Expression, purification, crystallization and preliminary X-ray analysis of Rv3117, a probable thiosulfate sulfurtransferase (CysA3) from Mycobacterium tuberculosis

Article (PDF Available)inActa Crystallographica Section F Structural Biology and Crystallization Communications 64(Pt 6):541-4 · July 2008with12 Reads
DOI: 10.1107/S1744309108014449 · Source: PubMed
Abstract
The gene product of open reading frame Rv3117 from Mycobacterium tuberculosis (Mtb) strain H37Rv is annotated as encoding a probable rhodanese-like thiosulfate sulfurtransferase (MtbCysA3). MtbCysA3 was expressed and purified and then crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.5 A. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 38.86, b = 91.43, c = 83.57 A, beta = 96.6 degrees . Preliminary diffraction data shows that two molecules are present in the asymmetric unit; this corresponds to a V(M) of 2.4 A(3) Da(-1).
crystallization communications
Acta Cryst. (2008). F64, 541–544 doi:10.1107/S1744309108014449 541
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Expression, purification, crystallization and
preliminary X-ray analysis of Rv3117, a probable
thiosulfate sulfurtransferase (CysA3) from
Mycobacterium tuberculosis
Sarah J. Witholt, Ramasamy
Sankaranarayanan, Craig R.
Garen, Maia M. Cherney,
Leonid T. Cherney and
Michael N. G. James*
Protein Structure and Function Group,
Department of Biochemistry, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada
Correspondence e-mail:
michael.james@ualberta.ca
Received 14 March 2008
Accepted 13 May 2008
The gene product of open reading frame Rv3117 from Mycobacterium tubercu-
losis (Mtb) strain H37Rv is annotated as encoding a probable rhodanese-like
thiosulfate sulfurtransferase (MtbCysA3). MtbCysA3 was expressed and puri-
fied and then crystallized using the sitting-drop vapour-diffusion method. X-ray
diffraction data were collected and processed to a maximum resolution of 2.5 A
˚
.
The crystal s belong to the monoclinic spa ce group P2
1
, with unit-cell parameters
a = 38.86, b = 91.43, c = 83.57 A
˚
, = 96.6
. Preliminary diffraction data shows
that two molecules are present in the asymmetric unit; this corresponds to a V
M
of 2.4 A
˚
3
Da
1
.
1. Introduction
Tuberculosis (TB) is a common and serious contagious disease caused
by Mycobacterium tuberculosis (Mtb). One third of the world’s popu-
lation is infected with the TB bacillus, leading to an estimated 4400
human deaths per day (World Health Organization, 2007). If not
treated, each person with active TB will infect 10–15 further people
each year (World Health Organization, 2007). Numerous drug-
resistant strains of TB have emerged owing to inconsistent or partial
drug treatments and limited drug availability. The TB Structural
Genomics Consortium was formed in 2000 with the goal of devel-
oping a basis for new TB drug development by determining the three-
dimensional structures of Mtb proteins (http://www.doe-mbi.ucla.edu/
TB/mission.php) with the view of eliminating TB as a public health
problem.
As members of the Mtb structural genomics consortium, we have
conducted molecular cloning and preliminary X-ray analysis on a
rhodanese-like thiosulfate sulfurtranferase protein (MtbCysA3).
Classical rhodanese proteins (EC 2.8.1.1) catalyze the transfer of a
sulfane sulfur from a donor substrate to the enzyme active site and
then in turn to a thiophilic acceptor (Westley, 1973; Nandi & Westley,
1998). It was initially believed that the primary function of this
enzyme was to convert cyanide to the less toxic thiocyanate (Lang,
1933). In fact, the best studied reaction in vitro has thiosulfate as the
donor to form a sulfur-substituted rhodanese-enzyme intermediate
(and sulfite), followed by transfer of the sulfane sulfur to cyanide,
which acts as the acceptor substrate. The products of this displace-
ment mechanism are thiocyanate and sulfur-free rhodanese. It has
been proposed that dihydrolipoate can act as a possible sulfur
acceptor that yields lipoate and inorganic sulfite as final products
(Villarejo & Westley, 1963; Volini & Westley, 1966). Other studies
have shown that by using reduced lipoate, rhodanese may be used to
reconstitute redox centres to aid in the formation of the characteristic
prosthetic group of iron–sulfur proteins (Pagani et al., 1984; Bonomi
et al., 1985). 24 rhodanese-like structures have been deposited in the
Protein Data Bank (PDB; Berman et al., 2000). These include nine
single rhodanese homology domain structures and 13 two-domain
structures. One three-domain mercaptopyruvate sulfur transferase
# 2008 International Union of Crystallography
All rights reserved
structure that contains similar N-terminal and central domains to
thiosulfate sulfurtransferase rhodanese has also been solved. Four of
the two-domain structures were used to compare sequence alignment
with MtbCysA3 (Fig. 1). The two sequences that show the greatest
similarities are 1uar (52% identity) from Thermus thermophilus
(RIKEN Structural Genomics/Proteomics Initiative, unpublished
work) and 1urh (26% identity) from Escherichia coli (Spallarossa et
al., 2004). Sequence alignments were performed using ClustalW
(Thompson et al., 1994).
2. Experimental methods
2.1. Cloning, expression and purification
The entire genome of the H37Rv strain was cloned into a bacterial
artificial chromosome (BAC) library at L’Institut Pasteur (Brosch et
al., 1998; Gordon et al., 1999). Polymerase chain reaction (PCR) was
used to amplify the Rv3117 protein, using the BAC library as the
template. PCR primers were designed for directional cloning of
inserts into the Gateway cloning system (Invitrogen). The primer
sequences were Rv3117F (5
0
-GGGACAAGTTTGTACAAAAAA-
GCAGGCTCCGGTCTG GTTCCGCG TGGTATGGCACGCTG C-
GATGTCCTGGTCT-3
0
) and Rv3117R (5
0
-GGGACCACTTTGTA-
CAAGAAAGCTGGGTCTCAGCTTCCCAACTCGATCGGGGC-
G-3
0
). The open reading frame encoding residues 1–277 of Rv3117
was cloned into the Gateway entry vector pDONR 221 (Invitrogen)
and then into the expression plasmid containing an amino-terminal
hexahistadine (His
6
) fusion tag (pDEST-17, Invitrogen). The pre-
sence of the gene insert was confirmed by DNA-sequence analysis
(DNA core facility, Department of Biochemistry, University of
Alberta, Canada).
His
6
-Rv3117 was expressed in E. coli BL21 (DE3) cells (Novagen).
Incubation of the transformed cells at 310 K was continued until the
OD
600 nm
reached 0.5. Subsequently, the temperature was shifted to
295 K and protein expression was induced by adding isopropyl -d-1-
thiogalactopyranoside (Fisher Scientific) to a final concentration of
0.5 mM. After overnight incubation, the cells were harvested by
centrifugation for 15 min at 9380g. Bacterial pellets were resus-
pended in 20 mM NaH
2
PO
4
pH 7.4, 300 mM NaCl, 10 mM imidazole,
2mM -mercaptoethanol containing Complete protease inhibitor
(Roche), 1 mM phenylmethylsulfonyl fluoride (Bioshop) and
10 mgml
1
hen egg-white lysozyme (Sigma). For purification, the
cells were lysed by freeze–thawing and then subjected to ultra-
sonication in the resuspension buffer. The lysate was cleared by
crystallization communications
542 Witholt et al.
Rv3117 Acta Cryst. (2008). F64, 541–544
Figure 1
Sequence comparison of selected sulfurtranferase proteins. Abbreviations and accession numbers are as follow s: BovRHD, bovine liver rhodanese (gi:135823); EcSseA,
E. coli strain K12 (gi:401186); TtRHD, T. thermophilus strain HB8 (gi:81600441); Rv3117, M. tuberculosis strain H37Rv (gi:15610254); AvRHD, A. vinelandii (gi:1729961).
ClustalW was used to perform sequence alignment (Thompson et al., 1994) and the figure was generated using the program ESPript (Gouet et al., 1999).
centrifugation (30 min, 20 000g) and the supernatant was then passed
through a 0.45 mM syringe filter (Millipore). The cleared supernatant
was loaded onto a 5 ml HisTrap FF column (GE Healthcare) pre-
equilibrated with 20 mM NaH
2
PO
4
pH 7.4, 300 mM NaCl, 10 mM
imidazole and 1 mM -mercaptoethanol. The His
6
-Rv3117 fusion
protein was eluted with a linear gradient of imidazole from 10 to
300 mM. The His
6
tag and the N-terminal recombination site were
removed by proteolytic cleavage using thrombin (Amersham Bio-
sciences), leaving an additional glycine residue at the N-terminus. The
site recognized by thrombin is encoded in the forward primer (itali-
cized in the primer sequence given). After dialysis against 20 mM
NaH
2
PO
4
pH 7.4, 300 mM NaCl, 10 mM imidazole and 1 mM
-mercaptoethanol, the cleaved protein mixture was once again
loaded onto a HisTrap column to remove the His
6
tag. The flow-
through fractions containing the digested MtbCysA3 were concen-
trated to 8 mg ml
1
using an Amicon Ultra (10 kDa cutoff; Milli-
pore). The protein was then dialyzed overnight against 5 mM Tris–
HCl pH 7.4, 100 mM NaCl and 1 mM dithiothreitol (Fisher Scien-
tific). The whole process of purification was performed at 277 K and
the results of each step were monitored using 16% SDS–PAGE.
2.2. Crystallization
Crystallization of native full-length MtbCysA3 was performed by
screening with various crystallization conditions using the sitting-
drop vapour-diffusion method in 96-well Intelliplates (Hampton
Research). Index Screen and Crystal Screens I and II (Hampton
Research) were used by mixing equal volumes (0.5 ml) of concen-
trated protein and precipitating solutions. After 3 d, preliminary
crystals were obtained from a variety of conditions. After optimiza-
tion of the best screening condition, X-ray diffraction-quality crystals
were grown in hanging drops in 24-well VDX plates (Hampton
Research) containing 0.5 ml protein solution at 8 mg ml
1
and 0.5 ml
precipitating solution and the drops were equilibrated against 1 ml
reservoir solution (25% PEG 3350, 0.1 M Tris–HCl pH 8.5, 0.2 M
MgCl
2
). The average dimensions of the MtbCysA3 crystals were
200 100 30 mm (Fig. 2).
2.3. Data collection
Crystals for synchrotron data collection were first rinsed in cryo-
protectant (25% glycerol in mother liquor) and then flash-cooled by
immersion in liquid nitrogen. Native data sets were collected on
beamline 8.3.1 at the Advanced Light Source (ALS) at the Lawrence
Berkeley National Laboratory, revealing a diffraction pattern to
2.5 A
˚
resolution (Fig. 3). The HKL-2000 program suite (Otwinowski
& Minor, 1997) was used to reduce, integrate and scale the collected
data. Crystallographic statistics of the native data are summarized in
Table 1.
crystallization communications
Acta Cryst. (2008). F64, 541–544 Witholt et al.
Rv3117 543
Figure 2
Preliminary MtbCysA3 crystals. Diffraction-quality crystals grew to approximate
dimensions of 200 100 30 mm in 25% PEG 3350, 0.1 M Tris–HCl pH 8.5, 0.2 M
MgCl
2
.
Table 1
Crystal parameters and data-collection statistics for Rv3117.
Values in parentheses are for the highest resolution shell.
Crystal parameters
Space group P2
1
Unit-cell parameters
a (A
˚
) 38.86
b (A
˚
) 91.43
c (A
˚
) 83.57
(
) 90.00
(
) 96.6
(
) 90.00
No. of molecules per unit cell 4
Data collection
Temperature (K) 100
Detector ADSC Q315
Wavelength (A
˚
) 1.214591
Resolution (A
˚
) 50.0–2.5 (2.59–2.50)
Unique reflections 20078 (2006)
Multiplicity 3.6 (3.4)
Mosaicity (
) 1.3–1.7
I/(I) 11.8 (2.1)
Completeness (%) 98.8 (98.0)
R
merge
0.104 (0.546)
Wilson B factor (A
˚
2
) 53.7
R
merge
=
P
hkl
P
i
jI
i
ðhklÞhIðhklÞij=
P
hkl
P
i
I
i
ðhklÞ, where I
i
(hkl) is the ith observa-
tion of reflection hkl and hI(hkl)i is the weighted average intensity for all observations i
of reflection hkl.
Figure 3
X-ray diffraction-pattern frame collected from MtbCysA3 crystals. The pattern
displays a maximum resolution of 2.5 A
˚
and space group P2
1
.
3. Results
Native data sets were collected from MtbCysA3 crystals. Based upon
the expected molecular weight of Rv3117 (31 014.2 Da), the
Matthews coefficient V
M
(Matthews, 1968) is calculated to be
2.4 A
˚
3
Da
1
, with an estimated solvent content of 48.3%. These
results suggest the presence of two molecules of Rv3117 per asym-
metric unit. In related structures, it has been found that this enzyme
exists in a dimeric form when biologically active (PDB codes 1dp2
and 1h4k). The program Phaser (McCoy et al., 2005) from the CCP4
suite of programs (Collaborative Computational Project, Number 4,
1994) confirmed the presence of a dimer; Phaser was used to deter-
mine the structure solution by molecular replacement for MtbCysA3
using the coordinates of PDB entry 1uar (T. thermophilus, 52%
identity). Crystallographic refinement and molecular-structure
analysis will be published in a future communication.
X-ray diffraction data were collected on beamline 8.3.1 at the
Advanced Light Source (ALS) at Lawrence Berkeley National
Laboratory under agreements with the Alberta Synchrotron Institute
(ASI). The ALS is supported by the National Institutes of Health and
operated by the Department of Energy. Beamline 8.3.1 was funded by
the National Science Foundation, the University of California and
Henry Wheeler. The ASI synchrotron-access program is supported
by grants from the Alberta Science and Research Authority (ASRA)
and the Alberta Heritage Foundation for Medical Research
(AHFMR). Research in the laboratory of MNGJ is supported by
Alberta Heritage Foundation for Medical Research (AHFMR);
MNGJ is the holder of a Canada Research Chair in Protein Structure
and Function.
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    • "Fourth group of rhodaneses are elongated active-site loop proteins like cdc25 phosphatase which is based on conserved residues in the active-site loop have also been characterized from Arabidopsis thaliana (Landrieu et al. 2004). Rhodaneses have been demonstrated and purified from bovine and bacterial sources including E. coli (Spallarossa et al. 2004), Azotobacter vinelandii (Colnaghi et al. 1996), Mycobacterium tuberculosis (Sarah et al. 2008) and P. aeruginosa (Cipollone et al. 2004). Metagenomics, developed over the past decade has been used to identify wide range of potential enzymes from uncultured microorganisms from various environments (Schloss and Handelsman 2003; Streit et al. 2004; Ranjan et al. 2005; Steele and Streit 2005; Rhee et al. 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Rhodanese is a multifunctional, sulfur transferase that catalyzes the detoxification of cyanide by sulphuration in a double displacement (ping pong) mechanistic reaction. In the present study, small-insert metagenomic library from soil sample collected from Ladakh (3,000–3,600 m.a.s.l) in northwestern Himalayas, India was constructed. Function-driven screening of ~8,500 colonies led to the isolation of one esterase-positive clone (clone-est) harboring 2.43 kb insert. Sequence analysis of the insert identified two ORF’s, phosM encoding phosphoesterase and rodM encoding rhodanese. The 800 bp rodM gene encoded a polypeptide of 227 amino acids (RodM). The RodM showed maximum homology with the rhodanese-like protein from Cyanobacterium synechococcus species with a score identity of only 51 %. Putative 3D structure of RodM developed by homology modeling resembles to homodimeric protein of SUD sulfur transferase of Wolinella succinogenes with properly structured active-site cysteine (Cys) residue. Rhodanese has been reported from few culturable microorganisms.
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