A flavin-dependent monooxygenase from Mycobacterium tuberculosis involved in cholesterol catabolism

Article abstract of DOI:10.1074/jbc.M109.099028

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17- one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_cat/K_m = 1000 ± 100 M ^-1 s^-1 versus 700 ± 100 M^-1 s ^-1). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_cat/K_m = 80 ± 40 M^-1 s ^-1). In the presence of 3-HSA the K_mapp for O₂ was 100 ± 10 μM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val³⁶⁷-Val³⁹⁴) could adopt two conformations differing by a rigid body rotation of 25° around Arg ³⁶⁶. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.

Full text of DOI:10.1074/jbc.M109.099028

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 29, pp. 22264–22275, July 16, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
A Flavin-dependent Monooxygenase from Mycobacterium
□
S
tuberculosis Involved in Cholesterol Catabolism*
Received for publication, December 24, 2009, and in revised form, April 6, 2010 Published, JBC Papers in Press, May 6, 2010, DOI 10.1074/jbc.M109.099028
Carola Dresen‡1,2, Leo Y.-C. Lin§1,3, Igor D’Angelo§3, Elitza I. Tocheva‡¶, Natalie Strynadka§4, and Lindsay D. Eltis‡§5
From the Departments of ‡Microbiology and Immunology and §Biochemistry and Molecular Biology, University of British
Columbia, Vancouver V6T 1Z3, Canada and the ¶California Institute of Technology, Pasadena, California 91125
Mycobacterium tuberculosis (Mtb),6 the most devastating
infectious agent of mortality worldwide (1), remains a primary
public health threat due to synergy with the human immuno-
deficiency virus and the emergence of extensively drug-resis-
tant strains. Mtb has the unusual ability to survive and replicate
in the hostile environment of the macrophage (2, 3) utilizing
largely unknown mechanisms. Genes essential to intracellular
survival have been identified using genome-wide mutagenesis
(4). A cluster of such genes was subsequently predicted to spec-
ify cholesterol catabolism (5). Targeted gene deletion studies
subsequently indicated that cholesterol catabolism occurs
throughout infection, contributing to the virulence of Mtb
(6–8). Although the exact role of cholesterol in infection
remains unclear, the gene deletion studies are consistent with
the high concentrations of cholesterol found within caseating
granulomas and the observed congregation of bacteria around
cholesterol foci (9).
Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii
RHA1 have similar cholesterol catabolic pathways. This pathway
contributes to the pathogenicity of Mtb. The hsaAB cholesterol
catabolic genes have been predicted to encode the oxygenase and
reductase, respectively, of a flavin-dependent mono-oxygenase
that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-
9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of
RHA1 did not grow on cholesterol but transformed the latter to
3-HSA and related metabolites in which each of the two keto
groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-
triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconan-
drost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-
9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase
(HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-
DHSA (apparent kcat/Km ؍ 1000 ؎ 100 M؊1 s؊1 versus 700 ؎ 100
M
؊1 s؊1). However, 3,9-DHSA was a poorer substrate than 3-hy-
droxybiphenyl (apparent kcat/Km ؍ 80 ؎ 40 M؊1 s؊1). In the pres-
ence of 3-HSA the Kmapp for O2 was 100 ؎ 10 ␮M. The crystal
The cholesterol catabolic pathway of Mtb is very similar to
that of other pathogenic and environmental actinomycetes
(10), as initially predicted in Rhodococcus jostii RHA1 (5). Side-
chain degradation is initiated by a cytochrome P450 and pro-
ceeds via a ␤-oxidative process (11). Degradation of the four-
ringed steroid nucleus is initiated by 3␤-hydroxysteroid
dehydrogenase (3␤-HSD), yielding 4-cholestene-3-one (12).
The extent to which side-chain and ring degradation occur
concurrently is unclear. Ring degradation involves the suc-
cessive actions of 3-ketosteroid-1⌬-dehydrogenase and
3-ketosteroid 9␣-hydroxylase, which result in the opening of
ring B with concomitant aromatization of ring A to yield a phe-
nol, 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione
(3-HSA) (13–17). The phenolic ring of the latter has been pre-
dicted to be hydroxylated by HsaAB to yield a catechol,
3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione
(3,4-DHSA) (Fig. 1) (5). The catechol is then subject to meta-
˚
structure of HsaA to 2.5-A resolution revealed that the enzyme
has the same fold, flavin-binding site, and catalytic residues as
p-hydroxyphenyl acetate hydroxylase. However, HsaA has a
much larger phenol-binding site, consistent with the enzyme’s
substrate specificity. In addition, a second crystal form of HsaA
revealed that a C-terminal flap (Val367–Val394) could adopt two
conformations differing by a rigid body rotation of 25° around
Arg366. This rotation appears to gate the likely flavin entrance to
the active site. In docking studies with 3-HSA and flavin, the
closed conformation provided a rationale for the enzyme’s sub-
strate specificity. Overall, the structural and functional data
establish the physiological role of HsaAB and provide a basis to
further investigate an important class of monooxygenases as
well as the bacterial catabolism of steroids.
* This work was supported by grants from the Canadian Institute for Health
Research (to L. D. E. and N. S., respectively) and the Michael Smith Founda-
tion for Health Research (MSFHR) Infrastructure (to N. S.) and Emerging 6 The abbreviations used are: Mtb, M. tuberculosis; HsaAB, 3-hydroxy-9,10-
Team programs.
seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase; 3-HSA, 3-hydroxy-
9,10-seconandrost-1,3,5(10)-triene-9,17-dione; 3,17-DHSA, 3,17-dihydroxy-
9,10-seconandrost-1,3,5(10)-triene-9-one; 3,9-DHSA, 3,9-dihydroxy-9,10-
seconandrost-1,3,5(10)-triene-17-one; 3,9,17-THSA, 3,9,17-trihydroxy-9,10-
seconandrost-1,3,5(10)-triene; 3,4-DHSA, 3,4-dihydroxy-9,10-seconand-
rost-1,3,5(10)-triene-9,17-dione; 3,4,17-THSA, 3,4,17-trihydroxy-9,10-seco-
nandrost-1,3,5(10)-triene-9-one; 3,4,9-THSA, 3,4,9-trihydroxy-9,10-seconan-
drost-1,3,5(10)-triene-17-one; 3-HB, 3-hydroxybiphenyl; TC-FDM, two-com-
ponent flavin-dependent monooxygenase; pHPAH, p-hydroxyphenyl acetate
hydroxylase from A. baumanii; FAD, flavin adenine dinucleotide; FMN, flavin
mononucleotide; HPLC, high-performance liquid chromatography; GC-MS,
gas chromatography/mass spectrometry; r.m.s.d., root mean square
The atomic coordinates and structure factors (codes 3AFE and 3AFF) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1–3, text, and references.
1 Both authors contributed equally to this work.
2 Recipient of
a
postdoctoral fellowship from the Deutsche
Forschungsgemeinschaft.
3 Recipients of a postdoctoral fellowship from MSFHR.
4 An HHMI International Scholar and an MSFHR Senior Scholar.
5 To whom correspondence should be addressed: 2350 Health Sciences Mall,
Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-0042; Fax: 604-822-6041;
E-mail: leltis@interchange.ubc.ca.
deviation;
BSTFA,
bis(trimethylsilyl)trifluoroacetamide;
TMCS,
trimethylchlorosilane.
22264 JOURNAL OF BIOLOGICAL CHEMISTRY
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HsaAB, a Steroid-degrading Flavin Monooxygenase
role in cholesterol catabolism and to
generate substrates. To substantiate
the role of the Mtb enzyme, each
component was heterologously pro-
duced, purified, and used to recon-
stitute the enzyme’s activity in vitro.
The enzyme’s specificity for a num-
ber of steroid metabolites was inves-
tigated. Finally, the crystal structure
of HsaA was determined and com-
pared with those of related oxygen-
ases. The results are discussed with
respect to TC-FDMs and the bacte-
rial catabolism of steroids.
MATERIALS AND METHODS
FIGURE 1. The role of HsaAB in the cholesterol catabolic pathway of actinomycetes. A, cholesterol is
Chemicals and Reagents—Chem-
icals were purchased from the
following suppliers: 3-hydroxybi-
phenyl (3-HB, IVA) from ASDI
(Newark, DE); 1,4-androstadien-
3,17-dione from Steraloids, Inc.
(Newport, RI); BSTFAϩTMCS
transformed to 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) via previous enzymatic
steps. HsaAB is proposed to catalyze the ortho hydroxylation of a phenol, 3-HSA, to a catechol, 3,4-dihydroxy-
9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). The latter is subsequently cleaved by HsaC to 4,5–
9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid (4,9-DSHA). B, other phenols that were
usedinthisstudy: a, 3,17-DHSA; b, 3,9-DHSA;and c, 3-hydroxybiphenyl(3-HB). ThesearetransformedbyHsaAB
to 3,4,17-THSA, 3,4,9-DHSA, and 2,3-dihydroxybiphenyl, respectively.
from Supelco Analytical (Bellefonte, PA); riboflavin and FMN
from Sigma; and FAD from MP Biomedicals Inc. (Solon, OH).
2,3-Dihydroxybiphenyl was a gift from Dr. Victor Snieckus.
Restriction enzymes and Expand High Fidelity PCR System
were purchased from New England Biolabs (Ipswich, MA) and
Roche Applied Science (Laval, Quebec), respectively. Oligonu-
cleotides were from Integrated DNA Technology. Crystalliza-
tion screening solutions were purchased from Hampton
Research, Emerald BioSystems, and Jena Bioscience. All other
reagents were of HPLC or analytical grade.
cleavage and C-C bond hydrolysis by HsaC (5, 6) and HsaD (18),
respectively. In Mtb and RHA1, the hsaACDB genes occur in a
single operon, and their respective products share 74–82%
amino acid sequence identity. In transposon mutagenesis stud-
ies, hsaA (3570c) was identified as one of 126 genes necessary
for survival of Mtb in macrophages under conditions that
model the immune response (4).
Flavin-dependent monooxygenases (FDMs) constitute an
important class of enzymes that utilize a reduced flavin to acti-
vate O2 and hydroxylate a variety of organic substrates. Two
broad classes are recognized based on whether the flavin is
reduced and used to hydroxylate the substrate at a single active
site (19) or whether flavin reduction and organic substrate
hydroxylation occur at different sites, in two separate polypep-
tides. In the latter, referred to as two-component (TC-) FDMs,
a reductase utilizes NADH to reduce the flavin, which is then
transferred to the oxygenase (20). The oxygenase generally has
a preference for FADH2 or FMNH2. Examples for the former
include 4-hydroxyphenyl acetate 3-monooxygenase (HpaB) of
Escherichia coli (21) and 2,4,5-trichlorophenol monooxygenase
Bacterial Strains and Culture Conditions—Escherichia coli
strains were grown in Luria-Bertani (LB) medium at 37 °C, 200
rpm. E. coli DH5␣ was used to propagate DNA. To produce
HsaB, E. coli GJ1158 (29) containing pT7HB was grown at 30 °C
in low salt LB medium supplemented with 100 ␮g mlϪ1 ampi-
cillin. Expression of hsaB was induced when the culture
attained an A600 of 0.6 by adding NaCl to a final concentration
of 0.3 M. Cells were further incubated for 16 h, harvested by
centrifugation, washed twice with 20 mM sodium phosphate
(pH 8.0) containing 10% glycerol, then frozen at Ϫ80 °C until
from Burkholderia cepacia (22). FMN-dependent systems are use. For metabolite analyses, R. jostii RHA1 ⌬hsaA mutant,
involved in the synthesis of antibiotics such as actinorhodin in RHA017 (30), was grown in W minimal medium (31) supple-
Streptomyces coelicolor (23), the bacterial degradation of poly- mented with 0.5 mM cholesterol as described previously (6). To
aminocarboxylates such as nitrilotriacetic acid (24), and the produce HsaA, RHA017 containing pTipHA was grown at
desulfurization of fossil fuels by rhodococci (25). pHPAH (EC 30 °C in LB supplemented with 25 ␮g/ml chloramphenicol.
1.14.13.3), a well characterized FMN-utilizing enzyme from Expression of hsaA was induced when the culture attained an
Acinetobacter baumanii, catalyzes the hydroxylation of p-hy-
A600 of 0.8 by adding thiostrepton to a final concentration of 50
droxyphenyl acetate (HPA) to 3,4-dihydroxyphenyl acetate ␮M. Cells were further incubated for 21 h prior to harvesting
(PDB entry 2JBT) (26). The reductase component, C1, requires and freezing as described above.
HPA for effective flavin reduction (27), and the oxygenase com-
Heterologous Expression of HsaAB—DNA was propagated,
ponent, C2, binds FMNH2 prior to HPA (28). HsaAB has been digested, ligated, and transformed using standard protocols
predicted to be a TC-FDM based on the ϳ32% amino acid (32). Plasmids were electroporated into RHA017 (30) and
sequence identity that it shares with C2 and C1 of pHPAH.
E. coli using a MicroPulser from Bio-Rad (Hercules, CA) with
Herein we describe the characterization of HsaAB of Mtb. A Bio-Rad 0.1-cm GenePulser Cuvettes. The hsaA (Rv3570c) and
⌬hsaA mutant of RHA1 was used to investigate the enzyme’s hsaB (Rv3567c) genes were amplified from Mtb H37Rv
JULY 16, 2010•VOLUME 285•NUMBER 29
JOURNAL OF BIOLOGICAL CHEMISTRY 22265

HsaAB, a Steroid-degrading Flavin Monooxygenase
genomic DNA by PCR using the Expand High Fidelity and phate, pH 8.0, containing 175 mM imidazole. The protein was
the following three-phase thermo-cycler program: 95 °C for exchanged into 20 mM HEPES, pH 7.5, 2 mM dithiothreitol, and
5 min; 25 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 10% glycerol via ultrafiltration, concentrated to ϳ20 mg/ml,
1.5 min; and a final phase at 72 °C for 7 min. The hsaA gene and flash frozen as beads in liquid N2.
was amplified using a forward primer (GCAATAGCATATG-
HsaB Was Purified Using Essentially the Same Protocol with
CATCACCATCACCATCACATCGAAGGTAGGACGTCC- the Following Modifications—E. coli cells containing pT7HB
ATTCAACACGTGATG), which introduced an NdeI site, six from 1 liter of culture were used as the starting material; cells
codons for histidine, and a Factor Xa recognition site, and a were disrupted using an Emulsiflex-05 homogenizer (Avestin,
reverse primer (GCCAAGCTTGGTAGCTGTCATTTCG- Ottawa, Ontario, Canada) operated at 10,000 p.s.i.; the manip-
CCTAGACC), which introduced a HindIII site. The ampli- ulations were performed aerobically. The yellow-colored
con was first cloned into pET41bϩ (Novogen, Oakville, protein was exchanged into 25 mM HEPES, pH 7.5, contain-
Ontario) as an NdeI/HindIII fragment, its nucleotide ing 5% glycerol, concentrated to ϳ20 mg/ml, and flash fro-
sequence was verified, and then subcloned in pTipQC2 (33) zen in liquid N2.
using the same restriction sites, yielding pTipHA. The hsaB
Identification of HsaB-bound Flavin—HsaB (20 ␮l, 86 ␮M)
gene was amplified using oligonucleotides (CTAGCTAGCGC- was diluted to 500 ␮l using 20 mM potassium phosphate, pH 7.0,
TCAGATCGATCCACGCACG and GCCAAGCTTCGTCT- prior to denaturation by heat. The mixture was incubated at
GCTCGCGCCACTAG) that introduced NheI and HindIII 100 °C for 3 min, rapidly cooled on ice, and centrifuged at
sites flanking the gene. The amplicon was cloned in pT7HP20 10,000 ϫ g for 30 min at 4 °C. The supernatant was passed
(34) as an NheI/HindIII fragment to yield pT7HB, from which through a 0.2-␮m filter and analyzed via HPLC using the
HsaB is produced as an N-terminally His-tagged protein, Ht- method of Faeder and Siegel (35). Solutions of riboflavin, FMN,
HsaB. The nucleotide sequences of cloned amplicons were con- and FAD were used as standards.
firmed by the Nucleic Acid Protein Service at University of
British Columbia.
Reconstitution of HsaB—Flavin-free HsaB was produced
anaerobically in the glove box by incubating a 500-␮l solution of
Metabolite Analysis—Production of metabolites in culture 86 ␮M HsaB and 10 mM NADH in 20 mM HEPES, pH 7.6, for 5
supernatant was monitored using GC-MS as described below. min, then immediately passing the sample over a 0.5- ϫ 5-cm
Samples were analyzed directly or after BSTFAϩTMCS- (99:1) column of Biogel P6 (Bio-Rad) equilibrated with 20 mM HEPES,
derivatization. When the metabolite concentration was judged pH 7.6. HsaB was reconstituted by aerobically incubating the
to be maximal, the culture supernatant was collected by centri- apoenzyme with 2 mM flavin for 1 h. Excess flavin was removed
fugation and then extracted twice with one volume of ethyl via ultrafiltration.
acetate. The ethyl acetate fractions were pooled, dried with
anhydrous magnesium sulfate, and evaporated to dryness with
Analytical Methods—TLC was performed using silica gel 60
F
254 (Merck). Compounds were detected at 254 nm after first
a rotary evaporator. Metabolites were purified by column chro- spraying plates with 50% sulfuric acid in ethanol and heating
matography (SiO2, n-hexane/ethyl acetate ϭ 2:3) and evapo- them for 5 min at 110 °C. Preparative column chromatography
rated to dryness. To identify reaction products, substrates were was carried out on silica gel 60 (mesh size 40–63 ␮m). NMR
dissolved to 205 ␮M in 220 ␮l of 20 mM sodium phosphate, pH spectra were recorded using a Varian Unity 500-MHz and an
7.0, containing 25.8 ␮M HsaA, 3 ␮M HsaBꢀFMN, and 1 mM Inova 600-MHz (Varian Inc., Mississauga, Ontario, Canada)
NADH. This mixture was incubated at 25 °C for 30 min then spectrometer. Chemical shifts (␦) are reported in ppm relative
extracted with 400 ␮l of ethyl acetate and either directly ana- to CHCl3 (1H: ␦ ϭ 7.24) and CDCl3 (13C: ␦ ϭ 77.0) as internal
lyzed via GC-MS or first dried under N2 and derivatized using standards. GC-MS was carried out using an HP 6890 series GC
BSTFAϩTMCS (99:1).
system fitted with an HP 5973 mass-selective detector and a
Protein Purification—To purify HsaA, the pellet of RHA017 30-m ϫ 250-␮m HP-5MS Agilent column. The operating con-
cells containing pTipHA obtained from 4 liters of culture was ditions were: TGC (injector), 280 °C; TMS (ion source), 230 °C;
resuspended in 40 ml of 20 mM HEPES (pH 7.5) containing 1 oven time program (T0 min), 104 °C; T2 min, 104 °C; T14.4 min
,
mM dithiothreitol at 4 °C. The cells were disrupted using 30 g of 290 °C (heating rate 15 °CꢀminϪ1); and T29.4 min, 290 °C. HPLC
0.1-mm zirconia/silica beads in a bead beater (Biospec Prod- analysis were performed using a Waters 2695 Separations
ucts, Bartlesville, OK) for five cycles of 2-min beating and HPLC module equipped with a Waters 2996 photodiode array
10-min incubation on ice. The chamber of the bead beater was detector and a 250 ϫ 4.60 mm C18 Prodigy 10␮ ODS-Prep
immersed in ice water throughout the whole procedure after column (Phenominex, Torrance, CA). SDS-PAGE was per-
which the cell suspension was kept under N2 and centrifuged formed using a Bio-Rad MiniPROTEAN III apparatus with a
(40,000 ϫ g, 45 min, 4 °C). HsaA was purified in a Labmaster 10% or 12% resolving gel. Gels were stained with Coomassie
Model 100 glove box (M. Braun, Inc., Peabody, MA) operated at Blue using standard protocols. Protein concentrations were
Ͻ5 ppm O2. The supernatant was passed through a 0.45-␮m determined using the Micro BCATM protein assay kit (Pierce)
filter (Sartorius AG, Go¨ttingen, Germany) and loaded onto and the Bradford method (36) using bovine serum albumin as a
nickel-nitrilotriacetic acid-agarose (Qiagen) column (1.8 cm ϫ standard.
5 cm) equilibrated with 20 mM sodium phosphate, pH 8.0, con-
Enzyme Activity—HsaAB activity was measured spectropho-
taining 10% glycerol. The purification was performed using an tometrically by following the hydroxylation of 3-HB in a cou-
imidazole step gradient according to the instructions of the pled assay with BphC (37) at 25 Ϯ 0.5 °C. Reactions were per-
manufacturer. HsaA was eluted using 20 mM sodium phos- formed in 125 ␮l of 20 mM potassium phosphate, pH 7.0,
22266 JOURNAL OF BIOLOGICAL CHEMISTRY
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HsaAB, a Steroid-degrading Flavin Monooxygenase
containing 1.6 mM 3-HB, 2.8 ␮M BphC, 4 ␮M HsaBꢀFMN, and glycol (5–20%) and flash frozen in liquid N2. In the second,
10.2 ␮M HsaA. Components were incubated for 30 s, then the crystals of protein purified anaerobically from RHA1 were
reaction was initiated by adding NADH to a final concentration grown anaerobically at 23 °C in the above-described glove box
of 600 ␮M and was monitored at 434 nm, the absorbance using the sitting drop, vapor-diffusion method and anaerobi-
maximum of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid cally purified HsaA. 1 ␮l of 11 mg/ml protein was mixed with 1
(⑀434 ϭ 16.9 mMϪ1 cmϪ1). Stock solutions were prepared fresh ␮l of crystallization solution (0.1 M Tris-HCl, pH 7.0, 20% (w/v)
daily. Absorbance was monitored using a Varian Cary 5000 polyethylene glycol-2000 MME). Thin plates were obtained
spectrophotometer controlled by WinUV software. One unit of within 3 days. Crystals were harvested directly from the mother
activity was defined as the amount of enzyme required to con- liquor and were flash frozen in liquid nitrogen in the anaerobic
sume 1 ␮mol of substrate per minute.
chamber prior to data collection.
Steady-state kinetic parameters were evaluated using stan-
X-ray Data Collection and Structure Determination—X-ray
dard conditions except that for 3-HSA and 3,17-DHSA, 11.7 data were collected under cryogenic conditions using a rotating
␮M HsaC was used instead of BphC. Substrate concentrations anode x-ray generator (CuK␣ radiation ␭ ϭ 1.542 Å) and syn-
were varied over the following ranges: 90–1600 ␮M for 3-HB chrotron radiation at the Canadian Light Source (beamline
and 30–1000 ␮M for each of 3-HSA and 3,17-DHSA. The molar CMCF1, ␭ ϭ 1.000 Å). Data were processed using XDS (39) or
absorptivities of 4,9-DSHA and 4,9-DSDHA were 2.41 and 2.40 HKL2000 (40). The initial model and phase information were
mMϪ1 cmϪ1, respectively, at 392 nm (pH 7.0). In these studies, obtained from aerobically grown crystals by molecular replace-
HsaA was thawed and stored under N2 in a sealed vial on ice. ment using the structure of substrate-free pHPAH (PDB entry
Aliquots were withdrawn using a gas-tight syringe. Kinetic 2JBS) and the program EPMR (41). Crystals grew in the space
parameters were evaluated by fitting the Michaelis-Menten group I222 with two copies of HsaA in the crystallographic
equation to the data using the least-squares fitting and dynamic asymmetric unit. Model building and structure refinement
weighting options of LEONORA (38).
were performed using CNS (42) and REFMAC (43). Anaerobi-
The steady-state kinetic parameters for O2 were determined cally, crystals grew in space group P21 with four molecules of
using a Clarke-type electrode (Hansatech Instruments Ltd., HsaA in the asymmetric unit. The structure was solved using
Norfolk, England) interfaced to a computer equipped with the the structure of aerobically grown HsaA as the starting model
Oxygraph Plus software. The reaction buffer was equilibrated and MOLREP (43), and was refined as described above.
for 20 min by vigorous bubbling with mixtures of N2 and O2
Data collection and refinement are summarized in Table 3.
prior to the reaction, and the reaction cuvette was continually Electron density maps were calculated using the CCP4 package
flushed with the same gas mixture during the reaction. The (44). Structural figures were made with the program PyMOL
assay was performed in 500 ␮l containing 660 ␮M 3-HSA, 0.2 (45). The coordinates for the two Mtb HsaA conformations
␮M HsaB, and 10.2 ␮M HsaA. The reaction was initiated by were deposited in the Brookhaven Protein Data bank with
adding NADH to 500 ␮M. After 60 s, 603 units of catalase was accession codes 3AFE and 3AFF.
added to the reaction mixture. The rate of O2 consumption
Molecular Docking Simulation and Modeling—3-HSA was
prior to catalase addition (vO2) was taken to represent the rate designed using PRODRG (46), energy was minimized using the
of substrate hydroxylation (vROH) plus the rate of adventitious Dreiding force field (47), and PM6 semi-empirical charges were
production of H2O2. The rate after catalase addition (vϩC) was calculated using MOPAC2007 (48). After removing water mol-
taken to represent vROH plus 50% of the rate of adventitious ecules from the HsaAO and HsaAC models, docking simula-
production of H2O2. Thus, vROH was calculated from 2vϩC Ϫ tions were initially performed using PATCHDOCK (49). Sig-
vO2
.
nificant clusters that placed the ligand molecule in the correct
Static Light Scattering—Purified HsaA in solution was ana- orientation with respect to the cofactor were picked and run
lyzed by the triple-angle light-scattering detector (Wyatt through Autodock version 4.0 (50) with active site residues held
MiniDAWN Tristar) connected on-line to a size-exclusion rigid and the initial torsion angles of 3-HSA randomly set.
chromatography column (Amersham Biosciences, Superdex
Solutions of the best free energy were minimized with strong
200 HR 10/30). The gel-filtration buffer contained 20 mM restraints (Force 200) until gradient norm convergence to 1.0,
HEPES, pH 7.5, 150 mM NaCl, and 10% glycerol (v/v). The over the entire system (heavy atoms only) with the exception of
molecular weights of HsaA fractions were calculated using the a sphere of 8 Å around the active site. Residues within this
ASTRA software (Wyatt Technology).
sphere were unrestrained to allow for minor side-chain re-ori-
Crystallization—HsaA was crystallized under two condi- entations. The cofactor was kept rigid during all calculations
tions. In the first, crystals of protein purified aerobically from and was positioned based on the closely similar binding site of
E. coli were grown aerobically at 18 °C using the hanging drop, pHPAH. Structural figures and graphics were rendered using
vapor-diffusion method using aerobically purified HsaA. 1 ␮l of PyMOL (45).
20–25 mg/ml protein was mixed with 1 ␮l of crystallization
RESULTS
solution (2 M sodium formate, 0.1 M sodium acetate, pH 4.6).
Single flattened rod-like crystals appeared in ϳ10 days and
Phenotype of the hsaA Deletion Mutant—Bioinformatic anal-
grew to their full size (250 ␮m ϫ 80 ␮m ϫ 50 ␮m) in ϳ2 weeks. yses indicate that HsaAB of the actinomycete cholesterol cata-
Prior to data collection HsaA crystals were briefly transferred to bolic pathway is a TC-FDM that transforms 3-HSA to
a series of solutions containing 80% of the equilibrated reservoir 3,4-DHSA (5). To test this prediction, we investigated the phe-
solution supplemented with increasing amounts of ethylene notype of RHA017, an hsaA deletion mutant of RHA1 (30).
JULY 16, 2010•VOLUME 285•NUMBER 29
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HsaAB, a Steroid-degrading Flavin Monooxygenase
Purification of HsaB from E. coli
(pT7HB) yielded 25 mg of protein per
liter of culture. The purified prepara-
tion was Ͼ95% HsaB as judged from
SDS-PAGE and had absorption max-
ima at 268, 370, and 448 nm consist-
ent with the presence of oxidized fla-
vin (supplemental Fig. 1A). HPLC
analyses of the acid-extracted flavin
revealed two species with retention
times and spectra corresponding
to FMN and FAD, respectively
(supplemental Fig. 1B). In subse-
quent experiments, HsaB was
reconstituted with either FMN or
FAD as solely bound cofactor.
HsaAB Catalyzes o-Hydroxyla-
tion—As noted above, RHA017 cul-
tures incubated with cholesterol
accumulated 3-HSA, 3,9-DHSA,
3,17-DHSA, and 3,9,17-THSA.
When the culture supernatant was
incubated for 30 min at room tem-
perature with 9 ␮M HsaA, 4 ␮M
HsaBꢀFMN, and 600 ␮M NADH,
3-HSA and 3,17-DHSA were re-
moved from the mixture and two
new compounds were detected by
GC-MS (Fig. 2B). Mass spectrome-
try and NMR spectroscopy identi-
fied these compounds as 3,4-DHSA
and 3,4,17-THSA (supplemental
FIGURE 2. GC-MS analyses of supernatant of the hsaA deletion mutant of R. jostii RHA1 (RHA017) incu-
bated in the presence of cholesterol. The traces A and B represent samples before and after treatment with
HsaAB as described under “Materials and Methods.” 3,4-tms-DHSA and 3,4,17-tms-THSA are the derivatized
catecholic transformation products of 3-HSA and 3,17-DHSA, respectively.
materials). Under similar condi-
tions, HsaAB transformed 3-HB
to 2,3-dihydroxybiphenyl, as con-
firmed by GC-MS (supplemental
Consistent with the enzyme playing a central role in cholesterol
catabolism, the deletion strain did not grow on cholesterol but
grew normally on pyruvate (data not shown). When pyruvate-
grown cells of RHA017 were incubated in the presence of cho-
lesterol, four major metabolites accumulated in the medium
(Fig. 2A). Purification of the three major ones afforded 3-HSA
and two keto-reduced versions thereof: 3,9-DHSA and 3,17-
DHSA. These were characterized using GC-MS and 1H-, 13C-,
(H,H)-COSY-, HSQC-, and HMBC-NMR as summarized in the
supplemental materials. The fourth metabolite was identified
as 3,9,17-THSA using GC-MS.
materials). These results demonstrate that HsaAB of Mtb is able
to catalyze the o-hydroxylation of a range of p-substituted phe-
nols to generate the corresponding catechols.
Steady-state Kinetic Analyses—To evaluate the specificity of
HsaAB for different substrates, a coupled spectrophotometric
assay was developed in which the catechol produced by the
HsaAB reaction was cleaved by an extradiol dioxygenase (HsaC
or BphC) to yield the yellow-colored meta-cleavage product.
Dioxygenase concentrations were adjusted so as not to be rate-
limiting. The concentration of HsaA was 2.5-fold higher than
that of HsaB to maximize coupling between these components
(see below). In all studies, the inferred initial rates of hydroxy-
lation displayed Michaelis-Menten kinetics (Fig. 3). The evalu-
ated kinetic parameters are considered to be apparent as they
are dependent on the ratio of HsaA and HsaB as well as the
concentration of O2.
Purification of HsaAB—HsaA produced in E. coli and puri-
fied aerobically had a very low specific activity. Production of
the protein in RHA017 using pTipHA increased the specific
activity by an order of magnitude; anaerobic purification fur-
ther increased this 2-fold. This approach typically yielded 25
mg of purified HsaA per liter of culture with an apparent purity
of Ͼ95% according to SDS-PAGE analysis and a specific activity
of 0.12 unit/mg using 3-HB in the standard assay. Proteolytic
The coupled assay was performed with 3-HSA to evaluate the
ability of HsaAB to utilize different flavins. In the presence of 4
␮M exogenously added FMN or FAD, no increase in enzyme
removal of the N-terminal His tag did not significantly affect activity was observed. By contrast, 4 ␮M riboflavin inhibited the
the specific activity. Accordingly, His-tagged HsaA was used in activity by ϳ50%. To further evaluate the ability of HsaAB to
this study.
utilize FMN and FAD, the reductase was reconstituted with
22268 JOURNAL OF BIOLOGICAL CHEMISTRY
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HsaAB, a Steroid-degrading Flavin Monooxygenase
TABLE 2
Crystallographic properties, X-ray diffraction data, phasing, and
refinement statistics for HsaA from M. tuberculosis
Diffraction data
HsaA (aerobic)
HsaA (anaerobic)
X-ray source
CMCF1
CuK␣
Wavelength (Å)
Space group
1.000
1.5418
I222
P21
Unit cell (Å, deg)
a ϭ 68.74, b ϭ 173.96,
c ϭ 178.07
␣ ϭ 90.0°, ␤ ϭ 90.0°,
␥ ϭ 90.0°
a ϭ 91.0, b ϭ 94.0,
c ϭ 98.1
␣ ϭ 90.0°, ␤ ϭ 94.0°,
␥ ϭ 90.0°
Resolution range (Å)
Highest shell (Å)
Unique reflections
I/␴I
40–2.00
50–2.40
2.0-2.05
2.44-2.40
72,634 (5,414)
26.4 (4.4)
64,589 (3,082)
16.2 (1.7)
R
sym(%)a
6.8 (34.2)
9.4 (54.3)
Completeness (%)
95.4 (97.4)
99.7 (96.0)
Refined model
Resolution range (Å)
No. reflections
20–2.0
68,987
18/22
31.24
50–2.4
64,589
20/27
35.24
R
factor/Rfree (%)b
Mean B-values (Å2)
r.m.s.d.
Bond lengths (Å)
Bond angles (deg)
0.020
1.77
0.020
1.847
a Rsym ϭ ⌺h⌺i I(hkl) Ϫ ͗I(hkl)͘/⌺h⌺iI(hkl).
b Rwork ϭ ⌺ʈFobs͉ Ϫ ͉Fcalcʈ/⌺͉Fobs͉. Rfree is the Rwork value for 5% of the reflections
excluded from the refinement. Data for the highest resolution shell are given in
parentheses.
each flavin as sole cofactor. As summarized in Table 1, the
apparent specificity of HsaAB for 3-HSA was remarkably sim-
ilar for assays containing HsaBꢀFMN and HsaBꢀFAD. However,
the apparent kcat and Km values were ϳ3-fold greater in the
presence of HsaBꢀFMN. All subsequent kinetic studies were
performed using HsaBꢀFMN and without supplementary flavin.
The coupled assay was then used to evaluate the specificity of
HsaAB for various p-substituted phenols. Of the purified ste-
roid metabolites, the mycobacterial enzyme had slightly higher
apparent specificity (kcat/Km) for 3-HSA versus 3,17-DHSA
(Table 1). By contrast, the apparent specificity for 3-HB was an
order of magnitude lower. Nevertheless, the apparent kcat val-
ues for these p-substituted phenols were within a factor of two.
Remarkably, the activity of 3,9-DHSA was too low to evaluate
kinetic parameters. However, in an assay using 205 ␮M 3,9-
DHSA and 25.8 ␮M HsaA, GC-MS analyses demonstrated that
the phenol was transformed to 3,4,9-THSA and that this reac-
tion occurred with one-third the efficiency of the 3-HB
transformation.
FIGURE 3. Steady-state kinetic analysis of HsaAB-catalyzed transforma-
tions. The dependence of the initial velocity on 3-HSA (A) and 3,17-DHSA (B)
concentrations in air-saturated buffer with fitted parameters Km ϭ 200 Ϯ 40
␮M and 350 Ϯ 90 ␮M, and Vmax ϭ 1.9 Ϯ 0.1 ␮M sϪ1 and 2.2 Ϯ 0.2 ␮M sϪ1
,
respectively. In these assays, the formation ring-cleaved products of
3,4-DHSA and 3,4,17-THSA, respectively, were followed. C, dependenceof the
initial velocity of O2 consumption on O2 concentration in the presence of 660
␮M 3-HSA with fitted parameters KmO2 ϭ 100 Ϯ 10 ␮M and Vmax ϭ 0.31 Ϯ 0.02
␮M sϪ1. O2 consumption was corrected for uncoupled consumption as
described in the text. The best fits of the Michaelis-Menten equation to the
data using LEONORA are represented as solid lines.
An oxygraph assay was developed to investigate the reactivity
of HsaAB with O2. In developing this assay, it was noted that O2
was consumed in the absence of HsaA. This HsaB-dependent
O2 consumption resulted in quantitative H2O2 production as
indicated by the addition of catalase (results not shown). To
minimize uncoupled O2 consumption, assays were performed
using 10-fold less HsaB than in the spectrophotometric assay.
The remaining H2O2 production was corrected for by mea-
TABLE 1
Apparent steady-state kinetic parameters of HsaAB from
M. tuberculosis
Substratea
Flavina
Km
␮M
kcat
s
k
cat/Km
Ϫ1
Ϫ1
M
Ϫ1 s
suring the rate of O2 consumption before (vO2) and after (v
)
the addition of catalase. The calculated rate of O2 consumptϩioCn
due to hydroxylation, vROH, was within 5% of that determined
using the spectrophotometric assay and the same concentra-
tion of HsaB. Using the oxygraph assay and saturating amounts
of 3-HSA, the apparent KmO2 was 100 Ϯ 10 ␮M (Table 2).
Overall Structure—The refined structure of HsaA purified
aerobically from E. coli contains two peptide chains (residues
3-HSAb
FAD
FMN
FMN
FMN
FMN
97 (4)
200 (40)
350 (90)
1440 (1420)
100 (10)
0.075 (0.001)
0.20 (0.01)
0.24 (0.03)
0.11 (0.06)
0.031 (0.002)
770 (20)
1000 (100)
700 (100)
80 (40)
3-HSAb
3,17-DHSAb
3-HBb
c
O2
320 (30)
a Indicates flavin used to reconstitute HsaB. All experiments were performed using
20 mM potassium phosphate, pH 7.0, at 25 °C.
b Coupled spectrophotometric assay.
c Oxygraph assay utilizing 660 ␮M 3-HSA and 20-fold less HsaB than in the spectro-
photometric assay. Values reported in parentheses represent Ϯ S.E.
JULY 16, 2010•VOLUME 285•NUMBER 29
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HsaAB, a Steroid-degrading Flavin Monooxygenase
However the former crystallized as
an octamer while the latter crystal-
lized as a tetramer. In HsaAC, the
two subunits of the asymmetric unit
are nearly identical as reflected by
an r.m.s.d. between the C␣ atoms of
less than 0.32 Å. Each protomer
forms an extended contact interface
with symmetry-related subunits
along the crystallographic and non-
crystallographic 2-fold axes to form
a
doughnut-shaped octameric
assembly that is 90–100 Å wide
with a central solvent-accessible
pore of 40–45 Å in diameter (Fig.
4C). Calculations performed using
the PISA server indicated that the
buried surface in each molecule is
1,138 Å2 and predicting that the
octamer is highly stable. In HsaAO,
the four protomers of the asymmet-
ric unit have an r.m.s.d. of 0.48 Å
over the 369 backbone C␣ atoms. In
comparison to the octameric
HsaAC, the tetrameric HsaAO has a
larger average buried surface area
per protomer of 2,616 Å2 (Fig. 4B).
FIGURE4. CrystalstructureofHsaAfromM.tuberculosis. A, ribbonrepresentationoftheHsaAO protomer.The
central ␤-barrel domain of this conserved fold is colored dark gray, and the flanking N- and C-terminal ␣-helix
bundles are colored red and orange, respectively. B, ribbon representation of the HsaAO tetramer viewed along
the2-foldsymmetryaxis. Subunitsarecoloreddifferentiallyinshadesof violet, cyan, yellow, andgreen. C, ribbon
representation of the HsaAC octamer complex. The four subunits that occur above the plane of view are colored Static light scattering analysis indi-
with different darker shades. The four subunits below the view plane are colored with lighter shades. D, expanded
cated that HsaA prepared aerobi-
cally from the E. coli host exists as a
mixture of dimers and octamers in
solution while HsaA prepared
anaerobically from the rhodococcal
view of the interactions between three subunits within the octamer. The C-terminal flaps (Val367–Val394) of two
subunits, colored in two hues of red, align in an anti-parallel fashion. This interface is in close proximity to the
hairpin loop (Gly261–Val281) of a third subunit (dark green). This loop, which is not visible in the electron density
and thus represented by black dots, would be projected next to the C-terminal flaps from below the view plane.
This interaction between the three HsaAC protomers repeats itself to form the octameric ring structure.
4–391), two formate ions, and 669 water molecules in the host exists as a tetramer (supplemental Fig. 2).
asymmetric unit (Table 2 and Fig. 4). Due to the lack of visible
Conformational States of the C-terminal Flap—Comparison
electron density, the model does not include three portions of of the tetrameric HsaAO and octameric HsaAC revealed differ-
the molecule: the N terminus (the His tag and residues 1–3), the ent contacts between HsaA protomers in the crystal lattice, as
C terminus (residues 391–394), and residues 261–282. As pre- reflected by the difference in buried surface area described
sented below, the Fo Ϫ Fc difference map revealed a ring-shaped above. In addition, two regions of the molecule displayed con-
6-␴ positive peak in the substrate-binding site of the enzyme. formational differences that likely further contribute to the dif-
This density was not assigned but likely represents an E. coli ference in crystal packing: an apical hairpin turn (Gly261
–
metabolite that co-purified with the enzyme. In comparison, Val281) and a C-terminal flap (Val367–Val394). In HsaAO (P21),
the refined structure of anaerobically prepared HsaA purified the C-terminal helix bundles (Fig. 4A) are clustered toward the
from RHA1 contains four peptide chains (residues 3–394) and center of the tetramer (Fig. 4A), forming a coiled-coiled com-
472 water molecules. N-terminal residues 1–3 and residues plex, with the ␤-barrel projecting outward in the tetrahedral
271–272 were not visible in any of the four molecules of the arrangement (Fig. 4B). At the center of the tetramer, a helix-
asymmetric unit. Comparison of the two structures revealed turn-helix motif (Glu317–Gly365) composed of residues imme-
major differences in an apical hairpin turn (Gly261–Val281) and diately preceding those of the C-terminal flap, stacks and aligns
the C-terminal segment (Val367–Val394) as discussed below. anti-parallel to the equivalent motif in the adjacent protomer.
The structures of aerobically and anaerobically prepared oxy- Moreover, the same cluster of residues from the two protomers
genases are henceforth referred to as HsaAC and HsaAO, for comprises the major direct noncovalent interactions at the
“closed” and “open” conformations, respectively.
interface: Phe341, Leu349, Trp359, and His363. A second helix
The overall fold of HsaA is conserved when compared with bundle motif, involving Phe278–Ala343 and the C-terminal flap,
other structurally characterized TC-FDM oxygenases. Thus, associates with equivalent motifs between dimers to form the
HsaA formed two distinct substructures: a ␤-barrel (Ser113
–
HsaAO tetramer. This second interface includes two equivalent
Arg206) and a bundle of ␣-helices (Fig. 4A). The latter are hydrogen bonds involving Arg328 and Arg332 of one molecule
derived from both termini (Arg7–Pro112 and Phe207–Val394). and Glu285 and Asp289 of an adjacent one. The C-terminal flap
The overall fold is maintained between HsaAC and HsaAO. also contributes to this second interface, mainly through hydro-
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HsaAB, a Steroid-degrading Flavin Monooxygenase
those of the surrounding residues (ϳ27.6 Å2) and are similar
throughout the flap.
We considered whether oxidation of a Cys residue might
influence the conformation of the C-terminal flap of HsaA.
Inspection of the two structures revealed that Cys63, Cys79, and
Cys145 all have low solvent accessibilities and are remote (Ͼ20
Å) from the C-terminal flap. Inspection of the electron density
revealed no evidence for oxidation. Thus, 2Fo Ϫ Fc and Fo Ϫ Fc
maps revealed no additional electron density around the Cys
thiols in the structure of the aerobically prepared enzyme.
Moreover, water molecules are ordered around the cysteines in
a similar fashion in both HsaAC and HsaAO. Overall, our data
indicate that oxidation of a Cys residue does not contribute to
the different conformation of the C-terminal flap.
The Flavin Binding (F-)Subsite—The structures of HsaA con-
tain a deep cavity that presumably sequesters the substrates:
reduced flavin, O2, and 3-HSA. This cavity is located at the
interface between the C-terminal helix bundle and the ␤-barrel.
Superposition of the structure of the pHPAHꢀflavinꢀpHPA
complex facilitates the description of the substrate-binding
pocket of HsaA (Fig. 6A). Notably, the pocket comprises two
adjacent subsites: the H-subsite binds the phenol, and the
slightly more solvent-accessible F-subsite binds the reduced
flavin. The F-subsite of HsaAO is remarkably similar to that of
pHPAH with all first shell residues being conserved (i.e. Trp84,
Trp141, Ser143, Trp182, Leu187, and His368). Based on this super-
imposed model (Fig. 6A), the isoalloxazine ring stacks between
the indole rings of Trp84 and Trp141, and is surrounded by the
hydrophobic side chains of Trp182 and Leu187. Ser143 forms a
hydrogen bond with flavin N5. Similarly to what was observed
in pHPAH, the imidazole group of His368 is on the re-face of the
isoalloxazine ring within 4 Å of N1, compatible with stabiliza-
FIGURE5.ConformationaldifferencesinHsaAO andHsaAC.Inthestructure
of HsaAC (pale red), the C-terminal flap (Val367-Val394), reorients and seals the
major entrance to the proposed 3-HSA binding site. The hairpin loop, high-
lighted in yellow, is present in the HsaAO structure, colored in cyan. Electron
density for this loop was not detected in the HsaAC structure.
phobic interactions involving Tyr377–Phe385 and Lys280
–
Ala284. More specifically, the side chain of Val378 and the back-
bone of Gly381 interact with the aliphatic side-chain atoms of
Lys280. The ⑀-amine group of the latter forms a hydrogen bond
with the side-chain amide oxygen of Asn382. Finally, the side
chains of Ile283 and Ala284 provide a hydrophobic platform for
Phe380 and Phe385 on the C-terminal flap. Overall, the apical
hairpin turn is stabilized by the C-terminal flap in the HsaAO
tetramer.
Ϫ
tion of a deprotonated N1 in the bound FMNH . Finally, the
entrance to the F-subsite is lined with charged residues and is
partially occluded by an adjacent protomer in the tetramer, as
observed in pHPAH. However, although residues contacting
the isoalloxazine moiety are largely conserved, side chains that
stabilize the phosphate group, located on a C-terminal helix,
are not (i.e. Asn371).
By contrast, the intersubunit interactions in the HsaAC octa-
mer are more intricate (Fig. 4C), with each protomer interact-
ing with three others. At one end, each protomer interacts with
its neighbor through their respective helix-turn-helix motifs in
much the same way as in HsaAO. At the other end, contact with
a third protomer involves hydrophobic interactions between
C-terminal flaps at the entrance of the active site with residues
The F-subsite in the HsaAC crystal form is very similar, but
differs in two respects. First, the entrance to the subsite is
occluded by the C-terminal flap. Second, the flavin binding site
is partially occupied by Trp139 and His368
.
of adjacent protomers, Ile376, Tyr374, Phe377, and Phe382
,
The 3-HSA Binding (H-)subsite—In contrast to the F-subsite,
involved. In comparison to its conformation in HsaAO, the flap
in HsaAC differs by a 25° rigid body rotation around Arg366 (Fig.
5C) enabling the described intersubunit association and effec-
tively occluding the entrance to the substrate-binding pocket of
HsaA. Finally, our structures suggest that the interface between
the C-terminal flaps is spatially positioned to potentially inter-
act with the apical hairpin turn (disordered in our HsaAC struc-
tures) of an adjacent protomer (Fig. 4D).
the H-subsite is buried in the core of the protein and is predom-
inantly lined with hydrophobic residues: Trp94, Pro238, Ile241
,
Tyr210, His237, and Trp233. These residues are conserved in
HsaA of R. jostii RHA1 but not in pHPAH. The size of the HsaA
H-subsite versus that of pHPAH is consistent with the former
accommodating a larger phenolic substrate.
To investigate the structural basis of binding of the phenolic
substrate, we modeled 3-HSA into the H-subsite of HsaAO and
HsaAC using molecular docking and energy minimization
methods. The high structural similarity of the F-subsites of
HsaAO, HsaAC, and pHPAHꢀflavinꢀpHPA allowed for position-
The two conformers also differ in the degree of flexibility of
their C-terminal segments, with the B-factors increasing
toward the C-terminal flap residues, reaching an average value
of 50.6 Å2 for the backbone C␣s of Pro388–Val394. This value is ing of flavin in the binding site. In both forms of HsaA, the
ϳ2-fold higher than that of the rest of the protein. By contrast, His368 and Trp141 side chains were the only ones of the F-sub-
the B-factors of the C-terminal flap in HsaAC are comparable to site that required minor reorientation to accommodate the fla-
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HsaAB, a Steroid-degrading Flavin Monooxygenase
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HsaAB, a Steroid-degrading Flavin Monooxygenase
vin. The latter molecule was kept rigid during docking and Pro370 and Pro119 both pack closely with the bicycloalkanone
energy minimization calculations. Positive clustering of docked moiety in the HsaAC model, while only Pro119 is present in the
solutions was readily observed using either HsaAO or HsaAC, HsaAO model. Overall, these differences underscore the possi-
with the best positional conformation placing the substrate in bility that conformational changes in the C-terminal flap may
close proximity to the flavin molecule. A solution where the C4 be important for binding and orientating the substrates.
atom of 3-HSA is within 4.2 Å to the C4a atom of the cofactor
DISCUSSION
was chosen as the strongest candidate for further energy mini-
The current study establishes that the role of mycobacterial
and rhodococcal HsaAB is to transform 3-HSA to 3,4-DHSA in
the catabolism of cholesterol. When incubated in the presence
of cholesterol, a ⌬hsaA mutant of RHA1 accumulated the
enzyme’s predicted substrate, 3-HSA, together with related
metabolites in which the keto groups on the bicycloalkanone
moiety are reduced. Consistent with its predicted role, recon-
stituted HsaAB from Mtb had the highest specificity for 3-HSA.
Moreover, the crystal structure of HsaA revealed a fold that is
typical of TC-FDM oxygenases and a substrate-binding pocket
that is well adapted to the steroid metabolite. The function of
HsaAB is consistent with transposon mutagenesis studies indi-
cating that hsaA (3570c) is necessary for survival of Mtb in
macrophages, but hsaB (3567c) is not (4), because it is likely that
flavin-reducing function of HsaB could be performed by
another enzyme.
mization. In docking 3-HSA, a number of aromatic side chains
interacting with the bicycloalkanone moiety underwent rear-
rangement during the docking and energy minimization
process.
In the modeled HsaACꢀflavinꢀ3-HSA complex, the phenol
ring of 3-HSA is orientated toward the flavin and the bicycloal-
kanone moiety toward the entrance to the active site, stacking
against Pro373 (Fig. 6B). The modeled complex shares at least
three important features with the pHPAHꢀflavinꢀpHPA com-
plex. First, the phenol ring is orthogonal to the flavin. Sec-
ond, conserved Ser118 in HsaA appears to interact with the
phenolic ring in such a way as to favor the deprotonated form
of the hydroxyl group located 2.4 Å away. Third, there is a
cavity between flavin C4a and phenol C4 that can accommo-
date O2 (Fig. 6C). More specifically, the distance between
flavin C4a and phenol C4 is 4.9 Å, compatible with the for-
mation of a C4a-hydroperoxyflavin intermediate on the re-
face of the isoalloxazine ring (Fig. 6C). In the modeled com-
plex, the probable O2-binding cavity is slightly larger than in
the pHPAHꢀflavinꢀpHPA complex (29 Å3 versus 18 Å3) due to
the presence of less bulky residues (e.g. Phe270 in pHPAH is
Ile241 in HsaA). Finally, the unassigned electron density in
HsaAC occupies a space corresponding to a portion of the
iso-alloxazine ring of the flavin and the phenol ring of the
3-HSA (Fig. 6D).
The metabolite analyses of the ⌬hsaA mutant suggest that at
least in RHA1, side-chain degradation largely precedes ring
degradation. More specifically, the identified metabolites rep-
resented either the end (C17 ketone) or penultimate (C17
hydroxy) product of side-chain degradation. This is consistent
with the recent finding that cytochrome P450125 initiates cho-
lesterol degradation in this strain by hydroxylating a terminal
carbon (11). Nevertheless, the ⌬hsaC mutant of RHA1 accumu-
lated three major catecholic metabolites possessing the ketone,
hydroxyl, as well a propionate group at C17,7 consistent with
some simultaneous degradation of the side chain and rings. The
different metabolites in the two mutants may reflect the rel-
ative specificities of the side chain-degrading enzymes for
the phenolic versus catecholic steroid metabolites. Finally,
the significance of the 9-hydroxy metabolites, 3,9-DHSA and
3,9,17-THSA, is unclear. Their relative lack of reactivity with
HsaAB suggests that they may have arisen adventitiously in
the mutant. The current studies do not rule out the possibil-
ity that the physiological substrate of HsaAB is 3-HSA with a
partially degraded side chain.
Two notable features of the modeled HsaACꢀ3-HSA interac-
tions involve C9 and C17 of the substrate (Fig. 6, B and E). The
C9 carbonyl is orientated toward two acidic residues: Asp372
and Glu374. Asp372 participates in an H-bond with His368, the
residue proposed to abstract a proton from the flavin N1. This
small acidic patch is in close proximity to Arg366, the pivotal
point of the C-terminal flap, which has distinct conformations
in HsaAO and HsaAC. For its part, the C17 carbonyl is orien-
tated toward a short hydrophobic channel lying between the
helix bundle and the ␤-barrel domains. This channel is ϳ9 Å
long and is lined with the hydrophobic side chains of Tyr377
,
Tyr210, Phe380, Phe385, and Leu387
.
Although the activity of HsaAB is lesser than that of other
characterized TC-FDMs, it is consistent with that of other cho-
lesterol catabolic enzymes of Mtb. For example, the apparent
specificity (kcat/Km) of a bacterial EDTA monooxygenase for
MgEDTA2Ϫ is 430-fold greater than that of HsaAB (51). Simi-
Several interactions between 3-HSA and the enzyme that are
present in the HsaAC model do not occur in the modeled
HsaAOꢀflavinꢀ3-HSA complex (Fig. 6F). First, Asp372 and Glu374
do not stabilize the C9 carbonyl, facing instead the bulk solvent.
Second, only Tyr207 is within hydrogen bonding distance of the
C17 carbonyl: the interaction with Tyr374 is absent. Finally, 7 C. Dresen, K. C. Yam, and L. D. Eltis, unpublished observation.
FIGURE 6. The substrate-binding site of HsaA from M. tuberculosis. A, the HsaAO structure, in which FMN was placed in the active site based on a superpo-
sition with the pHPAHꢀflavinꢀpHPA ternary complex (PDB code: 2JBT). B, the modeled HsaAꢀflavinꢀ3-HSA ternary complex showing the relative position and
orientation of the 3-HSA (orange) and FMNHϪ (yellow) in the active site. The two conformations of the C-terminal flap (Val367–Val394) are shown in different
shadesofred. C, therelativeorientationandgeometrybetweenthephenolicringof3-HSAandtheisoalloxazineringoftheflavininthemodeledHsaACꢀflavinꢀ3-
HSA complex. The distance between the phenolic C4 and flavin C4a atoms, 4.9 Å, is indicated by an orange dotted line. Predicted hydrogen bonds are indicated
by black dashed lines. D, the same orientation of the active site as in C with the unassigned electron density in HsaAC. The wire mesh represents an Fo Ϫ Fc map
contoured at 3␴. Stereo images of the bound substrates in modeled (E) HsaAC and (F) HsaAO complex highlight the interactions of the C9 and C17 ketones of
3-HSA. The depicted helix is part of the C-terminal flap.
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HsaAB, a Steroid-degrading Flavin Monooxygenase
larly, the apparent specificity of alkanesulfonate monooxygen- metabolically quiescent bacterial cells. Regardless of its func-
ase for alkanesulfonates was up to 112-fold greater (25). Never- tional significance, the orientation of the flap in the aerobically
theless, the relative activity of HsaAB is very similar to that of its purified HsaA from E. coli together with the presence of a
flanking enzymes in the cholesterol catabolic pathway, KshA ligand in the F-subsite of this preparation correlates with its
(17) and HsaC (6): the apparent kcat/Km of these enzymes are decreased catalytic activity.
also two or three orders of magnitude lesser than those of their
The structural data provide at least two intriguing insights
homologues. It is unclear whether this is characteristic of non- into the substrate specificity of HsaA (Fig. 6, E and F). First, the
mycobacterial steroid-degrading oxygenases. Although the predicted proximity of the C9 ketone with Asp372 (3.7 Å) may
KmO2app values of other TC-FDMs have not been reported, that explain why 3,9-DHSA is a poor substrate inasmuch as the
of HsaAB (100 Ϯ 10 ␮M) is remarkably similar to HsaC (90 Ϯ 20 9-OH substitution may disrupt the hydrogen bond between
␮M). Finally, the relative kcat/Km values of HsaAB and HsaC for Asp372 and His368. This may ultimately perturb the catalytic
Ϫ1
3-HSA (1000 Ϯ 100 M
s
Ϫ1) and 3,4-DHSA (15000 Ϯ 2000 function of the latter. Second, the channel that extends from the
M
Ϫ1 sϪ1), respectively, would help minimize intracellular con- C17 ketone suggests that the H-subsite can accommodate sub-
centrations of the latter, which is toxic to Mtb (6).
strates with partially degraded side chains. This raises the pos-
HsaA is similar to pHPAH oxygenase (52) in that both utilize sibility that the physiological substrates of the steroid ring-de-
FMNH2 and FADH2 with similar efficiencies and that exog- grading enzymes have not yet been identified, consistent with
enously added flavin does not increase the rate of product for- the relatively low specificity constants of these enzymes for side
mation by either enzyme. Nevertheless, the structure of HsaA chain degraded metabolites.
suggests that the F-subsite recognizes the isoalloxazine ring of
It remains unclear whether cholesterol catabolism is essen-
FMNH2 or FADH2 and does not accommodate the entire tial to the pathogenesis of Mtb (6–8). Nevertheless, transposon
FADH2 molecule: the adenyl moiety of the latter would pro- mutagenesis studies indicate that HsaAB activity is critical for
trude into bulk solvent (supplemental Fig. 3). In nitroalkane survival in macrophages (4). Further studies of HsaAB should
oxidase from Fusarium oxysporum (PDB entry 2C0U), the pro- help establish the role of cholesterol catabolism in the patho-
truding moiety mediates subunit interactions. Other bacterial genesis of Mtb as well as the catalytic mechanism of TC-FDMs.
TC-FDMs that transform aromatic compounds preferentially Indeed, characterization of the ligand bound to HsaAC may be
utilize one of the flavins: FMNH2 in the case of pristinamycin useful in both of these respects.
IIA synthase (53) and FADH2 in the case of chlorophenol-4-
hydroxylase (54), pyrrole-2-carboxylate monooxygenase (55),
and p-nitrophenol hydroxylase (56).
Acknowledgments—We thank the Canadian Light Source (Saska-
toon, Saskatchewan, Canada) for access to beamline CMCF1 for
X-ray synchrotron data collection. Jie Liu, Mark Okon, Gord Stewart,
and Christine Florizone provided skilled technical assistance. Dr. Vic-
tor Snieckus and Katherine Yam provided 2,3-dihydroxybiphenyl
and HsaC, respectively.
Although the closed conformation of the C-terminal flap
correlates with the presence of an active site ligand in HsaA, it is
unclear how the expression host (E. coli versus Rhodococcus)
and the exposure of the enzyme to O2 during purification con-
tribute to the presence of the ligand. Moreover, the functional
significance of the different conformations in HsaAO and
HsaAC and the octameric state of the latter remains unclear. In
the enzyme’s closed conformation, the C-terminal flap likely
prevents the entry of the reduced flavin, the first substrate to
bind in the related pHPAH (28). Nevertheless, modeling studies
indicate that the closed conformation better accounts for the
low specificity of HsaA for 3,9-DHSA, as described below. It is
possible that, in solution, a change in conformation in the
C-terminal flap favored by flavin binding might help direct the
subsequent entry of the organic substrate. The flap is present in
all TC-FDM oxygenases structurally characterized to date,
including pHPAH, HpaB (PDB entry 2YYG), HsaA from RHA1
(PDB entry 2RFQ), as well as two related enzymes: pig medium
chain acyl-CoA dehydrogenase (PDB entry 2JIF) and nitroal-
kane oxidase from F. oxysporum (PDB entry 2C0U). In all cases,
the enzymes were tetrameric and the flap was in the same con-
formation as in the HsaAO structure. However, alternate con-
formations of the flap may not have been observed in four of
these cases, because the enzymes were crystallized in the pres-
ence of reduced flavin and the corresponding organic substrate.
Structural flexibility in pHPAH (48) and HpaB (56) is suggested
by elevated B-factors of the C-terminal flap residues, as
observed in HsaAO. Finally, it is also possible that octameric
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