Activity of 3-ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis

Article abstract of DOI:10.1074/jbc.M111.289975

Mycobacterium tuberculosis (Mtb), a significant global pathogen, contains a cholesterol catabolic pathway. Although the precise role of cholesterol catabolism in Mtb remains unclear, the Rieske monooxygenase in this pathway, 3-ketosteroid 9α-hydroxylase (KshAB), has been identified as a virulence factor. To investigate the physiological substrate of KshAB, a rhodococcal acyl-CoA synthetase was used to produce the coenzyme A thioesters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4- en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnorchola- 1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The apparent specificity constant (k_cat/K_m) of KshAB for the CoA thioester substrates was 20-30 times that for the corresponding 17-keto compounds previously proposed as physiological substrates. The apparent K_mO2 was 90 ± 10 μM in the presence of 1,4-BNC-CoA, consistent with the value for two other cholesterol catabolic oxygenases. The Δ¹ ketosteroid dehydrogenase KstD acted with KshAB to cleave steroid ring B with a specific activity eight times greater for a CoA thioester than the corresponding ketone. Finally, modeling 1,4-BNC-CoA into the KshA crystal structure suggested that the CoA moiety binds in a pocket at the mouth of the active site channel and could contribute to substrate specificity. These results indicate that the physiological substrates of KshAB are CoA thioester intermediates of cholesterol side chain degradation and that side chain and ring degradation occur concurrently in Mtb. This finding has implications for steroid metabolites potentially released by the pathogen during infection and for the design of inhibitors for cholesterol-degrading enzymes. The methodologies and rhodococcal enzymes used to generate thioesters will facilitate the further study of cholesterol catabolism.

Full text of DOI:10.1074/jbc.M111.289975

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 47, pp. 40717–40724, November 25, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Activity of 3-Ketosteroid 9␣-Hydroxylase (KshAB) Indicates
Cholesterol Side Chain and Ring Degradation Occur
□
S
Simultaneously in Mycobacterium tuberculosis*
Received for publication, August 8, 2011, and in revised form, October 6, 2011 Published, JBC Papers in Press, October 10, 2011, DOI 10.1074/jbc.M111.289975
Jenna K. Capyk‡1,2, Israël Casabon§1, Robert Gruninger‡, Natalie C. Strynadka‡, and Lindsay D. Eltis‡§3
From the Departments of ‡Biochemistry and Molecular Biology and §Microbiology and Immunology, Life Sciences Institute,
University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
Background: Mycobacterium tuberculosis (Mtb) degrades cholesterol throughout its infection cycle.
Results: Cholesterol ring-degrading enzymes have higher activities with side chain degradation intermediates than with com-
pounds with fully degraded side chains.
Conclusion: Cholesterol side chain and ring degradation occur concurrently.
Significance: Understanding bacterial cholesterol catabolism facilitates the design of novel therapeutics and the production of
high value steroids.
Mycobacterium tuberculosis (Mtb), a significant global patho- ate thioesters will facilitate the further study of cholesterol
gen, contains a cholesterol catabolic pathway. Although the pre- catabolism.
cise role of cholesterol catabolism in Mtb remains unclear, the
Rieske monooxygenase in this pathway, 3-ketosteroid 9␣-hy-
Mycobacterium tuberculosis (Mtb)4 infects about one-third
of all people and causes nine million new cases of pulmonary
disease each year (1). The success of the bacterium as a patho-
gen is due in part to its remarkable ability to persist and repli-
cate inside human macrophages (2). Although the mechanisms
enabling Mtb to evade sterilization by the human immune sys-
tem are not well understood, recent studies have revealed that
the bacterium has a lipid-biased metabolic profile uniquely
adapted to its intracellular lifestyle (3). Studies further suggest
that genes necessary for lipid catabolism are preferentially
expressed during intra-macrophage growth (4, 5). Further-
more, Mtb has been shown to direct carbon flux away from the
tricarboxylic acid cycle (6), consistent with the hypothesis that
growth on fatty acids via the glyoxylate cycle is an important
component of Mtb virulence (7).
droxylase (KshAB), has been identified as a virulence factor. To
investigate the physiological substrate of KshAB, a rhodococcal
acyl-CoA synthetase was used to produce the coenzyme A thio-
esters of two cholesterol derivatives: 3-oxo-23,24-bisnorchol-4-
en-22-oic acid (forming 4-BNC-CoA) and 3-oxo-23,24-bisnor-
chola-1,4-dien-22-oic acid (forming 1,4-BNC-CoA). The
apparent specificity constant (kcat/Km) of KshAB for the CoA
thioester substrates was 20–30 times that for the correspond-
ing 17-keto compounds previously proposed as physiological
substrates. The apparent KmO was 90 ؎ 10 ␮M in the presence
2
of 1,4-BNC-CoA, consistent with the value for two other cho-
lesterol catabolic oxygenases. The ⌬1 ketosteroid dehydro-
genase KstD acted with KshAB to cleave steroid ring B with a
specific activity eight times greater for a CoA thioester than
the corresponding ketone. Finally, modeling 1,4-BNC-CoA
into the KshA crystal structure suggested that the CoA moi-
ety binds in a pocket at the mouth of the active site channel
and could contribute to substrate specificity. These results
indicate that the physiological substrates of KshAB are CoA
thioester intermediates of cholesterol side chain degradation
and that side chain and ring degradation occur concurrently
in Mtb. This finding has implications for steroid metabolites
potentially released by the pathogen during infection and for
the design of inhibitors for cholesterol-degrading enzymes.
The methodologies and rhodococcal enzymes used to gener-
A cholesterol catabolic pathway (8) (Fig. 1) enabling the bac-
terium to grow in vitro using this steroid as a sole source of
carbon (9) is proposed as a key component of Mtb lipid-biased
metabolic strategy. Analogous to cholic acid degradation in
Pseudomonas sp. Chol1 (10) and Rhodococcus jostii RHA1,5
cholesterol side chain degradation in Mtb is expected to pro-
ceed via CoA thioester intermediates in three cycles of ␤-oxi-
dation (11, 12). Degradation of the steroid rings A and B include
three successive oxygenase reactions (13–15) (Fig. 1). The cat-
alytic rates measured for these oxygenases using the predicted
* This work was supported by an operating grant from the Canadian Insti- 4 The abbreviations used are: Mtb, M. tuberculosis; 4-AD, 4-androstene-3,17-
tutes for Health Research (to L. D. E.).
dione; ADD, 1,4-androstadiene-3,17-dione; 4-BNC, 3-oxo-23,24-bisnor-
chol-4-en-22-oic acid; 1,4-BNC, 3-oxo-23,24-bisnorchola-1,4-dien-22-oic
acid; 4-BNC-CoA, 3-oxo-23,24-bisnorchol-4-en-22-oyl-coenzyme A thioes-
ter; 1,4-BNC-CoA, 3-oxo-23,24-bisnorchola-1,4-dien-22-oyl-coenzyme
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1 and 2.
1 These authors contributed equally to this work.
2 Recipient of a studentship from Natural Science and Engineering Research
Council.
A
thioester; 3-HSBNC, 3-hydroxy-9-oxo-9,10-seco-23,24-bisnorchola-
1,3,5(10)-trien-22-oic acid; 3-HSBNC-CoA, 3-hydroxy-9-oxo-9,10-seco-
23,24-bisnorchola-1,3,5(10)-trien-22-oyl-CoA thioester.
3 To whom correspondence should be addressed: 2350 Health Sciences Mall,
Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-0042; Fax: 5 W. W. Mohn, M. H. Wilbrink, I. Casabon, G. R. Stewart, J. Liu, L. D. Eltis, and R.
604-822-6041; E-mail: leltis@mail.ubc.ca.
van der Geize, in preparation.
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
JOURNAL OF BIOLOGICAL CHEMISTRY 40717

CoA Thioesters in Mtb Cholesterol Catabolism
FIGURE 1. Schematic of the proposed cholesterol degradation pathway of Mtb. Key carbons of cholesterol are numbered using the steroid numbering
convention. The processes of cholesterol side chain and partial ring degradation are shown as parallel pathways. Enzymes known to catalyze specific reactions
in Mtb are indicated. Non-enzymatic reaction is shown as a dashed arrow. Compounds 1, 2, and 3 represent 4-AD, ADD, and 3-hydroxy-9,10-seconandrost-
1,3,5(10)-trien-9,17-dione (3-HSA) (R ϭ ketone); 4-BNC, 1,4-BNC, and 3-HSBNC, (R ϭ isopropionate); 4-BNC-CoA, 1,4-BNC-CoA, and 3-HSBNC-CoA, (R ϭ
isopropionyl-CoA).
17-keto metabolites (Fig. 1) were less than their non-steroid- substrate for this enzyme may not be a 17-keto steroid but an
transforming homologues (13–15). Genetic knockouts of sev- intermediate of cholesterol side chain degradation (15). Delet-
eral of the cholesterol degradation genes have resulted not only ing either kshA or kshB completely abrogated the virulence of
in failure to grow on cholesterol in vitro but in attenuated path- Mtb in several mouse and macrophage infection models, lead-
ogenicity in various infection models (9, 11, 14, 16, 17).
ing to the suggestion that the enzyme reaction product has an
One important aspect of cholesterol catabolism in Mtb that immunomodulatory function (17).
has yet to be established is the sequence of the side chain and
Herein we explored the hypothesis that the physiological
ring degradation reactions with respect to each other. Metabo- substrates for cholesterol ring-degrading enzymes are interme-
lite accumulation studies for knockouts of kshA and kstD, diates of side chain ␤-oxidation bearing a CoA thioester. CoA
encoding the ring-degrading enzymes 3-ketosteroid 9␣-hy- thioester derivatives of the 17-isopropionate 3-keto steroids
droxylase and ⌬1 3-ketosteroid dehydrogenase, respectively, 3-oxo-23,24-bisnorchol-4-en-22-oic acid (4-BNC) and 3-oxo-
have suggested that the side chain is completely degraded to the 23,24-bisnorchola-1,4-dien-22-oic acid (1,4-BNC) (Fig. 2) were
17-ketone before any significant ring degradation occurs (18– synthesized using a rhodococcal acyl-CoA synthetase (CasI)
20). This conclusion was further supported by the accumula- and KstD from Mtb. The BNCs and their CoA thioesters were
tion of the 17-keto metabolites 4-androstene-3,17-dione used to elucidate the substrate specificity of KshAB. The
(4-AD) and 1,4-androstadiene-3,17-dione (ADD) in the super- enzyme substrate binding pocket was investigated using molec-
natant of Mtb cultures (11). Interestingly, the order of particu- ular modeling. The substrate preference of KstD was also inves-
lar reactions seems to be species-dependent. For example, tigated. The results are discussed in terms of microbial steroid
3␤-hydroxy-steroid dehydrogenase (3␤-HSD) of Mtb catabolism and the pathogenesis of Mtb.
CDC1551 and Mycobacterium bovis BCG are able to transform
EXPERIMENTAL PROCEDURES
cholesterol (21, 22), whereas the corresponding ring-degrading
enzyme of R. jostii RHA1 requires the activity of the first side
Chemicals and Reagents—ADD and 4-BNC were purchased
chain-transforming enzyme, 26-cholesterol hydroxylase from Steraloids, Inc. (Newport, RI). 4-AD was purchased from
(Cyp125) (23). Sigma-Aldrich (St. Louis, MO). Restriction enzymes and the
3-Ketosteroid 9␣-hydroxylase comprises a Rieske oxygenase, Expand High Fidelity PCR System were purchased from New
encoded by kshA, and a reductase, encoded by kshB (24). KshAB England Biolabs (Ipswich, MA) and Roche Applied Science,
utilizes O2 and NADH and acts in concert with KstD to effect respectively. Oligonucleotides for amplifying kstD were pur-
opening of ring B and aromatization of ring A in the Mtb cho- chased from Integrated DNA Technologies (San Diego, CA)
lesterol degradation pathway (15, 25) (Fig. 1). Studies of KshAB through the Nucleic Acid Protein Service Unit at the University
from Mtb (15) have probed its substrate specificity, suggesting of British Columbia. All other reagents were of HPLC or ana-
that ADD is its physiological substrate. Nonetheless, KshAB lytical grade. Water for buffers was purified using a Barnstead
exhibits specificity constants for the 17-keto steroids 4-AD and Nanopure DiamondTM system (Dubuque, IA) to a resistivity of
ADD that are 2–3 orders of magnitude less than those of other at least 18 megaohms.
characterized Rieske oxygenases (15). Additionally, this
Cloning of kstD—DNA was propagated, digested, ligated, and
enzyme exhibits an apparent KmO in excess of 1.2 mM in the transformed using standard protocols (26). Plasmid DNA was
2
presence of ADD. The relatively low reaction rates observed for purified as described previously (27) and was used to transform
KshAB have prompted speculation that the true physiological Escherichia coli by electroporation using a MicroPulser from
40718 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

CoA Thioesters in Mtb Cholesterol Catabolism
bated for a further 20 h at 16 °C and were then harvested by
centrifugation. Pellets from 100 ml of culture were resuspended
in 1 ml of 9 mM sodium phosphate, pH 8.0, containing 10%
glycerol, the Complete Mini Protease Inhibitor mixture, and
DNase I (Roche Applied Science) and then were lysed using a
bead beater and centrifuged. Proteins were purified from the
supernatant using a standard nickel-nitrilotriacetic acid proto-
col, exchanged into 25 mM HEPES, pH 7.5, with 50 mM potas-
sium chloride by dialysis at 4 °C, and concentrated to ϳ5 mg/ml
by ultrafiltration. Proteins were flash-frozen in liquid nitrogen
and stored at Ϫ80 °C. KshA and KshB were produced and puri-
fied as previously described (15).
Preparation of Substrates—4-BNC was dissolved to concen-
trations of 50 mM in 94% ethanol containing 60 mM NaOH.
4-AD, ADD, and 1,4-BNC were dissolved in ethanol up to 15
and 25 mM. CoA thioesters were stored and used as aqueous
solutions up to 6 mM. To produce 3-oxo-23,24-bisnorchol-4-
en-22-oyl-coenzyme A thioester (4-BNC-CoA), 5.52 ␮mol of
4-BNC was incubated with 0.75 mg of purified CasI for 7 h at
22 °C in 6 ml of 100 mM HEPES buffer, pH 7.5, containing 6.77
␮mol of CoASH, 30 ␮mol of MgCl2, and 15 ␮mol of ATP. To
produce 3-oxo-23,24-bisnorchola-1,4-dien-22-oyl-coenzyme
A thioester (1,4-BNC-CoA), 3.02 ␮mol of 4-BNC-CoA was
incubated with 0.6 mg (total protein) of KstD lysate for 2 h at
22 °C in 15 ml 50 mM Tris/HCl, pH 7.4, containing 6 ␮mol of
FIGURE 2. Compounds investigated as KshAB substrates. Shown are 4-AD
(A), ADD (B), 4-BNC (C), 1,4-BNC (D), and 1,4-BNC-CoA (E). Not shown is 4-BNC-
CoA, which differs from E by a 1–2 saturated bond on ring A.
Bio-Rad with Bio-Rad 0.1-cm GenePulser Cuvettes. The kstD
gene was amplified from Mtb H37Rv genomic DNA using for-
ward and reverse primers with the sequences 5Ј-TACGCTAG-
CACTGTGCAGGAGTTCGACGTCG-3Ј (the NheI site is
underlined) and 5Ј-CAGAATTCTCAGCGCTTTCCCGCCTG-3Ј
(the EcoRI site is underlined), respectively. Polymerase chain
reactions contained 20 ng of template DNA, 2 units of the
Expand High Fidelity PCR System polymerase, 20 ␮M each of
dNTP, and 30 pmol each of oligonucleotide in a volume of 100
␮l. Reactions were subject to 22 temperature cycles using a
Stratagene Robocycler Gradient 96 instrument (La Jolla, CA) as
follows: 95 °C for 40 s, 55 °C for 40 s, and 72 °C for 60 s. The kstD
amplicon was digested with NheI and EcoRI and ligated into
pET-28a(ϩ) (EMD) to yield pETKD1 such that the expressed
protein was produced with an N-terminal thrombin-cleavable
His6 tag. Nucleotide sequences were confirmed by the Nucleic
Acid Protein Service Unit.
ϩ
NAD . To produce 1,4-BNC, 4.06 ␮mol of 4-BNC was incu-
bated with 5 mg (total protein) of KstD lysate for 23 h at 22 °C in
ϩ
4 ml of 50 mM Tris/HCl, pH 7.4, containing 8 ␮mol of NAD .
The pH of the reaction solutions was adjusted to 7.4 with NaOH
or HCl (for reactions containing 4-BNC-CoA and 4-BNC,
respectively) before the addition of KstD lysate. After incuba-
tion, reaction mixtures were diluted 1:1 with methanol and
incubated on ice for 10 min. Methanol was evaporated at room
temperature under low pressure using a Savant SC110A Speed-
Vac concentrator (Thermo Electron Corp.). The mixture was
centrifuged at 4 °C (16,000 g ϫ 10 min). The supernatant was
recovered, treated a second time with methanol as described
above, filtered using a 0.2 ␮m PTFE filter unit (Millipore), and
stored on ice. Compounds were chromatographically purified
using a Waters 2695 Separations HPLC module (Milford, MA)
equipped with a Waters 2996 photodiode array detector and a
LUNA 3 ␮m PFP(2) 50 ϫ 4.6-mm column (Phenomenex, Tor-
rance, CA) using a solvent system of 0.1 M ammonium acetate,
pH 4.5 (Buffer A), and 90% methanol in 0.1 M ammonium ace-
tate, pH 4.5 (Buffer B). 500 ␮l (for 1,4-BNC) or 1 ml (for the CoA
thioesters) of the reaction mixture was injected onto the col-
umn, which was equilibrated with Buffer A. Eluates were mon-
itored at 248 nm. 4-BNC-CoA was eluted using a gradient of 0
to 100% Buffer B over 20 min at 1 ml/min (Gradient A). Under
these conditions, the retention times for 4-BNC and 4-BNC-
CoA were 18.7 and 16.7 min, respectively. 1,4-BNC and 1,4-
BNC-CoA were eluted using a biphasic gradient consisting of 0
to 70% Buffer B at 1 ml/min over 14 min followed by 70 to 100%
Buffer B at 0.5 ml/min over 12 min (Gradient B). Under these
conditions, the retention times for 4-BNC, 1,4-BNC, 4-BNC-
CoA, and 1,4-BNC-CoA were 24.3, 22.7, 19.8, and 18.0 min,
Protein Production—KstD was produced in E. coli
BL21(DE3) cells grown in LB with 25 ␮g/ml kanamycin at 20 °C
and 200 rpm. Expression of kstD was induced using 0.5 mM
isopropyl ␤-D-1-thiogalactopyranoside when cells reached an
A600 of 0.5. After 20 h, cells were harvested and washed by
centrifugation. The cell pellet from 0.3 liters of culture was sus-
pended in 0.5 ml of 50 mM Tris/HCl, pH 7.4, containing 0.175
mg/ml phenylmethanesulfonyl fluoride and DNase I and sub-
jected to five rounds of bead beating using an MP Biomedicals
FastPrep-24 bead beater (Solon, OH) set to 5.0 for 20 s with 4
min on ice between rounds. Cell debris was pelleted by centrif-
ugation (16,100 g ϫ 10 min) at 4 °C. The supernatant, referred
to hereafter as KstD-lysate, was separated from the pellet and
kept on ice until use.
CasI (Ro05822) and CasG (Ro05820) of R. jostii RHA1 were
produced in E. coli RosettaTM (DE3)pLysS containing pETCasI
and pETCasG.5 Cells were grown at 37 °C with shaking at 200
rpm in 100 ml of LB supplemented with 100 ␮g/ml ampicillin
and 34 ␮g/ml chloramphenicol. Expression of the cas genes was
induced using 1 mM isopropyl ␤-D-1-thiogalactopyranoside
when the culture attained an A600 of 0.6. The cells were incu- respectively. Methanol was removed under nitrogen flow, and
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 40719

CoA Thioesters in Mtb Cholesterol Catabolism
CoA thioesters were stored at Ϫ80 °C. Solutions of 1,4-BNC
were lyophilized and stored at Ϫ80 °C.
Analysis of Substrates and Products—Reaction substrates
and products were routinely verified by HPLC using the
conditions described above. Reactions containing 4-AD and
ADD were resolved using a gradient similar to Gradient A but
at pH 5.0. Under these conditions the retention times for 4-AD
and ADD were 17.1 and 16.2 min, respectively. CoA thioesters,
including 3-hydroxy-9-oxo-9,10-seco-23,24-bisnorchola-1,3,
5(10)-trien-22-oyl-CoA thioester (3-HSBNC-CoA) were iden-
tified by subjecting them to alkaline hydrolysis at pH 13 for 1 h
at 50 °C. The solutions were then acidified to pH ϳ 4.5 with 6 M
HCl and examined by HPLC to verify the generation of the
corresponding steroid carboxylic acid and CoASH. Products
from the hydrolyzed CoA thioester mixes were also extracted
with ethyl acetate and dried. These compounds as well as puri-
fied 1,4-BNC were derivatized with trimethylsilyldiazomethane
and subjected to gas chromatography-coupled mass spectrom-
etry (GC-MS) performed using an HP 6890 series GC system
fitted with an HP-5MS 30-m ϫ 250-␮m column (Hewlett-
Packard, Palo Alto, CA) and an HP 5973 mass-selective detec-
tor. Additionally, purified samples of 4-BNC-CoA and 1,4-
BNC-CoA were subjected to electrospray ionization-mass
spectrometry (ESI-MS) using a Bruker Esquire-LC ion trap
mass spectrometer equipped with an ESI ion source, which was
operated in the positive ion mode. Samples were dissolved 1:9
in methanol to a final concentration of 20–30 pmol/␮l. Five ␮l
were injected at a flow rate of 200 ␮l/min in 1:9 water:methanol.
The scanned mass range was m/z 50–2000 Da.
FIGURE3. Transformationof4-BNCto4-BNC-CoAbyCasI. HPLCchromato-
grams are shown of 50 ␮l of a CasI reaction (2 ␮M, dashed line) and no-enzyme
control (solid line) containing 0.9 mM 4-BNC, 1.1 mM CoASH, 5 mM MgCl2, and
2.5 mM ATP after 7 h. Peaks were resolved using a linear gradient of 0 to 90%
methanol in 0.1 M ammonium acetate, pH 4.5, over 20 min. Substrate peaks
are numbered: 1, ATP; 3, CoASH; 5, 4-BNC. Product peaks are numbered: 2,
AMP; 4, 4-BNC-CoA.
50 mM Tris/HCl, pH 7.4. For reactions containing 4-BNC-CoA,
the reaction pH was readjusted to 7.4 with NaOH before the
addition of the KstD-lysate. At various time points, 200 ␮l of
each reaction was stopped by rapid dilution into 200 ␮l of meth-
anol, evaporated to remove methanol, filtered, and analyzed by
HPLC.
Steroid concentrations were measured spectrophotometri-
cally using the following extinction coefficients for aqueous
Ϫ1
Ϫ1
solutions at 22 °C: ⑀248 ϭ 8,700 M cm for 4-BNC, ⑀248
ϭ
Ϫ1
Ϫ1
Ϫ1
Ϫ1
8,500 M cm for 1,4-BNC, ⑀248 ϭ 17,200 M cm for
4-BNC-CoA, and ⑀248 ϭ 16,900 MϪ1 cmϪ1 for 1,4-BNC-CoA.
The extinction coefficient for 4-BNC was measured using a
solution containing a gravimetrically determined amount of the
compound. That for 1,4-BNC was calculated from the ratios of
values for 4-AD and ADD and for 4-AD and 4-BNC assuming a
linear relationship for the contributions of the ⌬1 bond and
isopropionate group. The values for 4-BNC-CoA and 1,4-BNC-
CoA were calculated from linear sums of the values for the
steroid and CoA components. The extinction coefficients were
validated in an oxygraph assay as described below.
Modeling of Substrates into KshA Crystal Structure—Models
of 1,4-BNC and 1,4-BNC-CoA were generated using PRODRG
(29) and were docked to KshA (PDB 2ZYL) (15) with AutoDock
Vina (30) using default parameters. A grid of 20 ϫ 24 ϫ 20 Å
centered about the active site was defined as the searchable area
for placement of the ligand. The top 10 docked ligand confor-
mations were output, and the one with the lowest binding
energy was chosen as the best structure.
RESULTS
Production and Purification of Steroid CoA Thioesters—Two
Steady-state Kinetics—Steady-state kinetic analysis was per- potential CoA thioester side chain degradation intermediates
formed for KshA using an oxygraph system as described previ- were generated to test the hypothesis that such compounds are
ously (15). Activity assays were performed at 22 °C in a total the physiological substrates of KshAB. To generate the CoA
volume of 1 ml of air-saturated 0.1 M potassium phosphate, pH thioesters, two acyl-CoA synthetases, CasI and CasG, from
7.0, containing 430 ␮M NADH, 1.1 ␮M KshB, and 0.2 ␮M KshA. R. jostii RHA1 were tested for their ability to esterify 4-BNC.
The reaction was initiated by adding substrate after equilibra-
Transformation of 4-BNC and CoASH in the presence of
tion of all other components for 3 min. The apparent steady- ATP and Mg2ϩ by CasI resulted in the time-dependent deple-
state kinetic parameters for O2 were measured in the presence tion of all substrate peaks, and the formation of two new peaks
of 57 ␮M 1,4-BNC-CoA. Kinetic parameters were evaluated by resolved by HPLC using Gradient A (Fig. 3). The first product
fitting the Michaelis-Menten equation to the data using the peak had the same retention time (2.8 min) and spectrum as an
least-squares fitting and dynamic weighting options of AMP standard. The second product peak eluted at 16.7 min and
LEONORA (28).
had a spectrum with features characteristic of both the steroid
Measuring KstD Reaction—Reactions were performed at and CoA components. This product was purified and further
22 °C in a 1-ml volume containing 200 ␮M 4-AD or 4-BNC- characterized. Alkaline hydrolysis yielded 4-BNC and CoASH
ϩ
CoA, 400 ␮M NAD , and 0.04 mg (total protein) KstD-lysate in as determined by HPLC. The product identity of 4-BNC-CoA
40720 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

CoA Thioesters in Mtb Cholesterol Catabolism
was confirmed by electrospray ionization-MS (supplemental
Fig. 1A). Under the described reaction conditions, the product
yield was typically 75%. By contrast, CasG did not detectably
catalyze the thioesterification of 4-BNC.
To generate 1,4-BNC-CoA, purified 4-BNC-CoA was incu-
bated with lysate from cells containing KstD of Mtb. KstD has
previously been shown to catalyze the ⌬1-dehydrogenation of
4-AD,6 5␣-androstane-3,17-dione, 5␣-testosterone, and pro-
gesterone (31). Monitoring of the reaction by HPLC revealed a
decrease in the 4-BNC-CoA peak and appearance of a new peak
eluting at 18 min of Gradient B. This compound was analyzed
as described above and identified as 1,4-BNC-CoA (supple-
mental Fig. 1B). Transformation of 4-BNC-CoA to 1,4-BNC-
CoA by incubation with KstD-lysate resulted in 87% conversion
after 2 h. A greater total yield of 1,4-BNC-CoA could be
ϩ
obtained by adding KstD-lysate and NAD directly to the CasI
reaction mixture after 3 h, omitting the intermediate purifica-
tion step. Transformation of 4-BNC to 1,4-BNC by incubation
with KstD-lysate using a similar procedure resulted in a Ͼ90%
turnover after 23 h. The product identity was confirmed by
GC-MS analysis (supplemental Fig. 1C).
FIGURE 4. Steady-state kinetic analyses of KshAB with CoA thioesters.
Shown is the dependence of the initial velocity of O2 consumption on 1,4-
BNC-CoA (A) and 4-BNC-CoA (B) concentrations in air-saturated buffer with
fitted parameters Km ϭ 17 Ϯ 3 and 6.8 Ϯ 0.1 ␮M, and Vmax ϭ 0.56 Ϯ 0.04 and
0.146 Ϯ 0.007 ␮M sϪ1, respectively. The best fit of the Michaelis-Menten equa-
tion to the data using the least squares dynamic weighting options of
LEONORA is represented as a solid line.
KshAB Transforms CoA Thioester Steroids—Consistent with
KshAB of Mtb catalyzing the 9␣-hydroxylation of 4-AD and
ADD (15), each of 4-BNC, 1,4-BNC, 4-BNC-CoA, and 1,4-
BNC-CoA stimulated the depletion of oxygen in the presence
of KshA, KshB, and NADH in the standard oxygen electrode
activity assay. For each steroid substrate, the amount of oxygen
consumed corresponded with the calculated amount of sub-
strate added. These data are consistent with full coupling
between oxygen and steroid substrate consumption and indi-
cate that the extinction coefficients calculated for each of 1,4-
BNC, 4-BNC-CoA, and 1,4-BNC-CoA are accurate.
TABLE 1
Apparent steady-state kinetic parameters of KshAB for various
steroids
Experiments were performed using 0.1 M potassium phosphate, pH 7.0, at 22 °C.
Values reported in parentheses represent S.E.
Substrate
Km
␮M
kcat
s
0.07 (0.01)
0.80 (0.05)
0.08 (0.01)
0.25 (0.02)
0.61 (0.03)
2.7 (0.2)
K
cat/Km
Ϫ1
Ϫ1
Ϫ1s
4-ADa
24 (16)
110 (20)
3 (2)
3,000M(2,000)
ADDa
7,600 (700)
4-BNC
30,000 (10,000)
3,500 (400)
1,4-BNC
70 (10)
6.8 (0.1)
17 (3)
To confirm that KshAB catalyzes the 9␣-hydroxylation of the
thioester substrates, a reaction mixture of KshAB with 1,4-
BNC-CoA was examined by HPLC. The product peak had a
retention time (16.8 min using Gradient B) and a spectrum that
4-BNC-CoA
1,4-BNC-CoA
90,000 (10,000)
160,000 (20,000)
a Values presented in Capyk et al. (15).
was identical to those of 3-HSBNC-CoA.7 Moreover, alkaline cantly, the turnover numbers for the CoA thioester substrates
hydrolysis yielded 3-HSBNC and CoASH, as determined by were approximately nine and three times greater than for the
GC-MS (supplemental Fig. 1D) and HPLC analyses, confirming corresponding 17-keto steroids 4-AD and ADD, respectively.
the product identity.
The Km values for 4-BNC-CoA and 1,4-BNC-CoA were also 3.5
Steady-state Kinetic Parameters for KshAB—The specificity and 6.5 times less than those for their 17-keto derivatives.
of KshAB for each of the substrates discussed above was inves-
The reactivity of KshAB with O2 was investigated in the pres-
tigated using the oxygen electrode assay at ambient oxygen ence of 1,4-BNC-CoA. In the presence of 57 ␮M 1,4-BNC-CoA,
concentration (Fig. 4) (Table 1). Under these conditions, the the apparent steady-state kinetic parameters for O2 were Km ϭ
apparent specificity constants were 1,4-BNC-CoA Ͼ 4-BNC- 90 Ϯ 10 ␮M, kcat ϭ 2.5 Ϯ 0.1 sϪ1, and kcat/Km ϭ 29,000 Ϯ 2,000
Ϫ1
CoA Ͼ 4-BNC Ͼ 1,4-BNC. Significantly, apparent kcat/Km val-
M
Ϫ1 s
.
ues for all compounds except 1,4-BNC were greater than for
To further probe the role of the CoA moiety in KshAB reac-
either previously proposed 17-keto substrates, 4-AD and ADD tivity, we tested the ability of CoASH to inhibit enzymatic turn-
Ϫ1
(15). In fact, the value for 1,4-BNC-CoA (160,000 Ϯ 20,000 M
over of either ADD or 1,4-BNC-CoA. These assays were carried
s
Ϫ1) is more than 20 times greater than that for ADD, the pre- out under the conditions of the standard assay with the steroid
viously proposed physiological substrate.
substrates at concentrations corresponding to their respective
The turnover numbers were consistently greater for sub- Km values. No inhibition was observed at CoASH concentra-
strates with ⌬1,4 desaturated A rings: kcat ϭ 0.25 Ϯ 0.03 sϪ1 for tions up to 10-fold greater than substrate concentration (results
1,4-BNC versus 0.08 Ϯ 0.01 sϪ1 for 4-BNC and 2.7 Ϯ 0.2 sϪ1 for not shown).
1,4-BNC-CoA versus 0.61 Ϯ 0.03 sϪ1 for 4-BNC-CoA. Signifi-
Reaction of KstD with 4-AD and 4-BNC-CoA—To explore
further the possibility that CoA thioesters are the relevant sub-
strates for cholesterol ring catabolic enzymes in Mtb, we inves-
tigated the reaction rates of KstD with each of 4-AD and
6 J. K. Capyk and L. D. Eltis, unpublished observations.
7 I. Casabon and L. D. Eltis, unpublished observations.
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
JOURNAL OF BIOLOGICAL CHEMISTRY 40721

CoA Thioesters in Mtb Cholesterol Catabolism
FIGURE 5. Docking of 1,4-BNC-CoA in active site of KshA. 1,4-BNC-CoA is shown as a ball and stick representation. KshA (PDB ID 2ZYL (15)) is shown as a green,
semitransparent surface. KshA amino acids within 4 Å of the CoA group of 1,4-BNC-CoA are shown as sticks and labeled. Carbon atoms of amino acid residues
and the substrate are colored green and yellow, respectively. Oxygen, phosphorus, and nitrogen atoms are shown in red, orange, and blue, respectively. The
catalytic iron is shown as a rust-colored sphere. Residues mentioned in the text are labeled.
4-BNC-CoA (supplemental Fig. 2). Each compound was incu- 9␣-hydroxylation. The CoA moiety adopted more than one
bated with 40 ␮g (total protein) of KstD-lysate, and reaction conformation in different simulation experiments, all of which
progress was followed by HPLC analysis at various time points. had similar binding energies. These conformations show signif-
After 2 h, 87% of 4-BNC-CoA had been converted to 1,4-BNC- icantly different potential binding positions for CoA at the
CoA, whereas only 30% of 4-AD had been turned over. Com- mouth of the KshA active site channel. The conformation with
plete conversion was not observed over extended periods with- the lowest binding energy is shown.
out the addition of fresh enzyme, suggesting that there is a
The docked model shows the CoA group fitting snuggly into
time-dependent decrease in enzyme activity. Under these con- a pocket at the mouth of the active site channel. It also shows
ditions, the specific activity of 1,4-BNC-CoA was estimated at several predicted polar contacts including between the adenine
Ϫ1
195 ␮M mgϪ1 minϪ1, whereas that for 4-AD was 25 ␮M mg
moiety, Ser251, and the carboxylate of Tyr249; the ribose
minϪ1. Similar results were obtained using each of three differ- hydroxyl group and Asn244; the ribose phosphate, Ser253, and
ent concentrations of KstD-lysate.
Asn244; the phosphate proximal to the steroid group, Asp216
,
Modeling of 1,4-BNC-CoA in Active Site of KshA—Attempts and Tyr246. Most of the above-mentioned residues are not con-
to produce high quality KshA crystals with bound substrate by served in KshAs from Rhodococcus erythropolis SQ1, Rhodococ-
either co-crystallization or soaking experiments were not suc- cus rhodochrous DSM43269, and R. jostii RHA1, all of which are
cessful. To investigate the likely spatial implications of KshA predicted to be involved in steroid catabolism (20, 32, 33). How-
CoA thioester substrates, docking simulations were performed ever, the substrate specificities of the rhodococcal enzymes
using the published crystal structure of KshA (PDB ID 2ZYL) have not been defined, and it is likely that many do not act on
(15) and a structural model of 1,4-BNC-CoA.
CoA thioester substrates.
In a previous experiment ADD docked to KshA in two ori-
entations within the enzyme active site. One of these orienta-
tions was conducive to 9␣-hydroxylation of the molecule, albeit
DISCUSSION
The results we present suggest that steroid CoA thioesters
orienting the C17 ketone group toward the interior of the are the likely physiological substrates for Mtb KshAB. The
enzyme rather than pointing out of the active site channel (15). steady-state kinetic analyses demonstrate that KshAB trans-
Initial docking experiments of 1,4-BNC revealed a positioning forms the CoA thioesters more efficiently than 17-keto ste-
of the substrate that is inconsistent with the known outcome of roids, the previously proposed physiological substrates (15).
the reaction; C5 and C6 of ring A, rather than C9, were posi- This finding is especially dramatic in the enzyme reactivity with
tioned close to the catalytic iron. This orientation had a calcu- oxygen. The kcat/KmO in the presence of 1,4-BNC-CoA is an
2
lated binding energy of Ϫ8.6 kcal molϪ1. By contrast, simula- order of magnitude greater than that measured in the presence
tions using 1,4-BNC-CoA resulted in a single, well defined of ADD (15). Perhaps more significantly, the KmO of 90 Ϯ 10
2
conformation of the steroid moiety with a docking energy of ␮M is similar to that of other oxygenases in the cholesterol deg-
Ϫ11.1 kcal molϪ1 (Fig. 5). This conformation was conducive to radation pathway (13, 14) as well as in the lung tissues of healthy
40722 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 47•NOVEMBER 25, 2011

CoA Thioesters in Mtb Cholesterol Catabolism
rabbits (34). This is compared with the value in excess of 1.2 mM probable steroid molecules produced during cholesterol degra-
previously measured in the presence of ADD (15). The apparent dation in Mtb as well as for the design of small molecule inhib-
specificity constants for steroid metabolites were measured in itors for the cholesterol ring-degrading enzymes. KshAB has
air-equilibrated buffer, so the high KmO for the keto-substi- been implicated in the management of potentially immuno-
tuted steroid means O2 was well below sat2urating levels. In con- modulatory steroids suggested to play a role in early infection
trast, the lower KmO in the presence of a CoA thioester sub- (17), and our results broaden the range of potential compounds
2
strate means that in the presence of the latter, KshAB is closer in this category. Additionally, these results expand the potential
to being saturated with O2. Thus, the increased reactivity of substrate range and industrial potential of steroid ring-degrad-
KshAB for O2 in the presence of the CoA thioester substrates ing enzymes from other species. It remains unclear what degree
could account for the greater apparent substrate specificity of side chain degradation is completed before the activity of
compared with the 17-keto substrates.
each ring-degrading enzyme in vivo or whether physiologically
The steady-state kinetic parameters reveal that the turnover relevant parallel pathways exist. This study, in combination
numbers and Km values for 4-BNC-CoA and 1,4-BNC-CoA are with previous investigations (15, 17), strongly suggests the
significantly different but that the specificity constants for the existence of more than one functional pathway in Mtb.
two compounds differ by less than 2-fold. The kinetic differ-
The rhodococcal acyl-CoA synthetases provide useful tools
ences between the ⌬4 and ⌬1,4 thioesters mirror what was found for further study of the cholesterol degradation pathway in
previously for 4-AD and ADD (15). This trend of greater kcat Mtb. None of the FadDs in the Mtb cholesterol degradation
and Km for ⌬1,4 compounds than for their ⌬4 counterparts is cluster (fadD17, Rv3506; fadD19, Rv3515c; fadD3, Rv3561) was
also evident in the comparison of 4-BNC and 1,4-BNC notwith- able to catalyze CoA esterification of 4-BNC. This result is not
standing the relatively low specificity constant for the latter. unexpected as the proposed side chain degradation pathway is
The mechanistic determinants for the KshAB low turnover predicted to involve a single acyl-CoA synthetase activity that
number for 1,4-BNC compared with other ⌬1,4 substrates are forms the C26 CoA thioester; further CoA groups are incorpo-
not clear from the available data. The relative substrate speci- rated by thiolase enzymes. Although the physiological sub-
ficities of KshAB for ⌬4 and ⌬1,4 compounds appear to differ strates for R. jostii RHA1 CasI and CasG have not been deter-
between species, with KshAB from Mycobacterium smegmatis mined, the ability of enzymes from R. jostii RHA1 to transform
exhibiting a much lower capacity to transform ⌬4 steroids than steroids of intermediate side chain length coupled with the
Mtb KshAB, based on metabolite accumulation studies in methodology we developed to purify and manipulate these
⌬kstD knock-out cultures (18, 19).
compounds could be exploited for research in a variety of con-
This study suggests 4-BNC-CoA and 1,4-BNC-CoA as can- texts. Such compounds can be used to study potential choles-
didate physiological substrates for KshAB but does not address terol catabolic intermediates as substrates for a range of
the possibility of other CoA thioester substrates. Our molecular enzymes in Mtb and other bacteria and as standards to interro-
docking experiment demonstrates that there is room for this gate the occurrence of bacterial and mammalian in vivo
large molecule in the enzyme active site channel and that favor- metabolites.
able electrostatic interactions with the CoA moiety may exist
Our knowledge of the Mtb cholesterol degradation pathway
and could contribute to KshA substrate specificity. Nonethe- comprises a list of catabolic “limits” defining the biochemical
less, KshAB inhibition by CoASH was not observed, suggesting capacities of specific enzymes rather than their physiological
the significantly greater apparent specificity constants for the roles. Both the in vitro nature of experiments exploring this
CoA thioester substrates is not solely attributable to KshA pathway and a level of facultative promiscuity suggested by cur-
affinity for the CoA moiety. The side chain degradation path- rent data for some of the biochemically investigated enzymes
way proposed for Mtb (11) includes CoA thioester steroids with contribute to the limitations inherent in our understanding of
8-carbon and 5-carbon side chains in addition to the 3-carbon the pathway. Thus, it is established that 3␤-hydroxysteroid
isopropionyl-CoA thioester steroids we tested. Additionally, dehydrogenase (3␤-HSD) but not KstD can transform choles-
compounds with oxidized and hydrated side chains are pro- terol before Cyp125 catalyzes the first side chain reaction, and
duced during each round of ␤-oxidation. Further studies are that Cyp125 is capable of transforming cholesterol or 4-cho-
needed to determine whether KshAB preferentially hydroxy- lesten-3-one (21, 22, 36). Likewise, side chain degradation can
lates a particular thioester.
proceed to its conclusion before the action of either KstD or
A major implication of CoA thioesters as physiological sub- KshA (11), but both of these enzymes can transform interme-
strates for KshAB is concurrent cholesterol side chain and ring diates of side chain degradation.
degradation in Mtb. All previous biochemical experiments on
The current view of the metabolites occurring in the choles-
purified Mtb cholesterol ring-degrading enzymes have used terol degradation pathway derives from in vitro or gene knock-
compounds representing end products of side chain degrada- out studies that do not faithfully recapitulate the physiological
tion (13–15, 35). The CoA thioesters examined in this study conditions for Mtb in the human host. Indeed, even wild type
represent hypothetical intermediates in cholesterol side chain Mtb grown under laboratory conditions cannot be expected to
degradation, suggesting that the true physiological substrates be representative of a naturally occurring physiological state.
for some of these enzymes may also be side chain degradation This discrepancy in growth conditions may explain why 4-AD
intermediates, and countering the current paradigm that cho- and ADD were observed to accumulate in Mtb culture super-
lesterol side chain degradation in Mtb is completed before natants (11), whereas our results suggest that the KstD and
breakdown of the ring structure. This has implications for the KshA reactions cleaving ring B likely take place before forma-
NOVEMBER 25, 2011•VOLUME 286•NUMBER 47
JOURNAL OF BIOLOGICAL CHEMISTRY 40723

CoA Thioesters in Mtb Cholesterol Catabolism
15. Capyk, J. K., D’Angelo, I., Strynadka, N. C., and Eltis, L. D. (2009) J. Biol.
Chem. 284, 9937–9946
tion of the 17-ketone. Similarly, a previous study was not able to
detect the transformation of 4-AD by KstD (31), whereas we are
able to measure a reaction rate. Although the substrate speci-
ficities of purified enzymes are suggestive of physiologically rel-
evant reactions, the reaction conditions used for these assays
are far removed from those in the bacterial cytoplasm. Full
understanding of the cholesterol catabolites in Mtb, therefore,
requires us to probe these compounds in Mtb growing under
conditions representative of human infection.
16. Chang, J. C., Harik, N. S., Liao, R. P., and Sherman, D. R. (2007) J. Infect Dis.
196, 788–795
17. Hu, Y., van der Geize, R., Besra, G. S., Gurcha, S. S., Liu, A., Rohde, M.,
Singh, M., and Coates, A. (2010) Mol. Microbiol. 75, 107–121
18. Brzostek, A., Sliwiński, T., Rumijowska-Galewicz, A., Korycka-Machała,
M., and Dziadek, J. (2005) Microbiology 151, 2393–2402
19. Brzostek, A., Pawelczyk, J., Rumijowska-Galewicz, A., Dziadek, B., and
Dziadek, J. (2009) J. Bacteriol. 191, 6584–6591
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Acknowledgments—We thank Drs. Robert van der Geize and
Maarten Wilbrink for providing the construct for the casI and casG
genes. Jie Liu cloned and expressed kstD from H37Rv. We thank Drs.
Katherine Yam and Carola Dresen for helpful discussions.
21. Ouellet, H., Guan, S., Johnston, J. B., Chow, E. D., Kells, P. M., Burlingame,
A. L., Cox, J. S., Podust, L. M., and de Montellano, P. R. (2010) Mol.
Microbiol. 77, 730–742
22. Capyk, J. K., Kalscheuer, R., Stewart, G. R., Liu, J., Kwon, H., Zhao, R.,
Okamoto, S., Jacobs, W. R., Jr., Eltis, L. D., and Mohn, W. W. (2009) J. Biol.
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