nm during the
first 20 s of the reaction. The products were identified by HPLC
following a reaction containing purified protein (50–200 ␮g),
2
00 ␮M 1-(5␣)-AD dissolved in ethanol, and 200 ␮M DCPIP. No
activity was detected in control reaction mixtures lacking ste-
roids or purified enzyme or in extracts of E. coli with an empty
pET15b expression vector.
FIGURE 1. Conversion of 5␣-AD to ADD by ⌬1-KSTD (EC 1.3.99.4) and
⌬
4-KSTD (EC 1.3.99.5). The two double bonds introduced by the enzymes
are indicated. The intermediate products 1-(5␣)-AD and 4-AD are 1-(5␣)-an-
drostene-3,17-dione and 4-androstene-3,17-dione, respectively.
Crystallization, Data Collection, and Processing—⌬4-(5␣)-
KSTD was crystallized at 293 K as described before, with a pre-
The gene has been cloned, and the enzyme has been overex- cipitant consisting of 200 mM NH -acetate, 100 mM sodium
4
pressed as a His -tagged protein in Escherichia coli and purified citrate, pH 5.6, and 30% (w/v) PEG 4000 (10). Crystals were
6
(
10). Spectroscopic analysis confirmed that it binds FAD, and prepared for data collection by soaking them for ϳ30 s in
the purified enzyme was shown to have 3-ketosteroid ⌬4-(5␣)- mother liquor, supplemented with 20% (w/v) glycerol, followed
dehydrogenase activity. The enzyme has been crystallized, and by cryo-cooling in liquid nitrogen. The 4-AD soaks were pre-
its preliminary x-ray analysis has been published (10).
pared by soaking crystals for ϳ18 h in cryo-mother liquor,
Here we present the crystal structure of ⌬4-(5␣)-KSTD which had the 200 mM NH -acetate replaced by 200 mM NH Cl,
4
4
(
Ro05698) from R. jostii RHA1 in its steroid-free and product- and to which 10 ␮l of a saturated solution of 4-AD in ethanol
bound form. These first structures of a KSTD enzyme allowed had been added per 500 ␮l of cryo-mother liquor.
the identification of the active site residues involved in the
The diffraction data were collected at 100 K at the beamlines
dehydrogenation of 3-ketosteroid substrates and its reaction of the European Synchrotron Radiation Facility (Grenoble,
mechanism, which was confirmed through mutational France). The intensity data were processed using the programs
analyses.
MOSFLM (11) and SCALA (12) from the CCP4 package (13). A
summary of the data collection statistics is shown in Table 1.
Phasing—The FFAS03 server (14) was used for identifying
EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth Conditions—Rhodococcus suitable models for molecular replacement. The structure with
strains were cultivated in LBP medium containing 1% (w/v) the highest sequence identity (24%) was that of flavocyto-
bacto-peptone (Difco, Detroit, MI), 0.5% (w/v) yeast extract chrome c fumarate reductase from Shewanella frigidimarina
3
(
BD Biosciences) and 1% (w/v) NaCl at 30 °C and 200 rpm. in the open conformation (Protein Data Bank entry 1qo8) (15).
E. coli strains were grown in Luria-Bertani broth (Sigma) at Because of the low sequence identity, the molecular replace-
7 °C unless stated otherwise. ment model was enhanced by incorporating all-serine struc-
3
KSTD Activity Staining—Initial ⌬4-(5␣)-KSTD activity was tural information from various homologous proteins (17–24%
established by a native gel-based assay. Cell-free extracts of identity; Protein Data Bank entries 1d4c (16), 1e39 (17), 1kf6
three Rhodococcus strains (Rhodococcus erythropolis SQ1, Rho- (18), 1nek (19), and 1zoy (20)). This ensemble of all-serine mod-
dococcus rhodochrous, and R. jostii RHA1) were prepared as els was used to solve the structure of ⌬4-(5␣)-KSTD by molec-
described (5). These were loaded on native PAGE gels and ular replacement at 2.5 Å resolution using the program Phaser
stained for KSTD activity following published procedures (5).
(21). Several model building cycles, consisting of manual model
Protein Expression, Purification, and Characterization—⌬4- building using COOT (22) and density modification with
5␣)-KSTD was heterologously expressed in E. coli strain Resolve (23), were performed to improve the model obtained
(
BL21(DE3) and subsequently purified and crystallized as from Phaser until an R factor of 0.44%. Because of low sequence
described before (10). UV-visible spectra of purified enzyme similarity and variable positions of residues 291–426 (sub-
measured in the range of 200 to 700 nm (Cary 100, Varian) with strate-binding domain) in the homologous structures, no inter-
3
0976 JOURNAL OF BIOLOGICAL CHEMISTRY
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3-Ketosteroid-⌬4-(5␣)-dehydrogenase Reaction Mechanism
TABLE 1
TABLE 2
Data collection statistics
Refinement statistics
The values within parentheses are for the highest resolution shell, and the formulas
Steroid-free 4-AD
for R
and R
were taken from Weiss (52).
p.i.m.
merge
Resolution (Å)
1.6
1.6
a
Steroid-free
BM16
4-AD
ID14-1
Average B-factor
16.9
15.8
17.5
14.1
15.9
18.1
a
R
European Synchrotron
Radiation Facility
beamline
a
R
free
RMSD from target geometry
Bond lengths (Å)
0.012
1.44
4368
482
1
0.007
1.25
4353
482
1
Wavelength (Å)
Space group
Unit cell parameters
a (Å)
1.00
0.93
Bond angles (°)
C222
C222
1
1
Total number of atoms
Number of amino acids
99.2
99.8
Ϫ
Number of Cl ions
b (Å)
114.3
116.1
Number of acetate molecules
Number of glycerol molecules
Active site ligand
2
c (Å)
110.2
110.2
1
␣
␤
␥
(°)
(°)
(°)
90
90
4-AD
1
90
90
Number of FAD molecules
Number of water molecules
1
90
90
609
618
Resolution (Å)
merge
p.i.m
1.6 (1.69–1.60)
0.081 (0.319)
0.023 (0.138)
1.6 (1.69–1.60)
0.110 (0.373)
0.055 (0.201)
390,566 (38,365)
b
b
Ramachandran angles (%)
R
R
c
Favored
Allowed
Disallowed
a
95.4
4.1
95.5
4.1
Total number of
888,741 (80,282)
80,282 (9,999)
0.4
0.4
observations
Total number of unique
reflections
80,072 (8,955)
R ϭ ⌺ ʈF ͉ Ϫ ͉F ʈ/⌺ ͉F ͉. R is the R factor calculated with 5% of the
hkl obs
calc
hkl obs
free
reflections excluded from the refinement.
b
Mean I/␴(I)
22.6 (4.7)
97.0 (83.8)
11.1 (6.1)
10.5 (3.2)
95.2 (74.1)
4.9 (4.3)
Ramachandran statistics were obtained from COOT (22).
Completeness (%)
Multiplicity
a
b
c
Data collection statistics according to van Oosterwijk et al. (10).
͉I (hkl) Ϫ (I(hkl)͉/⌺hkl (hkl).
p.i.m. ϭ ⌺hkl(1/(N Ϫ 1)) ⌺ (hkl) Ϫ (I(hkl)͉/⌺hkl
Site-directed Mutagenesis—Site-directed mutagenesis was
R
merge ϭ ⌺hkl
⌺
i
i
i i
⌺ I
1
⁄
2
TM
R
i
͉I
i
⌺
i
I
i
(hkl).
performed using the QuikChange site-directed mutagenesis
kit (Stratagene) with plasmid pBluescript-ro05698 as template.
The PCR product of ro05698 (10) was cloned into the EcoRV
site of pBluescript (II) KS (Fermentas). Primers used for site-
directed mutagenesis are listed in supplemental Table S1. Suc-
cessful mutagenesis was confirmed by DNA sequencing. The
resulting mutant genes were subcloned following NdeI/BamHI
digestion into the NdeI/BamHI-digested pET15b (Novagen)
for protein expression and protein purification. Mutation
S468A in Ro05698 was obtained by ordering a synthetic DNA
fragment (487 bp, AGOWA) of ro05698 containing the desired
mutation. The synthetic fragment was digested with NotI/
BamHI and subcloned into the NotI/BamHI digested pET15b-
Ro05698 construct to obtain the full-length gene with the
desired mutation.
pretable density was observed for this region. However, manual
model building and density modification improved the phases
sufficiently to allow Arp/Warp (24) to build the complete
model using data to 1.6 Å resolution.
Refinement—The model obtained from Arp/wArp was used
as the starting model for the crystallographic refinement. First,
manual model building using COOT (25) was performed to
adjust minor discrepancies in the model. Then the model was
refined with Refmac using TLS refinement (26) to a final R
factor of 15.8% and an R of 17.5% at 1.6 Å resolution. Most
free
residues were visible in the electron density except for the first
2
5 residues (consisting of the His tag and 19 residues of the
6
6
His tag linker) and the last residue. Two differences were
found, A60T and T160A, compared with the published genome RESULTS AND DISCUSSION
sequence. The final model contains FAD, one glycerol mole-
cule, two acetate molecules, and 619 water molecules. Also 14
amino acids with double conformations were identified. In
addition, the structure contains a chloride ion, of which the
location was confirmed using a bromide MAD data set. The
secondary structure was assigned using DSSP (27).
Ro05698 from R. jostii RHA1 Is a ⌬4-(5␣)-KSTD Enzyme—
The genome of R. jostii RHA1 contains at least three probable
⌬
1-KSTD enzymes but only one single putative ⌬4-(5␣)-KSTD
enzyme, encoded by ro05698, based on its 31% sequence iden-
tity to the ⌬4-(5␣)-KSTD of R. erythropolis (1, 5). Indeed, cell-
free extracts of R. jostii RHA1 and two other Rhodococcus
strains showed ⌬4-(5␣)-KSTD activity in a native PAGE assay
The product-bound structure was solved by rigid body fitting
of the steroid-free structure followed by restrained refinement with 1-(5␣)-AD as substrate (supplemental Fig. S1). In R. jostii
with REFMAC5. The geometric restraints for the FAD and RHA1, this activity presumably originates from the product of
4
-AD models were generated using the PRODRG2 server (28). the ro05698 gene.
These ligands were manually built into 2F Ϫ F and F Ϫ F
To firmly establish that ro05698 codes for an enzyme with
o
c
o
c
electron density maps using COOT. The 4-AD model was ⌬4-(5␣)-KSTD activity, the gene was expressed in E. coli, and
refined to an R factor of 15.9% and an R value of 18.1% at 1.6 the Ro05698 protein was purified and incubated with the ste-
free
Å resolution. A summary of the refinement statistics and the roid substrate 1-(5␣)-AD. HPLC analysis of the product con-
geometric quality of the models is given in Table 2. The quality firmed the formation of 1,4-androstadiene-3,17-dione (ADD)
of the models was analyzed using the program MolProbity (29). (Fig. 2), indicating that ro05698 indeed codes for a ⌬4-(5␣)-
The figures were prepared using PyMOL (30). The atomic KSTD. As expected, no activity was observed with the product
coordinates and experimental structure factor amplitudes have 4-androstene-3,17-dione (4-AD) as substrate.
been deposited with the RCSB Protein Data Bank with Protein
Data Bank codes 4AT0 (steroid-free) and 4AT2 (4-AD).
The absorption spectrum of purified ⌬4-(5␣)-KSTD protein
is typical of that of a flavoprotein (31), with maxima at 461 and
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3
-Ketosteroid-⌬4-(5␣)-dehydrogenase Reaction Mechanism
S3) are poorly visible in the electron density map, which may
indicate flexibility. These loops may have a function in catalysis
by shielding the FAD and substrate from the solvent (see
below).
Comparison with Structurally Similar Proteins—A DALI
(
Distance-matrix ALIgnment) search (35) revealed that
⌬
4-(5␣)-KSTD is structurally most closely related to cyto-
chrome c fumarate reductase from S. frigidimarina (Z ϭ 42.6,
3
2
7% sequence identity), cytochrome c fumarate reductase from
Shewanella putrefaciens MR-1 (Z ϭ 42.2, 28% identical resi-
dues), quinol-fumarate reductase from E. coli (Z ϭ 32.6, 22%
identical residues), and L-aspartate oxidase from E. coli (Z ϭ
3
2.5, 21% sequence identity), which are all FAD-binding pro-
FIGURE2. HPLCanalysisofsubstrates/productsinareactionmixturecon-
taining purified ⌬4-(5␣)-KSTD (50–200 ␮g), the electron acceptor
DCPIP, and 1–5␣-AD as substrates. The conversion of 1–5␣-AD into ADD is
teins with a p-hydroxybenzoate hydroxylase-like fold. ⌬4-(5␣)-
KSTD has less, but still significant structural similarity to cho-
ϳ100%. Pure 1–5␣-AD and ADD served as references for substrate/product lesterol oxidase from Brevibacterium sterolicum (1coy, Z ϭ
identification.
1
6.2, 12% sequence identity), an enzyme that catalyzes a similar
reaction on a similar steroid substrate as ⌬4-(5␣)-KSTD.
3
89 nm (Fig. 3). The addition of 1-(5␣)-AD led to the rapid
The highest structural similarity is found for the F domain
disappearance of the maxima at ϳ389 and 461 nm, indicating with root mean square differences (RMSDs) on C␣ atoms of
that the flavin co-factor is reduced along with the dehydroge- 1.6–2.0 Å. The S-domain is less similar in structure, and only
nation of the steroid substrate. However, also a broad absorb- the fumarate reductases of S. frigidimarina and S. putrefaciens,
ance peak near 490 nm develops, which may indicate the for- two highly homologous proteins (59% sequence identity to each
mation of a charge-transfer complex of the electron-rich other), have a similar S domain with RMSDs of 1.9–2.0 Å (116
reduced FAD and the electron-poor oxidized substrate (32). residues of 135). Nevertheless, although the overall fold of all
This could suggest that the product does not dissociate from these proteins is highly similar and all of them display a similar
the reduced enzyme before the oxidizing substrate enters the FAD binding mode, their catalytic centers show a huge varia-
active site.
tion, consistent with the large variation of their substrates,
A SDS-PAGE gel electrophoresis assay showed that the flavin ranging from small compounds (e.g., fumarate) to sugars and
co-factor did not co-migrate with the protein, suggesting that it steroids.
is noncovalently bound to the Ro05698 protein. This was con-
FAD Binding—The FAD molecule binds noncovalently in a
firmed by the crystal structure of ⌬4-(5␣)-KSTD (see below).
long cleft in the F-domain (supplemental Fig. S2). Its ADP part
The Three-dimensional Structure of ⌬4-(5␣)-KSTD—The is bound by a motif resembling the ␤␣␤␣␤ dinucleotide binding
crystal structure of ⌬4-(5␣)-KSTD was elucidated by molecular motif (Rossmann fold), frequently observed in flavoproteins
replacement using an ensemble of six structures with 17–24% (36). The adenine moiety is bound by two hydrogen bonds to
sequence identity to ⌬4-(5␣)-KSTD as the starting model, and the backbone amide and carbonyl group of Val-205. The two
the resulting solution was refined to an R factor of 15.8% at 1.6 hydroxyl groups of the adenosine ribose are hydrogen-bonded
Å resolution (for details see Table 2). The crystals contain one to Glu-51. The two phosphate groups have hydrogen bonding
⌬
4-(5␣)-KSTD molecule per asymmetric unit. ⌬4-(5␣)-KSTD interactions with the backbone amides of Ile-31, Ala-32, Ala-
has an ellipsoid shape with dimensions of 80 ϫ 40 ϫ 35 Å (Fig. 59, Thr-60, and Arg-456 and with the hydroxyl group of Thr-
4
). The overall fold resembles that of p-hydroxybenzoate 60. In addition there is a salt bridge between one of the phos-
hydroxylase (33), a fold often observed in flavoenzymes (34). phates and His-268. The D-ribitol binds in an extended
The protein consists of two domains connected via a two- conformation and is hydrogen-bonded to several water mole-
stranded antiparallel ␤-sheet (Fig. 4). One domain (F) contains cules and to the side chains of Ser-471 and Thr-60.
a noncovalently bound FAD co-factor; it consists of a central
The isoalloxazine ring system is located close to the interface
five-stranded parallel ␤-sheet flanked on one side by four addi- of the F and S domains. Its N5 nitrogen atom is hydrogen-
tional ␤-strands and on the other side by three ␣-helices. The bonded to the backbone amide of Gly-64, and the main chain
other domain, or substrate-binding domain (S, residues 292– NHs of Ser-471 and Leu-472 have hydrogen bonding interac-
4
25) is an insert into domain F and divides it into two parts, F
tions with the O oxygen of the pyrimidine ring. Ser-471 and
1
2
(
residues 7–291) and F (residues 426–489). It consists of a Leu-472 are at the N-terminal end of the ϳ25 Å long C-termi-
2
four-stranded antiparallel ␤-sheet flanked on both sides by two nal ␣-helix formed by residues 470–489, and the dipole
-helices. The active site is located at the interface between the moment provided by this helix may stabilize the negative
F and S domains next to the isoalloxazine ring system of the charge on the O oxygen atom in the anionic hydroquinone
␣
2
FAD co-factor.
form of the FAD (37). Finally, the side chain hydroxyl group of
The ⌬4-(5␣)-KSTD structure is well defined, with the excep- Ser-471 points away from the N1 nitrogen of the flavin, but in a
tion of the His tag, the 19 residues of the linker, residues 1–6, different rotamer conformation it would be at hydrogen bond-
6
and the C-terminal residue 490. In addition, two loops near the ing distance to the N1 nitrogen and could be involved in proto-
active site (residues 170–182 and 256–268; supplemental Fig. nating it.
3
0978 JOURNAL OF BIOLOGICAL CHEMISTRY
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3-Ketosteroid-⌬4-(5␣)-dehydrogenase Reaction Mechanism
FIGURE 3. Absorption spectrum of purified ⌬4-(5␣)-KSTD from Rhodococcus jostii RHA1. The red line shows the absorption spectrum of ⌬4-KSTD with
substrate 1-(5␣)-androstene-3,17-dione dissolved in ethanol (200 ␮M). The green line shows the absorption spectrum of ⌬4-KSTD without steroid. The dotted
green line shows the absorption spectrum of the ⌬4-KSTD with ethanol as control. A ⌬4-(5␣)-KSTD concentration of 5.8 mg/ml in phosphate buffer with 10%
glycerol, pH 7.2, was used.
isoalloxazine ring system (Fig. 5 and supplemental Fig. S2). It is
bound in a partially hydrophobic environment (Phe-427 and
the dimethylbenzene ring), and it is held in place by the back-
bone nitrogen of Ala-264. A negative charge close to the di-
methylbenzene ring of the FAD may push electrons toward the
pyrimidine ring and thereby lower the redox potential (39).
Product Binding—The 1.6 Å resolution structure of ⌬4-(5␣)-
KSTD with bound 4-androstene-3,17-dione (4-AD; Table 2)
shows that 4-AD binds at the si face of the isoalloxazine ring
system, displacing three water molecules and the two acetate
molecules (Fig. 5). Binding of 4-AD does not cause significant
changes in the overall protein structure; the all-atom RMSD
between the structures is only 0.31 Å. The side chains of Trp-
1
36 and Ser-320, which display a correlated double conforma-
FIGURE 4. Overview of the ⌬4-(5␣)-KSTD structure. FAD, glycerol, and the
two acetate molecules bound in the active site are shown in yellow. The Cl
Ϫ
tion in the steroid-free structure, become ordered upon bind-
ion is shown in green. The inset shows the domain organization of ⌬4-(5␣)- ing of the steroid molecule. Trp-136 has a hydrophobic stacking
KSTD. Domain F is shown in blue (F1) and orange (F2), and the S domain is in
pink. Domain S is an insert into domain F, and the active site is located on the
interface between the two domains.
interaction with the steroid, but it is conserved in only a few
putative ⌬4-KSTDs (5). A W136F substitution caused only a
3
-fold reduction in apparent activity, but a W136A mutation
The isoalloxazine ring system is not planar; the butterfly resulted in inactive enzyme (Table 3), indicating that an aro-
angle between the pyrimidine and dimethylbenzene rings is matic residue capable of stacking is required at this position.
ϳ168°, similar to that observed in cholesterol oxidase (38). The Concomitantly with the rigidification of the Trp-136 side chain,
color of the crystals and the spectrum of the protein sample also the Ser-320 side chain becomes ordered.
used for crystallization indicate that the oxidized form of FAD
is present in the structure.
4-AD binds with its C3 keto group at the same location as one
of the acetate oxygen atoms in the steroid-free structure, form-
The Active Site—In the steroid-free ⌬4-(5␣)-KSTD structure, ing a hydrogen bond to Tyr-466 (Fig. 5). Acetate is more often
the active site is open to the solvent and contains a well ordered observed to mimic the binding of an oxygen atom of the sub-
network of water molecules and two acetate ions. A chloride ion strate (40). The C4 carbon atom of 4-AD is near the Ser-468
is located ϳ3.8 Å behind the dimethylbenzene ring of the hydroxyl group. Surprisingly, the C17 keto moiety of 4-AD has
SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37
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3
-Ketosteroid-⌬4-(5␣)-dehydrogenase Reaction Mechanism
FIGURE 6. The hydrogen bonding environment around Ser-468. The side
chains of Ser-468, Tyr-319, and Glu-290 are shown. Dashed red lines indicate
the hydrogen bonds and the contact between Ser-468 and the 4-AD C4 car-
bon. Distances are given in Å. The FAD and 4-AD are shown in yellow, and the
chloride ion is in green. A red arrow marks the proposed proton relay pathway
from the 4-AD C4 carbon to the solvent.
FIGURE 5. Product (4-AD) binding in ⌬4-(5␣)-KSTD. The most important
active site residues are displayed. Trp-136 and Ser-320 display double confor-
mations in the acetate bound structure but are fixed in the 4-AD bound struc-
ture. Trp-136 is stacking with the C and D ring of 4-AD.
is the only residue with a direct hydrogen bonding interaction
with the product in the crystal structure and thus may also be a
determinant for substrate recognition.
TABLE 3
Apparent kinetic parameters of the R. jostii Ro05698 ⌬4-(5␣)-KSTD
wild type and active site mutants using 1-(5␣)-AD as substrate
V
Km
␮M
k
s
k
s
cat/K
ꢀM
Ser-468 Is the Putative Catalytic Base—The Ser-468 hydroxyl
group is at 3.1 Å from the C4 atom of 4-AD, a position compat-
ible with a role of Ser-468 as the catalytic base, which abstracts
a proton from C4. The C4 protons have been labilized because
of keto-enol tautomerization of the keto group of the substrate
upon interaction with the Tyr-466 hydroxyl group. The Ser-468
hydroxyl group is part of an extensive hydrogen bonding sys-
tem, which can function as an effective system relaying protons
in a concerted way to the solvent and which includes the Tyr-
max
Ϫ1
cat
Ϫ1
m
Ϫ1
Ϫ1 Ϫ1
nmolꢀmg ꢀmin
a
2
2
6
Wild type 27 ϫ 10 Ϯ 1.5 ϫ 10 10 Ϯ 2
2.4 2.4 ϫ 10
b
W136A
W136F
Y319F
Y466A
Y466F
S468T
S468A
ND
ND
ND ND
2
2
4
8 ϫ 10 Ϯ 3 ϫ 10
66 Ϯ 25
ND
0.7 1 ϫ 10
ND
ND
ND ND
ND ND
ND
ND
ND
ND ND
2
2
1
1
3
19 ϫ 10 Ϯ 3.6 ϫ 10 17 ϫ 10 Ϯ 3.3 ϫ 10 1.6 9 ϫ 10
ND
ND
ND ND
a
Contains mutations A60T (nucleotide G178A) and T160A (nucleotide A478G),
but these mutations do not or hardly have an effect on activity.
ND, not detectable.
b
3
19 hydroxyl group, a water molecule, and the side chain of
Glu-290 (Fig. 6). Because such concerted proton transfers do
not involve high energy alkoxide intermediates (43), the proton
can easily be transferred from the C4 atom to the solvent.
Ser-468 is conserved in ⌬4-KSTD enzymes, including
Rv1817 from M. tuberculosis H37Rv, but not in ⌬1-KSTDs. Its
hydroxyl group is essential for activity, because a S468A
mutation abolished all catalytic activity, whereas a S468T
no interactions with the protein, but it is hydrogen-bonded to
two water molecules. Apparently, the substituent at this posi-
tion is not a very critical factor in substrate recognition,
although preliminary results show that an aliphatic tail at C17,
4
as in 5-␣-cholestan-3-one, is not accepted at this position.
Tyr-466 Is the Putative Catalytic Acid—The position of Tyr-
4
66 and its hydrogen bonding interaction with the C3 keto
mutant still retained activity. However, the apparent K of
m
group of 4-AD suggest that this residue may function as the
catalytic acid that protonates the C3 keto group, promoting
keto-enol tautomerization and labilization of the C4 hydrogen
atoms. Formation of the enolate at the C3 keto group has pre-
viously been suggested as the initial catalytic step for these pro-
teins (41, 42). Tyr-466 is conserved in all ⌬4 and ⌬1 KSTD
enzymes. Site-directed mutagenesis (Y466A and Y466F)
resulted in catalytically dead ⌬4-(5␣)-KSTD enzymes (Table 3),
confirming the importance of Tyr-466 and showing that the
hydroxyl group is critical for the reaction. Intriguingly, Tyr-466
this latter mutant protein was drastically increased, and the
apparent V
was somewhat lower than that of wild-type
max
enzyme (Table 3).
A Y319F mutant enzyme had also lost all activity on
1
-(5␣)-AD as substrate, showing the functional importance of
the hydroxyl group of this tyrosine for activity (Table 3). In
contrast, a E290Q mutation did not affect the catalytic activity
of the enzyme, indicating that this residue is not essential for
proton relay and that other relay routes may exist.
The Role of the FAD Co-factor in Catalysis—Flavoproteins
involved in dehydrogenation reactions often display a few
recurrent features, like the distance and angle between the FAD
N5 nitrogen atom and the site of oxidative attack and the pres-
4
J. Knol, D. van der Tuin, L. Dijkhuizen, and R. van der Geize, manuscript in
preparation.
3
0980 JOURNAL OF BIOLOGICAL CHEMISTRY
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3-Ketosteroid-⌬4-(5␣)-dehydrogenase Reaction Mechanism
ence of a hydrogen bond donor close to the N5 nitrogen (44). In
4-(5␣)-KSTD the N5 nitrogen is hydrogen-bonded to the
⌬
backbone amide of Gly-64. It is at a distance of 3.9 Å from the
C5 atom of the product, and the N10-N5-C5 angle is ϳ108°.
Both the distance and angle are in agreement with the values
observed in other proteins (44). These values clearly suggest
that the site of attack by the FAD is the C5 atom of the substrate.
The position of the FAD N5 atom above and of Ser-468 below
the 4-AD is in agreement with trans-dehydrogenation of the
substrate (42).
In many flavoproteins, catalysis takes place at a location
shielded from the solvent, which enhances the strength of polar
interactions and is instrumental to substrate activation (34, 44).
In some enzymes with the p-hydroxybenzoate hydroxylase fold
loops move in upon substrate binding to cover the active site,
and in others the complete S domain may relocate (15, 45). In
⌬
4-(5␣)-KSTD two loops close to the active site (supplemental
Fig. S3) show somewhat less defined electron density and may
be flexible. However, in the 4AD-bound structure, no signifi-
cant conformational differences are seen between the steroid-
free and product-bound states. At present we cannot exclude
that crystal contacts prevent large conformational changes or
that the loops move to restrict solvent access to the FAD. Bind-
ing of the steroid substrate may also already provide sufficient
shielding. Further research will be needed to resolve this
question.
Comparison with Cholesterol Oxidase—⌬4-(5␣)-KSTD is
functionally and structurally related to cholesterol oxidase (45).
The structures of the two proteins can be superimposed with an
RMSD of 2.6 Å for 213 of 483 C␣ atom positions with the high-
est similarity in the F-domain. Although ⌬4-(5␣)-KSTD cata-
lyzes the dehydrogenation of the C4-C5 bond of steroids, cho-
lesterol oxidase catalyzes the oxidation of steroids containing a
3␤-hydroxyl group with concomitant isomerization of the ⌬5
double bond to the ⌬4 position. However, whereas in ⌬4-(5␣)-
KSTD the substrate/product binds at the si face of the isoallox-
azine ring system, in cholesterol oxidase the substrate binds in a
pocket in front of the N5 nitrogen. This different substrate-
binding mode positions the substrate such that in ⌬4-(5␣)-
KSTD the C5 hydrogen atom is near the FAD N5 nitrogen, but
in cholesterol oxidase it is the C3 hydroxyl group that is near the
N5 atom of the FAD. This explains the different reaction spec-
ificity and regioselectivity of the two enzymes.
Cholesterol oxidase also contains an extra domain, which FIGURE 7. Proposed reaction mechanism of ⌬4-(5␣)-KSTD. Panel 1, cataly-
sisisinitiatedbytheinteractionoftheO3ketogroupofthesubstratewiththe
binds the apolar aliphatic tail connected to the C17 atom of the
hydroxyl group of Tyr-466, which promotes keto-enol tautomerization and
cholesterol. In the 4AD-bound structure of ⌬4-(5␣)-KSTD, the labilization of the C4 hydrogen atoms. Ser-468, acting as a base, abstracts a
proton from the C4 atom of the substrate, with Tyr-319 serving as a proton
relay systemtothesolvent. Panel 2, next, theFAD abstractsahydrideion from
the C5 carbon of the substrate, which, with a concomitant rearrangement,
results in the formation of a double bond between C4 and C5. Panel 3, the
product is formed and leaves the active site, perhaps only after the oxidizing
substrate enters the active site. The negative charge on the reduced FAD is
stabilized by the dipole moment of the C-terminal helix. Finally, the oxidized
C17 keto group of the substrate/product is exposed to the sol-
vent, in agreement with the wider range of substituents at this
4
position that are accepted by ⌬4-(5␣)-KSTD.
The Catalytic Mechanism—The oxidation of 5␣-AD or
1
-(5␣)-AD requires the abstraction of two hydrogen atoms and
the transfer of two electrons as a hydride ion to the FAD. It is FAD is regenerated by an as yet unknown electron acceptor.
not known whether these proton and hydride transfers occur in
a concerted or stepwise manner. The two electrons will finally relays its own proton to the solvent via Tyr-139 (Fig. 7). The
be transferred to a currently unknown electron acceptor, for resulting deprotonated state of the substrate can be stabilized
example menaquinone (46).
by the delocalization of the negative charge over the C3 keto
The crystal structure indicates Ser-468 as the putative base group. In addition, Tyr-466, functioning as an acid, can stabilize
that abstracts the C4 ␣-hydrogen proton from the substrate and the ensuing enolate by hydrogen bonding to the C3 oxygen
SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37
JOURNAL OF BIOLOGICAL CHEMISTRY 30981

3
-Ketosteroid-⌬4-(5␣)-dehydrogenase Reaction Mechanism
atom. The FAD N5 atom is in a good position to abstract a
hydride ion from the C5 atom of the enolate intermediate. In
synchrony, the lone pair electrons of the negatively charged C3
oxygen atom move back toward C3 and the double bond
between C3 and C4 shifts to the C4-C5 position, generating the
product. The negative charge on the N5 of FAD can be delocal-
(2008) 3-Keto-5␣-steroid ⌬ -dehydrogenase from Rhodococcus erythro-
1
polis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are
highly specific enzymes that function in cholesterol catabolism. Biochem.
J. 410, 339–346
6
. Fujii, C., Morii, S., Kadode, M., Sawamoto, S., Iwami, M., and Itagaki, E.
(
1999) Essential tyrosine residues in 3-ketosteroid-⌬ -dehydrogenase
1
from Rhodococcus rhodochrous. J. Biochem. 126, 662–667
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. Matsushita, H., and Itagaki, E. (1992) Essential histidine residue in 3-ke-
ized over the N1-C ϭ O region, but also the rest of the isoallox-
2
tosteroid-⌬ -dehydrogenase. J. Biochem. 111, 594–599
1
azine ring may contribute (47). Two backbone amides stabilize
8
. Van der Geize, R., Yam, K., Heuser, T., Wilbrink, M. H., Hara, H., Ander-
ton, M. C., Sim, E., Dijkhuizen, L., Davies, J. E., Mohn, W. W., and Eltis,
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nomycete provides insight into Mycobacterium tuberculosis survival in
macrophages. Proc. Natl. Acad. Sci. U.S.A. 104, 1947–1952
the negative charge on O by hydrogen bonding, and the dipole
2
moment of the C-terminal helix is also directed toward O . The
2
generated reducing equivalents may be donated to a respiratory
chain, similar to what has been proposed for the ⌬1-KSTD of
Arthrobacter globiformis (48), but further studies are required
to confirm this.
9
. Rosas-Magallanes, V., Stadthagen-Gomez, G., Rauzier, J., Barreiro, L. B.,
Tailleux, L., Boudou, F., Griffin, R., Nigou, J., Jackson, M., Gicquel, B., and
Neyrolles, O. (2007) Signature-tagged transposon mutagenesis identifies
novel Mycobacterium tuberculosis genes involved in the parasitism of hu-
man macrophages. Infect. Immun. 75, 504–507
The Importance of Ketosteroid Dehydrogenases for Pathogenic
Organisms—⌬4-(5␣)-KSTD is the first ketosteroid dehydro-
genase of which a crystal structure has been elucidated. The 10. van Oosterwijk, N., Knol, J., Dijkhuizen, L., van der Geize, R., and Dijkstra,
enzyme has an important role in the desaturation of the steroid
A ring, which is a key step in the microbial degradation of sat-
urated steroids. It has been proposed that saturated steroid
intermediates are formed during cholesterol catabolism. Sev-
eral pathogenic bacteria, including M. tuberculosis, Rhodococ-
cus equi, and Mycobacterium bovis, contain a cholesterol cata-
B. W. (2011) Cloning, overexpression, purification, crystallization and
preliminary x-ray analysis of 3-ketosteroid ⌬ -(5␣)-dehydrogenase from
4
Rhodococcus jostii RHA1. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Com-
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1
1. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R., and Leslie, A. G.
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bolic pathway similar to that of R. jostii RHA1 (8, 49). In 12. Evans, P. (2006) Scaling and assessment of data quality. Acta Crystallogr. D
6
2, 72–82
M. tuberculosis this pathway and in particular the ⌬1-KSTD
enzyme (Rv3537, which shows 28% sequence identity to R. jostii
RHA1 ⌬4-(5␣)-KSTD) have been implicated to be important
for growth of the intracellular pathogen in the hostile environ-
ment of macrophages (50, 51). This makes the cholesterol cat-
1
3. Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans,
P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G., McCoy, A., McNicholas,
S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read,
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FFAS03. A server for profile-profile sequence alignments. Nucleic Acids
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Res. 33, W284–W288
RHA1 ⌬4-(5␣)-KSTD structure may facilitate the design of
1
5. Bamford, V., Dobbin, P. S., Richardson, D. J., and Hemmings, A. M. (1999)
Open conformation of a flavocytochrome c3 fumarate reductase. Nat.
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potent ketosteroid dehydrogenase inhibitors, as a first step
toward the development of new antituberculosis drugs.
1
6. Leys, D., Tsapin, A. S., Nealson, K. H., Meyer, T. E., Cusanovich, M. A., and
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BM16 and ID14-1 (European Synchrotron Radiation Facility,
Grenoble, France) for help during data collections.
1
7. Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkin-
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