Acta Crystallographica Section F
Editors: H. M. Einspahr and M.S.Weiss
Crystallization and preliminary X-ray diffraction analysis of
the high molecular weight ketoacyl reductase FabG4
complexed with NADH
Debajyoti Dutta, Sudipta Bhattacharyya and Amit Kumar Das
Acta Cryst. (2012). F68, 786–789
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Acta Cryst. (2012). F68, 786–789Dutta et al. · FabG4
Acta Cryst. (2012). F68, 786–789
Acta Crystallographica Section F
Crystallization and preliminary X-ray diffraction
analysis of the high molecular weight ketoacyl
reductase FabG4 complexed with NADH
Debajyoti Dutta, Sudipta
Bhattacharyya and Amit Kumar
Department of Biotechnology, Indian Institute of
Technology, Kharagpur, Kharagpur 721 302,
Received 12 January 2012
Accepted 5 May 2012
FabG4 from Mycobacterium tuberculosis belongs to the high molecular weight
ketoacyl reductases (HMwFabGs). The enzyme requires NADH for ?-ketoacyl
reductase activity. The protein was overexpressed, purified to homogeneity and
crystallized as a FabG4–NADH complex. A mountable FabG4:NADH complex
crystal diffracted to 2.59 A˚resolution and belonged to space group P1, with
unit-cell parameters a = 63.07, b = 71.03, c = 92.92 A˚, ? = 105.02, ? = 97.06,
? = 93.66?. The Matthews coefficient suggested the presence of four monomers
in the unit cell. In addition, a self-rotation function revealed the presence of two
twofold NCS axes and one fourfold NCS axis. At ? = 180?the highest peak
corresponds to the twofold NCS between two monomers, whereas the second
peak corresponds to the twofold NCS between two dimers.
Complex circuits of lipid metabolism in Mycobacterium tuberculosis
enable the organism to synthesize several unusual lipid derivatives
for its virtually impenetrable cell envelope. Nonpolar lipid derivatives
resist the entrance of deleterious molecules generated by the host
into the bacterial cell. The bacteria also keep changing their cell-
envelope composition in response to environmental conditions
(Makinoshima & Glickman, 2005). Lipid metabolism, including fatty-
acid metabolism, thus plays a significant role in this organism.
Fatty-acid synthesis in M. tuberculosis is classified into two types:
FAS-I and FAS-II (Takayama et al., 2005). The eukaryotic-like single
enzyme multi-domain system FAS-I synthesizes fatty-acyl chains of
up to 20 carbons in length. FAS-II, which is a set of enzymes,
subsequently elongates the fatty-acyl chain up to 70–80 carbons in
length. The elongation module of the FAS-II pathway consists of the
enzymes ketoacyl synthase (KasA/KasB; Mdluli et al., 1998; Schaeffer
et al., 2001; Gao et al., 2003), ketoacyl reductase (FabG1 or MabA;
Marrakchi et al., 2002), hydroxyacyl dehydratase (HadAB/HadBC;
Sacco et al., 2007), enoyl reductase (InhA; Dessen et al., 1995) and an
acyl carrier protein (AcpM; Wong et al., 2002). FabG1, which cata-
lyses the second step in the FAS-II module, is also known as ?-
ketoacyl reductase. The M. tuberculosis genome encodes more than
one copy of the ?-ketoacyl reductase gene. The annotated FabG
genes are fabG1 (Rv1483), fabG2 (Rv1350), fabG3 (Rv2002), fabG4
(Rv0242c), fabG5 (Rv2766c) and fabG6 (Rv3502c). Ketoacyl
reductases belong to the short-chain dehydrogenase/reductase
(SDR) family and mostly have a core structure of 250–350 amino
acids in length (Oppermann et al., 2003). All of the FabG gene
products of M. tuberculosis are 247–317 amino acids in length, with
the exception of FabG4. FabG4 is unusually long (454 amino acids)
and contains two distinguishable domains (Dutta et al., 2011). The
N-terminal domain is a flavodoxin-type domain and the C-terminal is
a ketoreductase domain. Thus, this protein, along with its sequence
homologues, is categorized as a high molecular weight FabG
(HMwFabG). Although the role of FabG4 in mycobacteria is yet to
# 2012 International Union of Crystallography
All rights reserved
be addressed, some reports have recently identified FabG4 as an
essential gene in mycobacteria (Beste et al., 2009; Gurvitz, 2009;
Sharma et al., 2010).
The C-terminal domain (215–454) of FabG4 shares 32% sequence
identity with M. tuberculosis FabG1 (Cohen-Gonsaud et al., 2002),
35% with Plasmodium falciparum FabG (Wickramasinghe et al.,
2006), 36% with Brassica napus FabG (Fisher et al., 2000), 38% with
Staphylococcus aureus FabG1 (Dutta et al., 2012) and Rickettsia
prowazekii FabG (Subramanian et al., 2011), 39% with Bacillus
anthracis FabG (Zaccai et al., 2008), 40% with Aquifex aeolicus FabG
(Mao et al., 2007) and 41% with Escherichia coli FabG (Price et al.,
2001). Unlike all of these FabGs, FabG4 utilizes NADH for catalysis.
In the cases of E. coli FabG and M. tuberculosis FabG1, NADPH has
been hypothesized to reorient the catalytic residues in position for
catalysis (Price et al., 2004; Cohen-Gonsaud et al., 2005). These holo
structures also maintain two loops near the catalytic residues in stable
conformations. In some FabG structures the stability of these two
loops does not depend on the coenzyme (Wickramasinghe et al.,
2006). It has beenproposed that the amino-acid residue preceding the
catalytic serine is responsible for holding the catalytic loop in its
proper position (Poncet-Montange et al., 2007). We have recently
determined the crystal structure of apo FabG417–448, truncating the
six C-terminal residues
C-terminal residues are involved in a hydrophobic interaction with
the corresponding catalytic loop in FabG4, thereby stabilizing it. This
clearly indicates the role of the conserved C-terminus in HMwFabGs.
Intriguingly, a conserved C-terminus is also observed for FabG1s
among mycobacterial species (Cohen-Gonsaud et al., 2005).
Although much study has been carried out on FabGs, the nature
of the HMwFabGs has remained obscure, including their coenzyme
specificity. The present work reports the overexpression, purification,
crystallization and preliminary X-ray diffraction studies of the
449QAMIGA454(Dutta et al., 2011). The
2. Materials and methods
2.1. Purification of FabG417–454
The cloning of the FabG4 gene from the M. tuberculosis H37Rv
genome in pQE30 expression vector and the overexpression of
recombinant His6-FabG417–454in E. coli M15 (pREP4) cells have
previously been reported (Dutta et al., 2011). The entire FabG4 gene
was not soluble. The protein became soluble upon truncation of the
first 16 amino-acid residues.
The cells from 2 l culture were resuspended in buffer A (10 mM
Tris–HCl pH 7.9, 300 mM NaCl, 10 mM imidazole, 5% glycerol)
containing 0.1 mM each of leupeptin, aprotinin and pepstatin and
0.02 mM phenylmethylsulfonyl fluoride. The following steps were
carried out at 277 K. The suspension was lysed by ultrasonication
and the resulting lysate was centrifuged at 22 000g for 30 min. The
performance affinity matrix (GE Healthcare Biosciences) pre-
equilibrated with buffer A. The column was then extensively washed
with buffer A. An intermediate buffer B (10 mM Tris–HCl pH 7.9,
300 mM NaCl, 100 mM imidazole, 5% glycerol) was passed through
the column to remove any nonspecifically bound contaminant.
Finally, the protein was eluted with buffer C (10 mM Tris–HCl pH 7.9,
300 mM NaCl, 300 mM imidazole, 5% glycerol). The eluted protein
was subjected to gel-filtration chromatography using Superdex 200
prep-grade matrix in a 16/70C column (GE Healthcare Biosciences)
pre-equilibrated with buffer D (10 mM Tris–HCl pH 7.9, 200 mM
NaCl, 5% glycerol) on an A¨KTAprime Plus system. The flow rate was
Ni–NTA Sepharose high-
set to 1 ml min?1. The fractions containing the desired protein were
pooled together and kept for further use. The purity of the protein
sample was assessed by SDS–PAGE and the protein concentration
was measured using both the Abs280 and the Bradford method
(Bradford, 1976). The absence of NAD+cofactor was confirmed by
the OD280/OD260ratio as described previously (Krimsky & Racker,
2.2. Crystallization of the FabG417–454:NADH binary complex
Purified FabG4 was concentrated to 20 mg ml?1using a 10 kDa
cutoff Vivaspin 20 concentrator (GE Healthcare). Concentrated
protein was mixed with NADH (Sigma) in a 1:5 molar ratio followed
by an incubation period of 30 min at 300 K. The mixture was
subjected to crystallization using the previously reported crystal-
lization condition for the C-terminally truncated mutant of apo
FabG4 (Dutta et al., 2011). Despite several trials, no crystals appeared
from this condition. The mixture was thus subjected to preliminary
crystallization trials using Crystal Screen, Crystal Screen 2 and Index
from Hampton Research by mixing 2 ml protein/NADH mixture with
an equal volume of reservoir solution using the sitting-drop vapour-
diffusion method. Initial hits were only obtained using a condition
consisting of 0.1 M bis-Tris pH 6.5, 45%(v/v) polypropylene glycol
P400. The single crystals that appeared from the initial crystallization
condition were too tiny to mount (Fig. 1a). The initial crystallization
condition was further fine-screened using the hanging-drop vapour-
diffusion method to optimize the crystal size and quality. After a
number of screening trials with different buffering reagents and
Acta Cryst. (2012). F68, 786–789Dutta et al.
Crystals of the FabG417–454:NADH complex. (a) Initial crystals obtained from the
preliminary crystallization screening. (b) Mountable crystals obtained from 0.1 M
MES pH 6.5, 45%(v/v) polypropylene glycol P400.
precipitating reagents, mountable crystals were obtained from the
condition 0.1 M MES pH 6.5, 45%(v/v) polypropylene glycol P400
2.3. Data collection and processing
For data collection, an FabG417–454:NADH crystal was directly
mounted from the mother liquor and flash-cooled in a nitrogen
stream at 100 K. X-ray diffraction data were collected using an in-
house X-ray diffraction facility. The facility was equipped with a
Rigaku MicroMax-007 HF rotating-anode generator as a Cu K?
X-ray source and a Rigaku R-AXIS IV++image-plate detector. The
crystal-to-detector distance was maintained at 200 mm and the crystal
was rotated 360?with 1?rotation per frame. The crystal diffracted to a
resolution of 2.59 A˚. The images were processed with XDS (Kabsch,
2010) in space group P1 and scaled with SCALA (Evans, 1993) from
the CCP4 suite (Winn et al., 2011). The final statistics of data
collection and processing are tabulated in Table 1.
3. Results and discussion
Recombinant FabG417–454was successfully purified to homogeneity.
The N-terminal residues (1–16) were found to hinder solubility and
hence were truncated without altering the activity (Dutta et al., 2011).
FabG417–454:NADH complex crystals were obtained from 0.1 M MES
pH 6.5, 45%(v/v) polypropylene glycol P400 and diffracted to 2.59 A˚
resolution. The crystals belonged to space group P1, with unit-cell
parameters a = 63.07, b = 71.03, c = 92.92 A˚, ? = 105.02, ? = 97.06,
? = 93.66?, which differed from those of the FabG417–448crystals. The
Matthews coefficient (2.21 A˚3Da?1) confirmed that there were four
monomers in the unit cell (Matthews, 1968). The presence of a single
fourfold NCS axis was also indicated by the self-rotation function plot
at ? = 90?(Fig. 2a). The NCS axis makes an ?30?angle with the
crystallographic y axis. The self-rotation function at ? = 180?(Fig. 2b)
also indicated two twofold NCS axes. The highest peak (peak 1;
Rf = 508.2) is attributed to the dimeric axis between two monomers,
whereas the second peak (peak 2; Rf = 338.7) is attributed to the
dimeric axis between two dimers.
The structure was solved by the molecular-replacement method
with MOLREP (Vagin & Teplyakov, 2010), using a monomer of the
apo FabG4 structure (PDB entry 3lls; Seattle Structural Genomics
Center for Infectious Disease, unpublished work) as a search model.
A promising result with a homotetrameric assembly has been found
with a final score of 0.71; the resulting R factor was 37.4%. The model
was subsequently subjected to rigid-body refinement followed by
restrained refinement using REFMAC5 (Murshudov et al., 2011).
Structural analysis is currently in progress.
This work was carried out using the protein X-ray crystallography
facility funded by the Department of Biotechnology, Government of
India and housed at the Central Research Facility (CRF) of the
Indian Institute of Technology (IIT), Kharagpur. DD acknowledges
the Department of Science and Technology, Government of India for
Dutta et al.
Acta Cryst. (2012). F68, 786–789
Data-collection and processing statistics.
Values in parentheses are for the highest resolution shell.
Unit-cell parameters (A˚,?)
a = 63.07, b = 71.03, c = 92.92,
? = 105.02, ? = 97.06, ? = 93.66
Total No. of observations
No. of unique reflections
Molecules per asymmetric unit (Z)
Matthews coefficient (A˚3Da?1)
ijIiðhklÞ ? hIðhklÞij=P
iIiðhklÞ, where Ii(hkl) is the ith observed
intensity of reflection hkl and hI(hkl)i is the mean intensity over all i measurements.
Self-rotation function plot obtained from the FabG417–454:NADH complex data for
(a) ? = 90?and (b) ? = 180?. Two peaks (peak 1 and peak 2) are indicated by red
a fellowship. SB thanks IIT Kharagpur for an individual institute Download full-text
Beste, D. J., Espasa, M., Bonde, B., Kierzek, A. M., Stewart, G. R. &
McFadden, J. (2009). PLoS One, 4, e5349.
Bradford, M. M. (1976). Anal. Biochem. 72, 248–254.
Cohen-Gonsaud, M., Ducasse, S., Hoh, F., Zerbib, D., Labesse, G. & Quemard,
A. (2002). J. Mol. Biol. 320, 249–261.
Cohen-Gonsaud, M., Ducasse-Cabanot, S., Quemard, A. & Labesse, G. (2005).
Proteins, 60, 392–400.
Dessen, A., Que ´mard, A., Blanchard, J. S., Jacobs, W. R. & Sacchettini, J. C.
(1995). Science, 267, 1638–1641.
Dutta, D., Bhattacharyya, S. & Das, A. K. (2012). Proteins, 80, 1250–1257.
Dutta, D., Bhattacharyya, S., Mukherjee, S., Saha, B. & Das, A. K. (2011). J.
Struct. Biol. 174, 147–155.
Evans, P. R. (1993). Proceedings of the CCP4 Study Weekend. Data Collection
and Processing, edited by L. Sawyer, N. Isaacs & S. Bailey, pp. 114–122.
Warrington: Daresbury Laboratory.
Fisher, M., Kroon, J. T., Martindale, W., Stuitje, A. R., Slabas, A. R. & Rafferty,
J. B. (2000). Structure, 8, 339–347.
Gao, L.-Y., Laval, F., Lawson, E. H., Groger, R. K., Woodruff, A., Morisaki,
J. H., Cox, J. S., Daffe, M. & Brown, E. J. (2003). Mol. Microbiol. 49, 1547–
Gurvitz, A. (2009). Mol. Genet. Genomics, 282, 407–416.
Kabsch, W. (2010). Acta Cryst. D66, 125–132.
Krimsky, I. & Racker, E. (1963). Biochemistry, 2, 512–518.
Makinoshima, H. & Glickman, M. S. (2005). Nature (London), 436, 406–409.
Mao, Q., Duax, W. L. & Umland, T. C. (2007). Acta Cryst. F63, 106–109.
Marrakchi, H., Ducasse, S., Labesse, G., Montrozier,H., Margeat, E., Emorine,
L., Charpentier, X., Daffe ´, M. & Que ´mard, A. (2002). Microbiology, 148,
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.
Mdluli, K., Slayden, R. A., Zhu, Y., Ramaswamy, S., Pan, X., Mead, D., Crane,
D. D., Musser, J. M. & Barry, C. E. III (1998). Science, 280, 1607–1610.
Murshudov, G. N., Skuba ´k, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A.,
Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst.
Oppermann, U., Filling, C., Hult, M., Shafqat, N., Wu, X., Lindh, M., Shafqat,
J., Nordling, E., Kallberg, Y., Persson, B. & Jo ¨rnvall, H. (2003). Chem. Biol.
Interact. 143–144, 247–253.
Poncet-Montange, G., Ducasse-Cabanot, S., Quemard, A., Labesse, G. &
Cohen-Gonsaud, M. (2007). Acta Cryst. D63, 923–925.
Price, A. C., Zhang, Y.-M., Rock,C. O. & White, S. W. (2001). Biochemistry, 40,
Price, A. C., Zhang, Y.-M., Rock, C. O. & White, S. W. (2004). Structure, 12,
Sacco, E., Covarrubias, A. S., O’Hare, H. M., Carroll, P., Eynard, N., Jones,
T. A., Parish, T., Daffe ´, M., Ba ¨ckbro, K. & Que ´mard, A. (2007). Proc. Natl
Acad. Sci. USA, 104, 14628–14633.
Schaeffer, M. L., Agnihotri, G., Volker, C., Kallender, H., Brennan, P. J. &
Lonsdale, J. T. (2001). J. Biol. Chem. 276, 47029–47037.
Sharma, P., Kumar, B., Singhal, N., Katoch, V. M., Venkatesan, K., Chauhan,
D. S. & Bisht, D. (2010). Indian J. Med. Res. 132, 400–408.
Subramanian, S., Abendroth, J., Phan, I. Q. H., Olsen, C., Staker, B. L., Napuli,
A., Van Voorhis, W. C., Stacy, R. & Myler, P. J. (2011). Acta Cryst. F67, 1118–
Takayama,K., Wang, C. & Besra, G. S. (2005).Clin. Microbiol. Rev.18, 81–101.
Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22–25.
Wickramasinghe, S. R., Inglis, K. A., Urch, J. E., Mu ¨ller, S., van Aalten, D. M.
& Fairlamb, A. H. (2006). Biochem. J. 393, 447–457.
Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242.
Wong, H. C., Liu, G., Zhang, Y.-M., Rock, C. O. & Zheng, J. (2002). J. Biol.
Chem. 277, 15874–15880.
Zaccai, N. R., Carter, L. G., Berrow, N. S., Sainsbury, S., Nettleship, J. E.,
Walter, T. S., Harlos, K., Owens, R. J., Wilson, K. S., Stuart, D. I. & Esnouf,
R. M. (2008). Proteins, 70, 562–567.
Acta Cryst. (2012). F68, 786–789Dutta et al.