Bioinformatic Analysis of Leishmania donovani Long-Chain Fatty Acid-CoA Ligase as a Novel Drug Target.

SAGE-Hindawi Access to Research
Molecular Biology International
Volume 2011, Article ID 278051, 14 pages
doi:10.4061/2011/278051
Research Article
Bioinformatic Analysis of Leishmania donovani Long-Chain
Fatty Acid-CoA Ligase as a Novel Drug Target
Jaspreet Kaur, Rameshwar Tiwari, Arun Kumar, and Neeloo Singh
Drug Target Discovery & Development Division, Central Drug Research Institute (CSIR), Chattar Manzil Palace,
Lucknow 226001, India
Correspondence should be addressed to Neeloo Singh, neeloo888@yahoo.com
Received 14 January 2011; Revised 29 March 2011; Accepted 13 April 2011
Academic Editor: Hemanta K. Majumder
Copyright © 2011 Jaspreet Kaur et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Fatty acyl-CoA synthetase (fatty acid: CoA ligase, AMP-forming; (EC 6.2.1.3)) catalyzes the formation of fatty acyl-CoA by a
two-step process that proceeds through the hydrolysis of pyrophosphate. Fatty acyl-CoA represents bioactive compounds that
are involved in protein transport, enzyme activation, protein acylation, cell signaling, and transcriptional control in addition
to serving as substrates for beta oxidation and phospholipid biosynthesis. Fatty acyl-CoA synthetase occupies a pivotal role in
cellular homeostasis, particularly in lipid metabolism. Our interest in fatty acyl-CoA synthetase stems from the identification of
this enzyme, long-chain fatty acyl-CoA ligase (LCFA) by microarray analysis. We found this enzyme to be differentially expressed
by Leishmania donovani amastigotes resistant to antimonial treatment. In the present study, we confirm the presence of long-chain
fatty acyl-CoA ligase gene in the genome of clinical isolates of Leishmania donovani collected from the disease endemic area in
India. We predict a molecular model for this enzyme for in silico docking studies using chemical library available in our institute.
On the basis of the data presented in this work, we propose that long-chain fatty acyl-CoA ligase enzyme serves as an important
protein and a potential target candidate for development of selective inhibitors against leishmaniasis.
1. Introduction
Leishmaniasis is a disease caused by protozoan parasites
of the Leishmania genus. Visceral leishmaniasis (VL), also
known as kala-azar, is the most severe form of leishmaniasis
(http://www.dndi.org/diseases/vl.html). With no vaccine in
sight, treatment for kala-azar relies primarily on chemother-
apy [1].
Phylogenetics suggests that Leishmania is relatively early
branching eukaryotic cells and their cell organization differs
considerably from that of mammalian cells [2, 3]. Hence, the
biochemical differences between the host and parasite can be
exploited for identification of new targets for rational drug
design. It is also imperative that the probability of developing
drug resistance should be less with these targets. This can be
achieved by targeting an essential cellular process, which has
the pressure to remain conserved and cannot be bypassed by
using alternative pathway.
One interesting target which emerged from our microar-
ray experiments [4] was long-chain fatty acid-CoA ligase
(EC 6.2.1.3) (GenBank Accession No. XM 001681734), a
key enzyme involved in the metabolism of fatty acids in all
organisms [5–9]. Fatty acyl-CoA has multiple roles involved
in protein transport [10, 11], enzyme activation [12], protein
acylation [13], cell signaling [14], transcriptional regula-
tion [15], and particularly β-oxidation and phospholipid
biosynthesis. Especially in Leishmania, long-chain fatty acids
are predominant precursors of total lipid composition (the
combination of phospholipids, sphingolipids, and ergos-
terol). Long-chain fatty acyl-CoA ligase is critical enzyme
processing long-chain fatty acid acylation which is essential
for lysophosphatidylinositol (lyso-PI) incorporation into
glycosyl phosphatidylinositols (GPIs) [16, 17]. These GPIs-
anchors are the major surface virulent factors in Leishmania
and have received considerable attention [18]. De novo
sphingolipid biosynthesis starts with the condensation of
serine and the product of long-chain fatty acyl-CoA ligase. L.
major preferentially incorporates myristoyl-CoA (C14) over
palmitoyl-CoA (C16) into their long-chain base [19, 20].
This selection of specific long-chain fatty acyl-CoA reflects

2 Molecular Biology International
120000 125000 130000 135000 140000
LmjF13.0400 LmjF13.0410 >LmjF13.0420< LmjF13.0430 MKK2 LmjF13.0450
LmjF13.0420 X
Putative protein Putative protein
conserved
Long-chain fatty
acid-CoA ligase
protein, putative
Flagellar
radial spoke
protein,
putative
Mitogen-activated
protein kinase
kinase-2
Hypothetical
protein
conserved
Long-chain fatty acid-CoA
ligase protein, putative
Figure 1: Graphical representation of long-chain fatty acyl-CoA ligase (LCFA) gene (in Artemis) on chromosome 13 of Leishmania major.
the presence of myristoyl-specific long-chain fatty acyl-CoA
ligase in Leishmania [21].
Gaining new knowledge on fatty acid metabolism will
not only provide fundamental insight into the molecular
bases of Leishmania pathogenesis but also reveal new targets
for selective drugs. Enzymes involved in fatty acid and sterol
metabolism have been shown to be important pharmaceu-
tical targets in Leishmania and other kinetoplastida [22].
Triacsin C, a specific inhibitor of long-chain fatty acyl-CoA
synthetase, was shown to have an inhibitory effect on the
growth of Cryptosporidium parvum in vitro [23].
Four fatty acyl-CoA synthetases have been described
previously in Trypanosoma brucei, displaying different chain-
length specificities [24, 25]. The whole genome sequence
of three Leishmania spp. (L. major, L. infantum, and L.
braziliensis) has been sequenced, and the availability of
putative long-chain fatty acyl-CoA ligase genes was present
in all three Leishmania spp. at chromosome 13, which would
be required for initiation of β-oxidation and fatty acid
metabolism.
In the present study we confirm the presence of long-
chain fatty acyl-CoA ligase gene in Leishmania donovani
clinical isolate collected from, the state of Bihar India [26–
29], which alone accounts for 50% of the total burden of
visceral leishmaniasis worldwide [30]. Further progress in
the understanding of this enzyme is likely to be achieved
through the whole genome sequence (WGS) project of
these clinically important isolates [26–29], underway in our
laboratory (http://www.leishmaniaresearchsociety.org/).
2. Material and Methods
2.1. Collection of Clinical Isolates. The clinical isolates of
L. donovani were collected from two kala-azar patients
selected from Muzaffarpur, Bihar. The criterion for visceral
leishmaniasis diagnosis was the presence of Leishman-
Donovan (LD) bodies in splenic aspirations performed,
which was graded to standard criteria [30]. Response to
sodium antimony gluconate (SAG) treatment was evaluated
by repeating splenic aspiration at day 30 of treatment.
Patients were designated as antimonial responsive (L. dono-
vani isolate 2001) based on the absence of fever, clinical
improvement with reduction in spleen size, and absence
of parasites in splenic aspirate while patients who showed
presence of parasites in splenic aspiration were considered to
be antimonial unresponsive (L. donovani isolate 39) [26–29].
2.2. Sample Collection and Nuclear DNA Isolation. L. dono-
vani isolates 2001 (SAG-sensitive) and 39 (SAG-resistant)
used in the present study, were maintained in culture as
described previously in [26–29]. For nuclear DNA isolation
10–15 mL log-phase culture was taken and centrifuged at
5,000 rpm for 8 min at 4◦C. The supernatant was decanted;
cell pellet was resuspended in 3–6 mL NET buffer and
centrifuged at 5,000 rpm for 8 min at 4◦C. The supernatant
was discarded, and the pellet was redissolved in 750 μL
NET buffer, 7.5 μL proteinase K (10 mg/mL stock) (MBI,
Fermentas, Cat No. EO0491), and 50 μL of 15% sarkosyl.
Sample was incubated at 37◦C overnight for proteinase
K activity. The cell lysate was centrifuged at 18,000 rpm
for 1 hr at 4◦C. The supernatant containing nuclear DNA
was transferred to a fresh tube and given RNase treatment
(20 μg/mL) (MBI, Fermentas, Cat No. EN0531) at 37◦C
for 30 min. DNA was extracted first with one volume phe-
nol/chloroform/isoamyl alcohol (25 : 24 : 1) and finally with
chloroform. Nuclear DNA was precipitated with 2.5 volumes
of prechilled absolute ethanol, dissolved in nuclease-free
water and stored at 4◦C for future use.
2.3. Primer Design, PCR Amplification, and Sequencing of
Long-Chain Fatty Acyl-CoA Ligase Gene. PCR amplification
was carried out using Pfu DNA polymerase (MBI, Fermentas,
Cat No. EP0501). Reactions were carried out in a Perkin
Elmer GeneAmp PCR system with 2001 nuclear DNA (10–
50 ng) as template. The following oligonucleotide primers
were designed on the basis of available gene sequence of
L. major (GenBank Accession No. XM 001681734): for-
ward primer: 5′GGGCCATATGCTGCAGCG 3′ (18 mer) and
reverse primer: 5′GGCCTCGAGCTAAAACAAATCATCG3′
(25 mer). The amplification conditions were initial denatu-
ration at 95◦C for 10 min, denaturation at 95◦C for 30 sec,
annealing at 65◦C for 1 min, extension at 72◦C for 2 min,
and final extension at 72◦C for 10 min; 30 cycles. The PCR
product was purified from agarose gel using MBI Fermentas
DNA Extraction kit (MBI, Fermentas, Cat No. K0513) and
further for DNA sequencing by Bangalore Genei, India.

Molecular Biology International 3
LCFA (2 Kb)
( )
( )
2791 bp 762 bp 2791 bp
2460 bp 2312 bp
3848 bp
M M M M LCFA
50
150
500
750
1000
1500
2000
50
150
500
750
1000
1500
21226
A B C D
2001 39 2001 39 2001 39
PvuII
BamHI
XhoI
P
vu
II
B
am
H
I
X
ho
I
P
vu
II
B
am
H
I
X
ho
I
P
vu
II
B
am
H
I
X
ho
I
P
vu
II
B
am
H
I
X
ho
I
P
vu
II
B
am
H
I
X
ho
I
P
vu
II
B
am
H
I
X
ho
I
Figure 2: Determination of long-chain fatty acyl-CoA ligase gene copy number. The nuclear DNA of L. donovani 2001 and 39 promastigotes
was isolated and 16 μg was digested with different restriction enzymes. (A) Nuclear DNA digest stained with ethidium bromide (B) Southern
blot of “A” with α-tubulin gene probe. (C) Southern blot of “A” with long-chain fatty acyl-CoA ligase probe. (D) PCR amplification of
long-chain fatty acyl-CoA ligase gene (M: Marker, LCFA: 2010 bp of long-chain fatty acyl-CoA ligase gene).
Table 1: Selected ortholog for Leishmania donovani long-chain fatty acyl-CoA ligase gene in kinetoplastida: ORTHOMCL4080
(http://www.genedb.org/).
Systematic ids Organism Product
LbrM13 V2.0240
L. braziliensis
MHOM/BR/75/M2904
Fatty acid thiokinase (long chain), putative; acyl-CoA synthetase, putative;
long-chain-fatty acid-CoA ligase protein, putative
LinJ13 V3.0300 L. infantum JPCM5
Fatty acid thiokinase (long chain), putative; long-chain-fatty acid-CoA
ligase protein, putative; acyl-CoA synthetase, putative
LmjF13.0420 L. major strain Friedlin
Long-chain fatty acid-CoA ligase protein, putative; acyl-CoA synthetase,
putative; fatty acid thiokinase (long chain), putative
Tb11.02.2070 T. brucei 927
Long-chain fatty acid-CoA ligase protein, putative; fatty acid thiokinase
(long chain), putative; acyl-CoA synthetase, putative
Tc00.1047053504089.40 T. cruzi
Long-chain fatty acid-CoA ligase protein, putative; acyl-CoA synthetase,
putative; fatty acid thiokinase, long chain, putative
TvY486 1104610 T. vivax Long-chain fatty acid-CoA ligase protein, putative
2.4. Characterization of Long-Chain Fatty Acyl-CoA Ligase
Gene. L. donovani nuclear DNA (16 μg for each reaction) of
two different clinical isolates, drug (SAG) sensitive 2001 and
drug (SAG) resistant 39, were digested with 40-unit three
different restriction enzymes (PvuII, BamHI, and XhoI),
which were cut overnight and separated on 0.8% agarose
gel by electrophoresis at 50 V. In order to improve transfer
efficacy, DNA in agarose gel was treated with 0.25 N HCl
for 15 min (partial depurination), rinsed with autoclaved
water 3x, and treated with 0.4 N NaOH (breaking backbone
at depurinated region) for 30 min. DNA was transferred
to nylon membrane by conventional downward capillary
transfer method for 5 h using 3 mm Whatman paper wick
[8]. The efficiency of transfer was assessed by visualizing
DNA by methylene blue staining. After transfer on nylon
membrane the DNA was neutralized in 0.5 M Tris (pH
7.4), 1.5 M NaCl, 2x for 5 min at room temperature. The
membrane was then washed in 2X SSC, 2x for 15 min. Nylon
membrane was incubated with 2.5 mL of prehybridization
buffer (0.6 M NaCl, 0.5 M Tris-HCl (pH 7.5), 0.008 M
EDTA, 1% sodium pyrophosphate, 0.2% SDS, and 50 μg/mL
heparin) and incubated in a hybridization oven at 65◦C for
2 h. Radioactive probe was prepared by labeling 25 ng of
the DNAs with [α-32P] dCTP by random priming method
(BRIT/BARC, India) and purified using a desalting column
(sephadex G-50). The radioactivity was checked with a
Geiger Muller Counter (dosimeter) and stored at −20◦C.
The probe was added to the prehybridization buffer and
incubated at 65◦C overnight in hybridization oven. Mem-
brane was washed twice with 2X SSC, 0.1% SDS (15 min
each) at 65◦C and then washed with 2X SSC, 0.1% SDS for
30 min at 65◦C to reduce background signals. Hybridized

4 Molecular Biology International
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
10
35
13
10
37
39
12
74
23
10
46
78
35
110
60
41
84
117
45
119
98
47
94
156
82
157
136
84
130
195
121
196
175
123
169
234
155
235
211
157
204
273
11
11
36
14
38
40
13
75
24
11
47
79
36
111
61
42
85
118
46
120
99
48
95
157
83
158
137
85
131
196
122
197
176
124
170
235
1 MEGERMNA- - - - F - - - - - - - - - - - - - - - P - - - - - - - - - -
1 MQAHE L F R - - - - Y F RMP E L VDF R QCVT L P T NT L MGF GAF
1 MVAQY T VP - - - - VGKA- - - - - - - - - - - - - - - - - - - - - - A
1 MKKVWL NR - - - - Y P - - - - - - - - - - - - - - - - - - - - - - - - -
1 ML QR L S HR S VL GY HGAP QVAAL R CS L R F GT NI - - GGS Y Y
1 *
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S T
S R R L T T FWR P R HP KP L KP PWHL S MQS VE VAGS GGAR R S A
NE H- - - - - - - - - - - - - - - - - - - - - - - - E - - - - - T AP R R N
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
QE S - - - - - - - - - - - - - - - - - - - - - - - - D - - - - - T AG- T H
M- - - - - - - - - - MDE - E L NLWDF L E R AAAL F GR KE - - - - -
L L DS DE P L VY F Y DD- VT T L Y E GF QR GI QVS NNGP CL G- -
Y QCR E KP L VR P P NT KCS T VY E F VL E CF QKNKNS NAMG- -
- - - ADVP T E I NP DR - Y QS L VDMF E QS VAR Y ADQ- - P A- -
QMNNDI R L GL VI KP - QS T L VE LWE QAL KA P T R R F L GS K
. . : : .
VVS R L H- - - - - - - - - - - - - - - - - - - - - - - - - - - - T G- E V
- S R KP DQ- - - - - - - - - - - - - - - - - - - - - - - - - - - - - P Y E
- WR DVKE I HE E S KS VMKKVDGKE T S VE KKWMY Y E L S HY H
- F VN- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MG- - E
MWVR GK- - - - - - - - - - - - - - - - - - - - - - - - - - - - L G- Y V
HR T T Y AE VY QR AR R L MGGL R A- - L GVGVGDR VAT L GF NH
- WL S Y KQVAE L S E CI GS AL I QKGF KT AP DQF I GI F AQNR
- Y NS F DQL T DI MHE I GR GL VKI GL KP NDDDKL HL Y AAT S
- VMT F R KL E E R S R AF AAY L Q- QGL GL KKGDR VAL MMP NL
- WAT Y E S I NT E VE AMR T L L HS - - MGVE KGS R VVVI S E NR
: : . : . : * : . . : .
F R HL E A Y F A V P GMGA V L HT A NP R L S P K E I A Y I L NHA E DK
P EWV I I E QGC F A Y S MV I V P L Y DT L GNE A I T Y I V NK A E L S
HKWMKMF L GA QS QGI P V V T A Y DT L GE K GL I HS L V QT GS K
L QY P V A L F GI L R A GMI V V NV NP L Y T P R E L E HQL NDS GA S
Y EWV V V HF A TMQL GA HF V A L P T NV T P S E A QL V V K S T QS K
. . . . : : .
V L L F DP NL - L - P L V E A I R GE L - K T V QHF V VMDE K A P E - -
L V F V DK P E K A K L L L E GV E NK L I P GL K I I V VMDS Y GS E L V
A I F T DNS L - L P S L I K P V Q- A - A QDV K Y I I HF DS I S S E DR
A I V I V S NF - A HT L E K V V D- K - T - A V QHV I L T R - MGDQL S
V L F V E T K A - S Y A A I K GWI GR V - GQL E HA I C F E DQV GE - -
: . : : : : :
W
(a)
Figure 3: Continued.

Molecular Biology International 5
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
156
236
212
158
205
274
166
257
249
195
220
313
201
293
287
231
255
352
238
332
324
269
292
391
267
359
357
299
321
430
289
395
395
318
358
469
300
433
432
332
397
508
165
256
248
194
219
312
200
292
286
230
254
351
237
331
323
268
291
390
266
358
356
298
320
429
288
394
394
317
357
468
299
431
432
331
396
507
336
471
469
369
433
546
- - - GY - - - - - - - - - - - - - - - - - L A Y - - E E A L - - - - - - G-
E - R GQ- - - - - - - - - - - - - - - - - R C GV E V T S MK AME DL GR
R QS GK I Y QS A HDA I NR I - - K E V R P DI K T F S F DDI L K L GK
T A K GT V V NF V V K Y I K R L V P K Y HL P D- - A I S F R S A L HNGY
- - - GS - - - - - - - - - - - - - - - - - Y A V - - A I S I - - A A DV P E
* : :
- - - E E A DP V - R V P E R A A C GMA Y T T GT T GL P K GV V Y S HR A
- - A NR R K P K P P A P E DL - A V I C F T S GT T GNP K GAMV T HR N
E S C NE I DV HP P GK DDL - C C I MY T S GS T GE P K GV V L K HS N
R - MQY V K P E - L V P E DL - A F L QY T GGT T GV A K GAML T HR N
- - K T L A R T D- V R A E DT - AMI V F T A GT T GP P K GVML S HK S
: . : : * * : * * . * * . : . *
L V L HS L A A S - L V DGT A L S - E K DV V L P V V P MF HV NAWC L P
I V S DC S A F V K A T E NT V NP C P DDT L I S F L P L A HMF E R V V E
V V A GV GGA S - L NV L K F V G- NT DR V I C F L P L A HI F E L V F E
ML A NL E QV N- A T Y GP L L HP GK E L V V T A L P L Y HI F A L T I N
I V A NV S S V Y - A S L GE A L T - HS DMF MS L C S WC V A GA L T T D
: : : . : .
- Y A A T L V - G- A K QV L - - - - - - P - GP R L DP A S L V E L F DGE
- C VML C H- G- A K I GF - - - - - - F QGDI R L L MDDL K V L - - -
- L L S F YW- G- A C I GY A T V K T L T S S S V R NC QGDL QE F - - -
- C L L F I E L GGQNL - - - - - - - L - I T NP R DI P GL V K E L A K Y
L Y QA L C K - G- A C V C I - - - - - - P - P E I L E GF QDL P L V NP -
* : .
P-loop
G-motif
GVT F T A GVP T VWL A - - - - L A DY L E S T - - - - - - - - - - - - -
QP T VF P VVP R L L NRMF DR I F GQA NT - - T VKRWL L DF A S -
KP T I MVGVA A VWE T VR KGI L NQI DNL P F L T KKI FWT A Y -
P F T A I T GVNT L F NA - - - - L L NNK- - - - - - - - - - - - - - - -
- - S VI VS VA QP F QR A Y A NI VDDI L NR R S F T KDL T R F VVG
: * : .
- - - - - - - - - - - - - - - - - - - - GHR L - - - - - - - - KT L R R L V
KR KE A DVR S GI - I R NNS LWDR L I F HKVQS S L GGR VR L MV
NT KL NMQR L H- - I P GGGA L GNL VF KKI R T A T GGQL R Y L L
- - - - - - - - - - - - - - - - - E F QQL DF S - - - - - - - - S L HL S A
R I T E NR L MF KKP GR T L QA F S HA L L GKF KA QF GS E L R VA I
: : :
VGGS A A P R S L I A R F E R - MGVE VR QGY GL T E T S P V- VVQN
T GA A P VS A T VL T F L R A A L GCQF Y E GY GQT E CT A GCCL T M
NGGS P I S R DA QE F I T NL - I CP ML I GY GL T E T CA S T T I L D
GGGMP VQQVVA E RWVKL T GQY L L E GY GL T E CA P - L VS VN
I I GHQL T KDQS E L MA D- L DVF VVNT Y GF ME A GGL - VA T D
. . * * *
A-motif
(b)
Figure 3: Continued.

6 Molecular Biology International
F VKS HL E S L S E E E KL T L KAKT GL P I P L VRL RVAD- - E E G
P G- - DW- - - - - - - - - T - T GHVGAPMP CNL I KL GWQL E EM
P A- - NF - - - - - - - - - E - L GVAGDL T GCVT VKL VD- VE E L
P Y DI DY - - - - - - - - - H- S GS I GL P VP S T E AKL VD- - DDD
V- DVP - - - - - - - - - - - - QRL KA- - L P GL E VRVVN- - E KN
. : : : .
RP VP KDGKAL GE VQL KGPWI T GGY Y G- - - NE E AT RS AL T
NYMAS E G- - E GE VCVKGP NVF QGY L K- - - DP AKT AE AL D
GY F AKNN- - QGE VWI T GANVT P E Y Y K- - - NE E E T S QAL T
NE VP P GQ- - P GE L CVKGP QVML GYWQ- - - RP DAT DE - I I
E I VAP GD- - L GE I L I E AP NAMQGY F DVHI DP E E AKNS L V
. . * * : : . . * : . :
P D- - - GF F RT GDI AVWDE E GY VE I KDRL KDL I K- S GGEW
KD- - - GWL HT GDI GKWL P NGT L KI I DRKKHI F KL AQGE Y
S D- - - GWF KT GDI GEWE ANGHL KI I DRKKNL VKT MNGE Y
KN- - - GWL HT GDI AVMDE E GF L RI VDRKKDMI L - VS GF N
E Y GS RT F VRS GDY GT L - T GGWI T VKGNKDVL I T L ANS KT
: . : : * * . * : : . . . : . .
L-motif
I S S VDL E NAL MGHP KVKE AAVVAI P HP KWQE R P L AVVVP
I AP E KI E NI YMR S E P VAQVF VHGE - - - S L QAF L I AI VVP
I AL E KL E S VY R S NE Y VANI CVY AD- - - QS KT KP VGI I VP
VY P NE I E DVVMQHP GVQE VAAVGVP S GS S GE AVKI F VVK
VNP L E VE AAL T KS P F I KQVF I Y GNR - - - - HP Y VVAL VVA
: . : * : : . . : *
R GE KP T P E - - - - - - - - - - - - - - - - - E L - - - - - - - - - - NE
DVE T L CSWAQKR GF - - - E GS F - - - E E L CR NKDVKKAI L E
NHAP L T KL AKKL GI ME QKDS S I NI E NY L E DAKL I KAVY S
KDP S L T E E - - - - - - - - - - - - - - - - - S - - - - - - - - - - - - -
NT KAI AAHL R KV- - - E R R DG- V- - - P L VNDR E KADCI R A
HL L KA- - - - GF AKWQL P DAY VF AE E - I P - R - - - - T S AGK
DMVR L GKDS GL KP F E QVKGI T L HP E L F S I DNGL L T P TMK
DL L KT GKDQGL VGI E L L AGI VF F DGEWT P QNGF VT S AQK
- L - VT F CR R QL T GY KVP KL VE F R DE L P - - - - - - KS NVGK
E L R R VS Q- - DL P P R AHVR R F AF VDE - F T L ANGF MT VKMG
: : : :
F L KR - - - - AL R E QY KNY - - - - - - - - - - - - - - - Y GGA
AKR P E L R NY F R S QI DDL Y S I - - - - - - - - - - - - - I KV
L KR KDI L NAVKDKVDAVY S - - - - - - - - - - - - - - - S S
I L R R E L R DE AR GKVD- - - N- - - - - - - - - - - - - - - KA
Y AR QKI E DHY VHY F E AL Y DE T P KF Y GF AVDDY DDL F
: .
337
472
470
370
434
547
374
499
496
397
456
586
410
533
430
530
493
625
445
569
566
465
531
664
484
605
602
504
566
703
496
638
641
513
598
742
525
677
680
544
634
781
373
498
495
396
455
585
409
532
529
429
492
624
444
568
565
464
530
663
483
604
601
503
565
702
495
637
640
512
597
741
524
676
679
543
633
780
541
699
700
561
669
816
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
ttLC-FACS
LCFA_HUMAN
LCF1_YEAST
LCFA_ECOLI
LdLCFA
cons
(c)
Figure 3: Amino acid sequence alignments of long-chain fatty acyl-CoA synthetases. The amino acid sequence of Leishmania LCFA
(LdLCFA) was aligned with LC-FACS from T. thermophilus (ttLC-FACS, Q6L8FO), human (LCFA HUMAN, P41215), yeast (LCF1 YEAST,
P30624), and E. coli (LCFA ECOLI, P29212). The boxed areas denoted with bold letters correspond to conserved motifs of long-chain fatty
acyl-CoA ligase: G, A, and L motifs as well as the P-loop. Filled squares, open circles, filled circles, and filled triangles indicate residues believed
to be involved in dimer formation, fatty acid binding, magnesium ion binding, and adenylate binding, respectively.

Molecular Biology International 7
membrane was layered over a wet Whatman paper sheet to
soak extra solution and covered with Saran Wrap (cellophane
paper) and exposed to X-ray film. After 4–18 h exposure in
an exposure cassette at −70◦C, X-ray film was developed for
analysis.
2.5. Phylogenetic Analysis. The amino acid sequence of
Leishmania long-chain fatty acyl-CoA ligase, obtained
from our microarray experiments [4], was compared with
sequences available in GeneDB ORTHOMCL4080 database
(http://www.genedb.org/) to identify the nearest ortholog
of this sequence in kinetoplastida. Multiple sequence
alignments were performed using Clustal W version 1.8
(http://www.ebi.ac.uk/clustalw) and T-cofee [31]. To cal-
culate evolutionary distances of kinetoplastida long-chain
fatty acyl-CoA ligases with human acyl CoA synthetases
(ACSs) [32], phylogenetic dendrograms were constructed by
neighbor-joining method and tree topologies were evaluated
by performing bootstrap analysis of 1000 data sets using
MEGA 3.1 (Molecular Evolutionary Genetics Analysis) [33].
All 26 human ACSs amino acid sequences were selected
[32], along with their transcript variants which are aligned
with different long-chain fatty acyl-CoA ligase ortholog
present in kinetoplastida family, to define the clade difference
with Trypanosome and Leishmania long-chain fatty acyl-CoA
ligase, and human acyl-CoA synthetases.
2.6. Homology Modeling of Leishmania Long-Chain Fatty
Acyl-CoA Ligase. The amino acid sequence of Leishmania
long-chain fatty acyl-CoA ligase was retrieved from the NCBI
database (GenBank Accession No. XM 001681734). It was
ascertained that the 3D structure of Leishmania long-chain
fatty acyl-CoA ligase protein was not available in Protein
Data Bank (PDB); hence, the present exercise of developing
the 3D model of this protein was undertaken. cBLAST
(http://www.ncbi.nlm.nih.gov/Structure/cblast/cblast.cgi)
and PSI-BLAST search was performed against PDB with the
default parameter to find suitable templates for homology
modeling. The sequence alignment of Leishmania long-chain
fatty acyl-CoA ligase and respective templates was carried
out using the CLUSTALW (http://www.ebi.ac.uk/clustalw)
and MODELLER9V8 programs [34, 35]. The sequences that
showed the maximum identity with high score and lower
e-value were used as a reference structure to build a 3D
model.
The retrieved sequences of Thermus thermophilus (PDB
Accession Code: 1ULT, 1V25, 1V26) [36] and Archaeoglobus
fulgidus (PDB Accession Code: 3G7S) long-chain fatty acyl-
CoA ligases served as template for homology modeling based
on its maximum sequence similarity to Leishmania long-
chain fatty acyl-CoA ligase. The alignment was manually
refined at some loops region of the templates. The result-
ing alignment was used as an input for the automated
comparative homology modeling for generating 3D model
structure of Leishmania long-chain fatty acyl-CoA ligase.
The academic version of MODELLER9V8 was used for
model building. The backbones of core region of the protein
were transferred directly from the corresponding coordinates
of templates. Side chain conformation for backbone was
generated automatically. Out of 50 models generated by
MODELLER, the one with the best DOPE score, minimum
MOF (Modeller Objective Function), and best VARIFY 3D
profile was subjected to energy minimization. In order to
assess the stereochemical qualities of 3D model, PROCHECK
analysis [37] was performed and Ramachandran plot was
drawn.
3. Results
3.1. Metabolism of Long-Chain Fatty Acyl-CoA Ligase Enzyme.
Three types of fatty acyl CoA ligase have been defined
with respect to the length of the aliphatic chain of the
substrate: short (SC-EC 6.2.1.1), medium (MC-EC 6.2.1.2),
and long-chain (LC-EC 6.2.1.3) fatty acyl-CoA ligase. These
utilize C2-C4, C4-C12, and C12-C22 fatty acids as substrates,
respectively [9]. Fatty acid activation step involves the linking
of the carboxyl group of the fatty acid through an acyl bond
to the phosphoryl group of AMP. Subsequently, a transfer of
the fatty acyl group to the sulfhydryl group of CoA occurs,
releasing AMP [38–40]. This magnesium-dependent two-
step acylation of fatty acid by fatty acyl CoA synthetases
was defined as unidirectional Bi Uni Uni Bi Ping-Pong
mechanism [36, 39].
Genome analysis suggests that L. major oxidizes fatty
acids via β-oxidation in two separate cellular compartments:
the glycosome and mitochondria [41]. An argument for
the involvement of glycosome in lipid metabolism is the
fact that in each of three trypanosomatid genomes three
genes called half ABC transporters (GATI 1-3) have been
found identical with peroxisomal transporters involved in
fatty acid transport. In T. brucei, it was conformed that these
transporters are associated with glycosomal membrane [42].
These transporters might be coupled with fatty acyl-CoA
ligase in glycosome, which can provide activated form of fatty
acids to these transporters like oleoyl-CoA, and also other
acylated fatty acids.
In T. brucei, little β-oxidation was observed in mito-
chondria. However, T. brucei contains at least two enzymes
involved in β-oxidation of fatty acid (2-enoyl-CoA hydratase
and hydroxyacyl-dehydrogenase, encompassed in a single
protein) with glycosome localization [43]. The presence of
a PTS (Peroxisomal Targeting Sequence) on T. brucei and T.
cruzi carnitine acetyl transferase, catalysing the last peroxi-
somal step in fatty acid oxidation, suggests that the major
β-oxidation processes are situated in glycosomes [44]. In
L. donovani, one of the β-oxidation enzyme 3-hydroxyacyl-
CoA dehydrogenase has been localized to glycosomes [45].
The hypothetical localization of Leishmania long-chain fatty
acyl-CoA ligase was predicted in mitochondria or glycosome
but, with the reference of other organisms, the specialized
localization of specific long-chain fatty acyl-CoA ligase
family protein needs to be taken into account in future.
As mentioned in a previous study β-oxidation has been
found to be unregulated in Leishmania’s amastigotes then
in promastigote stage [46–48]. This specialized increase was
described so that, in infectious stage, energy requirement

8 Molecular Biology International
Table 2: Results of protein structure by PROCHECK and VERIFY 3D.
Leishmania long-chain fatty
acyl-CoA ligase
T. thermophilus long-chain fatty
acyl-CoA synthetase (1ULTA)
Residues in most favoured regions 521 (87.9%) 405 (90.0%)
Residues in additional allowed regions 62 (10.5%) 43 (9.6%)
Residues in generously allowed regions 7 (1.2%) 2 (0.4%)
Residues in disallowed regions 3 (0.5%) 0 (0.0%)
Number of nonglycine and nonproline residues 593 450
Number of end-residues (excl. Gly and Pro) 2 155
Number of glycine residues (shown as triangles) 48 48
Number of proline residues 26 34
Total number of residues 669 687
Residues with Verify 3D Score >0.2 52.24% 96.63%
Errat overall quality factor 44.154 89.655
G F F R T G DI A VWDE E G Y V E I K DR L K
GWL HT G DI G KWL P NG T L K I I DR K K
GWF K T G DI G EWE A NG HL K I I DR K K
GWL HT G DI A VMDE E G F L R I V DR K K
T F V R S G DY G T L - T G G WI T V K G NK D
M A Y T T G T T G L P K G V V Y S H R A
I C F T S G T T G N P K G A M V T H R N
I M Y T S G S T G E P K G V V L K H S N
L Q Y T G G T T G V A K G A M L T H R N
I V F T A G T T G P P K G V M L S H K S
R Q G Y G L T E T
Y E G Y G Q T E C
L I G Y G L T E T
L E G Y G L T E C
V N T Y G F M E A
ttLC-FACS
LCFA_HUMAN
LCFA_YEAST
LCFA_ECOLI
LdLCFA
ATP/AMP FACS
200–275 aa 150–170 aa 70–75 aa 110–130 aa
Figure 4: Domain organization of amino acid sequence alignments of ATP-AMP and fatty acyl CoA synthetase (FACS) motif from T.
thermophilus (ttLC-FACS), human (LCFA HUMAN), yeast (LCF1 YEAST), E. coli (LCFA ECOLI), and Leishmania (L LCFA).
was supplemented to utilize fatty acid as carbon and energy
source rather than glucose [47]. Long-chain fatty acyl-CoA
ligase is the key enzyme involved in β-oxidation of fatty
acids, and its compartmentation in glycosome supports
a strong evidence of the involvement of this enzyme in
cellular biogenesis and its importance at particular stage
of Leishmania life cycle. In the same way upregulation of
long-chain fatty acyl-CoA ligase with combination of other
enzymes involved in fatty acid catabolism might play a
crucial role in cell survival at infectious stage of Leishmania,
and these analyses must be supplemented with experimental
biology.
3.2. Characterization of Leishmania Long-Chain Fatty Acyl-
CoA Ligase Gene. The presence of L. donovani long-chain
fatty acyl-CoA ligase gene in the clinical isolates was
ascertained by PCR amplification. The putative long-chain
fatty acid-CoA ligase gene of L. major is present in the
Leishmania Genome Databank (http://www.genedb.org/) on
chromosome 13 (Figure 1). Specific 2010 bp size amplified
product was obtained, showing the presence of long-chain
fatty acyl-CoA ligase gene in the L. donovani clinical isolate
(Figure 2(D)). The amplified product was sequenced and
confirmed to be long-chain fatty acid-CoA ligase gene
by performing NCBI-BLAST identity with L. major gene.
NCBI-BLAST result showed 96% sequence similarity and
1% gaps with L. major long-chain fatty acyl-CoA ligase gene
(GenBank Accession No. XM 001681734). The starting 18
nucleotides and 19 nucleotides from the end sequence were
missed due to direct amplified product sequencing. These
nucleotides were collected from its maximum similar L.
major long-chain fatty acyl-CoA ligase sequence (GenBank
Accession No. XM 001681734).
For the determination of long-chain fatty acid-CoA ligase
gene copy number, nuclear DNA from the L. donovani
clinical isolates (2001, 39) was digested with various restric-
tion enzymes. The restriction map was designed from the
complete putative long-chain fatty acyl-CoA ligase gene and
the flanking region present in chromosome 13 of L. major
(Figure 2). Southern hybridization was performed using the
2010 bp long-chain fatty acid-CoA ligase gene PCR product
as probe (Figure 2(C)). The same blot was also probed with
alpha tubulin gene probe as an internal control, showing
equal loading (Figure 2(B)). Complete digestion resulted in
a single copy within the L. donovani genome, as BamHI
enzyme showed only one band of approximately 3848 bp,
except PvuII which was cut once into the gene sequence and
XhoI which was cut twice into the gene sequence, which

Molecular Biology International 9
ACS
M2A
ACSM2B
ACSM1
ACSM
4
A
C
SM
5
A
C
SM
3 v1
A
C
SM
3 v2
ACS
S2 v1
CSS2 v2
ACS
S1
AC
SS3
AC
SF
1
ACSVL2 v2A
CS
VL
2 v
1
AC
SV
L1A
C
SV
L3
A
C
SV
L6
A
C
SV
L5
A
C
SV
L4
A
C
SF2
A
C
SF3
TcLC
FA
T
bLC
FA
TvLCFALbLCFALiLCFA
Ld
LC
FA
ACSBG2
ACSBG1
ACSL4 v2
ACS
L4 v1
ACS
L3 v2
A
C
SL
3
v1
ACS
L5 v
3
AC
SL
5
v1
A
C
SL
5
v2
A
C
SL
1
A
C
SL
6
v2
AC
SL
6 v
1
Medium-chain clade
Short-chain
clade
Very long-chain
clade
ACSF2 clade
ACSF3 clade
Kinetoplastid
clade
Bubblegum
clade
Long-chain
clade
ACSF4 clade
A
C
SF4
0.2
Figure 5: Phylogenetic trees based on human acyl CoA synthetases (ACSs) gene sequences [38] showing the relationship of all Leishmania
long-chain fatty acyl-CoA ligase orthologs (Table 1), with their nearest phylogenetic relatives. Phylogenetic trees were constructed by the
neighbour-joining method as well as the maximum likelihood method as implemented in MEGA4 software. Numbers at nodes are bootstrap
values (ML/NJ; xx represents no bootstrap value in NJ tree where nodes differ in both dendrograms;—represents value <50). The bar
represents 0.02 substitutions per alignment position. The bar represents substitutions per alignment position.
exhibited two and three hybridizing bands, respectively.
The results showed that long-chain fatty acid-CoA ligase is
present as a single copy gene in the L. donovani genome.
The restriction pattern also verifying the restriction pattern
of L. donovani and L. major long-chain fatty acyl CoA ligase
coding region is almost the same.
3.3. Identification of Conserved Domains and Structure-
Function Correlation in Leishmania Long-Chain Fatty Acyl-
CoA Ligase. Leishmania long-chain fatty acyl-CoA ligase
gene was translated from full length ORF on the basis
of its nucleic acid sequence. Long-chain fatty acyl-CoA
synthetase from T. thermophilus, yeast, and E. coli and
all 26 distinct human acyl-CoA synthetases were sub-
jected to phylogenetic analysis to facilitate the evalua-
tion of conserved motif with relationship of reference
Leishmania long-chain fatty acyl-CoA ligase amino acid
sequence. The amino acid sequence of Leishmania long-
chain fatty acyl-CoA ligase (LdLCFA) was aligned with
LC-FACS from T. thermophilus (TtLC-FACS,1ultA), human
(LCFA HUMAN, P41215), yeast (LCF1 YEAST, P30624),
and E. coli (LCFA ECOLI, P29212) on the basis of PSI-
BLAST (Figure 3). However the overall similarity of Leish-
mania long-chain fatty acyl-CoA ligase (LdLCFA) with other

10 Molecular Biology International
fatty acyl-CoA synthetases family proteins is low, about
17% with TtLC-FACS, 15% with LCFA HUMAN, 14% with
LCF1 YEAST, and 13% with LCFA ECOLI. Based on the
crystal structure of TtLC-FACS and alignment with other
long-chain fatty acyl-CoA synthetases [36], the amino acid
sequence of Leishmania long-chain fatty acyl-CoA ligase
shows conserve region corresponding to the linker (L),
adenine (A), and gate (G) motifs as well as the P-loop, the
phosphate-binding site. Previous studies [32, 36] put for-
ward different motifs which can give insight to enhance our
understanding of predicted structure-function relationships
in Leishmania. P-loop is the Motif I which is also known
as AMP-binding domain found in a close proximity to the
adenosine moiety and helps to maintain the substrate in
the proper orientation. The consensus sequence of Motif I,
[Y,F]TSG[T,S]TGXPK shows high level of conservation with
respect to Leishmania long-chain fatty acyl-CoA ligase, that
is, 237-FTAGTTGPPK-246. Motif II contains the L-motif
(432-DRLKDL-437) that acts as a linker between the large
N-terminal domain and the smaller C-terminal domain in
TtLC-FACS. The linker region is thought to be critical for
catalysis function as it facilitates a conformational change
upon ATP binding that permits subsequent binding of the
fatty acyl and/or CoA substrates. In Leishmania long-chain
fatty acyl-CoA ligase, this linker region (517-GNKDVL-522)
is less similar compared with other organisms and is likely
to be critical in enzyme activity. Motif III was found to be
in all acyl CoA synthetases and a part of A-motif (adenine
motif). This region has been described as an ATP/AMP-
binding domain in other acyl-CoA synthetases [49–51]. The
conserved consensus sequence of A-motif is YGXTE, a highly
conserved motif with respect to Leishmania long-chain fatty
acyl-CoA ligase region, that is, YGFME. From the crystal
structure of TtLC-FACS, it was proposed that Y324 was an
adenine-binding residue [42] and also conserved throughout
all organisms including Leishmania. The crystal structure of
S. enterica acetyl-CoA synthetase revealed that the glutamate
residue of A-motif is positioned near oxygen O1 of the AMP
phosphate [52]. This region was predicted to be involved in
substrate binding or stabilization, conserved in Leishmania
long-chain fatty acyl-CoA ligase also. Motif IV comprises the
first five residues of the nine-amino acid G-(or gate) motif
(226-VPMFHVNAW-234) of ttLC-FACS (36), showing less
sequence similarity with Leishmania long-chain fatty acyl-
CoA ligase (281-CSWCVAGAL-289). From the crystal struc-
ture of TtLC-FACS, it was proposed that the indole ring
of W234 acts as a gate and blocks the entry of fatty acids
into its substrate binding tunnel unless ATP is first bound,
resulting in a conformational change that swings the gate
open (36). However, a tryptophan residue corresponding
to W234 was not found in any Leishmania, human, yeast,
and E. coli fatty acyl-CoA synthetase sequences. In contrast,
although no highly conserved sequences were identified, a
corresponding gate residue may be located elsewhere in the
structure of Leishmania long-chain fatty acyl-CoA ligase.
The fatty acyl-CoA synthetases are part of a large family
of proteins referred to as the ATP-AMP-binding proteins. A
common feature of enzymes in this family is that they all
form an adenylated intermediate as part of their catalytic
cycle. This group of enzymes is diverse in catalyzing the acti-
vation of a wide variety of carboxyl-containing substrates,
including amino acids, fatty acids, and luciferin. Sequence
comparison of members of the ATP-AMP-binding protein
family has identified two highly conserved sequence ele-
ments, [53] Y[T]S[GTTG]X[PKGV]· · ·G[YG]XT[E] (the
bracket shows the conserved sequence in Leishmania long-
chain fatty acyl-CoA ligase), which encompass the ATP-AMP
signature motif (Figure 4).
In fatty acyl-CoA synthetases family proteins, there was a
third sequence element defined as FACS signature motif that
was less conserved and partially overlaps the FACS signature
motif, which is involved in both catalysis and specificity
of the fatty acid substrate [54]. There are a number of
notable features within the FACS signature motif: (i) this
region contains two invariant glycine residues (at positions
2 and 7) and a highly conserved glycine at position 16,
Leishmania long-chain fatty acyl-CoA ligase shares glycine
residue with other FACSs at position 7 and 16 but Tyr
instead of Gly was found in position 2. (ii) This region
contains additional six residues that are invariant in the fatty
acyl-CoA synthetases: W[3], T[6], D[8], D[22], R[23], and
K[25], but in Leishmania long-chain fatty acyl-CoA ligase
these residues are F[3], S[6], D[8], G[22], N[23], and D[25].
(iii) The residue in the fourth position is hydrophobic and
is a leucine, a methionine, or phenylalanine. However, in
Leishmania long-chain fatty acyl-CoA ligase hydrophobic
residue valine was situated in position 4. (iv) This region of
enzyme contains hydrophobic residues (leucine, isoleucine,
or valine) at positions 4, 9, 18, 20, and 21. These residues, in
addition to tryptophan or phenylalanine residues at position
3, may comprise part of a fatty-acid-binding pocket. All of
these five conserved regions from FACS signature motif are
having similarity among them except Leishmania long-chain
fatty acyl-CoA ligase, with some variable regions. These
less conserved regions in Leishmania long-chain fatty acyl-
CoA ligase-FACS signature motif were predicted to adopt
inconsistent specificity and catalytic activities of the fatty acid
substrate compared to other fatty acyl CoA synthetases.
3.4. Phylogenetic Analysis of Leishmania Long-Chain Fatty
Acyl-CoA Ligase and Human Acyl-CoA Synthetases Sequences.
We performed phylogenetic analysis to infer evolutionary
relationships of all available sequences from kinetoplastida
long-chain fatty acyl-CoA ligases (Table 1) and human (host)
ACSs family sequences. This experiment was performed to
validate that the parasite enzyme is unquestionably different
from the human enzyme, and this aspect merits further
study to validate this enzyme as a drug target. We obtained
comparable results using the neighbor-joining distance-
based algorithm as well as maximum parsimony. We found
9 clades, including kinetoplastida clade (one set of six kine-
toplastida long-chain fatty acyl-CoA ligase protein family)
forming a clade with high bootstrap support (Figure 5).
kinetoplastida clade was highly dissimilar and distinct from
all ancestral nodes with other human ACSs family proteins
and showing distinctiveness of kinetoplastida long-chain
fatty acyl-CoA ligases, including Leishmania long-chain fatty

Molecular Biology International 11
(a) (b)
Figure 6: Leishmania long-chin fatty acyl-CoA ligase model. (a) The larger left hand-side domain is the N-terminal domain and the smaller
one is the C-terminal domain which is connected by a linker chain. (b) Superposition of the modeled structure of Leishmania long-chain
fatty acyl-CoA ligase (Orange) with the crystal structure of the T. thermophilus long-chain fatty acyl-CoA synthetase (PDB code: 1ult A)
(Blue).
B
A
L
b
a
l
pb
b
THR 241
PRO 305
ARG 358
ARG 393
LEU 413
ASP 435
ARG 497
TRP 512
LEU 623
VAL 630
VAL 415
−180 −135 −90 −45 0 45 90 135 180
−135
−90
−45
0
45
90
135
180
φ (degrees)
ψ
(d
eg
re
es
)
∼p∼b
∼a
∼l
∼b
∼b
∼b
(a)
B
A
L
b
a
l
pb
b
HIS 230 (A)
GLU 328 (A)
0
−180 −135 −90 −45 0 45 90 135 180
−135
−90
−45
45
90
135
180
φ (degrees)
ψ
(d
eg
re
es
)
∼p
∼a
∼l
∼b
∼b
∼b
(b)
Figure 7: Ramachandran plot of (a) modeled structure Leishmania long-chain fatty acyl-CoA ligase (b) and the crystal structure of the
Tt0168 (PDB code: 1ult A).
acyl-CoA ligase. This divergence of Leishmania long-chain
fatty acyl-CoA ligase with respect to the homologous human
enzymes may be an important protein as a potential target
candidate for chemotherapeutic antileishmanial drugs.
3.5. Homology Modeling of Leishmania Long-Chain Fatty
Acyl-CoA Ligase Protein. The backbone root-mean-square-
deviation (RMSD) values between final model and template
crystal structure used are 1.04 A˚ with Thermus thermophilus
(PDB Accession Code: 1ULT, 1V25, 1V26) and 1.40 A˚ with
Archaeoglobus fulgidus (PDB Accession Code: 3G7S) long-
chain fatty acyl-CoA ligase. Small RMSD can be interpreted
as structures share common structural homology and the
generated structure is reasonable for structural similarity
analysis (Figure 6). The final modeled structure of Leish-
mania long-chain fatty acyl-CoA ligase was evaluated for
overall quality using available analyses procedures. These
analysis compare specific properties of the model with those
of known high-quality protein structures using programs
like PROCHECK, Verify3D, and WHATIF (Table 2). An
important indicator of the stereochemical quality of the
model is distribution of the main chain torsion angles phi
and psi in Ramachandran plot (Figure 7). The plot clearly
shows the vast majority of the amino acids in a phi-psi
distribution consistent with right α-helices, and the remain-
ing fall into beta configuration. Only three residues fall
outside the allowed regions. Plots comparison shows that the
structure is reasonable overall because the space distribution

12 Molecular Biology International
for the homology-modeled structure was similar to the X-
ray structure of the Thermus thermophilus long-chain fatty
acyl-CoA ligase (PDB Accession Code: 1ULTA). The results
showed that our modeled structure was reasonably good at
that much less sequence identity.
4. Discussion
Earlier during the course of work, microarray analysis was
performed on the same clinical L. donovani isolates (2001
and 39) in order to identify differential gene expression [4].
Out of all genes found differentially expressed, significant
upregulation of long-chain fatty acyl-CoA ligase gene in
SAG unresponsive clinical isolate [33] was found to be
intracellular amastigote specific and has confirmed the
involvement of long-chain fatty acyl-CoA ligase in resistance.
Similarly, it has been proven before that the rate limiting
enzyme, long-chain fatty acyl-CoA ligase of β-oxidation,
was found to be upregulated in amastigotes derived from
cloned line of L. donovani ISR because, during late stages of
differentiation, the parasites shift from glucose to fatty acid
oxidation as the main source of energy, and thereby there
is increase in enzyme activity associated with β-oxidation
capacity [47, 48]. Early in vivo studies showed that enzymatic
activities associated with β-oxidation of fatty acids were sig-
nificantly higher in L. mexicana amastigotes [47]. Addition-
ally microarray experiments with intracellular amastigotes
hybridized onto Affymetrix Mouse430 2 GeneChips showed
that several genes involved in fatty acid biosynthesis pathway
were found to be upregulated [55]. Presently studies are
ongoing in our laboratory on microarray analysis using
intracellular amastigotes hybridized to Affymetrix GeneChip
human genome U133 Plus 2.0 array which will further yield
useful information towards the fatty acid/lipid metabolism
within this clinical isolate. A very recent study by Yao et
al., 2010, on differential expression of plasma membrane
proteins in logarithmic versus metacyclic promastigotes of
L. chagasi has also identified long-chain fatty acyl-CoA
synthetase [56].
As mentioned before, long-chain fatty acid-CoA ligase is
present in both prokaryotes and eukaryotes. This divergence
of Leishmania long-chain fatty acyl-CoA ligase with respect
to the homologous human enzymes may be an important
protein as a potential target candidate for chemotherapeutic
antileishmanial drugs. Many differences exist between host
and parasite pertaining to the structure and arrangement of
this enzyme. However, Leishmania has significant divergence
and adaptation to specific environmental conditions between
its two life stages, in the insect vector and human host. This
can affect the parasites metabolic machinery in terms of
presence of certain pathways, their subcellular localization
and expression at different developmental stages, and the
interplay between scavenging and synthesis of key metabo-
lites. It has been argued previously [57] that successful targets
for metabolic intervention are most likely to be found among
enzymes exerting strong control of flux through metabolic
pathways. These control points are likely to be species and
development dependent. Even if a unique or highly divergent
enzymatic process is found in the parasites, this does not
necessarily mean it can be developed as a target for useful
inhibitors. On the other hand, enzymes that are present in
both the parasites and their animal hosts will often differ
sufficiently in their sequence for inhibitors to be specific.
Finally, even orthologous enzymes functioning in the same
pathway and in the same subcellular compartment of the
parasites may have different inhibitor binding properties,
leading to variability in the effectiveness and specificity of
inhibitors targeting any particular enzyme.
The detection of the long-chain fatty acid-CoA ligase
gene in the genome of L. donovani clinical isolate, in
the present study, deserves a full exploration with respect
to its potential as a drug target. Changes in membrane
lipids/deficiency of certain fatty acids and disease association
have been documented [34, 58]. Modulation of enzymes
involved in lipid synthesis and of others possibly involved
in cell wall metabolism may modify access of drug to the
plasma membrane. Moreover, our microarray experiment
indicated that this enzyme was amastigote specific making
it all the more important to study it further and test if it can
be exploited as a validated drug target. We have also shown
earlier in our laboratory [34] that modification of lipid
composition on the plasma membrane of the parasite might
have important implications towards generating susceptibil-
ity/resistance to antileishmanial drugs. As this enzyme stipu-
lates several important cellular processes in Leishmania like
stage-specific expression [47, 48], host-parasite interaction
[55], cell membrane composition [17, 18], phospholipid
biosynthesis [16, 21], and drug resistance [4], the present
study proposed further evaluation of Leishmania long-chain
fatty acyl-CoA ligase as a candidate drug target.
Acknowledgments
This work was supported by Department of Biotechnology,
New Delhi, India (Grant No. BT/PR5452/BRB/10/430/2004,
BT/PR9266/BID/07/221/2007 and BT/PR13384/MED/29/
166/2009). J. Kaur and R. Tiwari contributed equally to the
present work.
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