See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/281311915

Structure-Based Design of Inhibitors of the Crucial Cysteine Biosynthetic

Pathway Enzyme O-Acetyl Serine Sulfhydrylase

Article  in  Current Topics in Medicinal Chemistry · August 2015

DOI: 10.2174/1568026615666150825142422 · Source: PubMed

CITATIONS READS

12 578

2 authors:

Mohit Mazumder Samudrala Gourinath

Pine Biotech Jawaharlal Nehru University
85 PUBLICATIONS   339 CITATIONS    230 PUBLICATIONS   2,199 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Actin binding protein View project

Molecular aspects of calcium recognition in EF hand containing calcium binding proteins View project

All content following this page was uploaded by Mohit Mazumder on 05 October 2015.

The user has requested enhancement of the downloaded file.

 Send Orders for Reprints to reprints@benthamscience.ae
Current Topics in Medicinal Chemistry, 2016, 16, 000-000 1

Structure-Based Design of Inhibitors of the Crucial Cysteine Biosynthetic

Pathway Enzyme O-Acetyl Serine Sulfhydrylase

Mohit Mazumder and Samudrala Gourinath*

School of life sciences, Jawaharlal Nehru University, N.Delhi-110067, India

Abstract: The cysteine biosynthetic pathway is of fundamental importance for the growth, survival,
Please provide
and pathogenicity of the many pathogens. This pathway is present in many species but is absent in corresponding author(s)
photograph
mammals. The ability of pathogens to counteract the oxidative defences of a host is critical for the size should be 4" x 4" inches
survival of these pathogens during their long latent phases, especially in anaerobic pathogens such as
Entamoeba histolytica, Leishmania donovani, Trichomonas vaginalis, and Salmonella typhimurium.
All of these organisms rely on the de novo cysteine biosynthetic pathway to assimilate sulphur and
maintain a ready supply of cysteine. The de novo cysteine biosynthetic pathway, on account of its
being important for the survival of pathogens and at the same time being absent in mammals, is an
important drug target for diseases such as amoebiasis, trichomoniasis & tuberculosis. Cysteine biosynthesis is catalysed
by two enzymes: serine acetyl transferase (SAT) followed by O-acetylserine sulfhydrylase (OASS). OASS is well studied,
and with the availability of crystal structures of this enzyme in different conformations, it is a suitable template for
structure-based inhibitor development. Moreover, OASS is highly conserved, both structurally and sequence-wise, among
the above-mentioned organisms. There have been several reports of inhibitor screening and development against this
enzyme from different organisms such as Salmonella typhimurium, Mycobacterium tuberculosis and Entamoeba
histolytica. All of these inhibitors have been reported to display micromolar to nanomolar binding affinities for the open
conformation of the enzyme. In this review, we highlight the structural similarities of this enzyme in different organisms
and the attempts for inhibitor development so far. We also propose that the intermediate state of the enzyme may be the
ideal target for the design of effective high-affinity inhibitors.
Keywords: Conformational changes, cysteine biosynthetic pathway, Inhibitors, O-acetyl serine sulfhydrylase, pathogens.

1. INTRODUCTION pathogens during their long latent phases, especially in

Cysteine plays a vital role in organic sulphur metabolism.
Utilisation of sulphur from cysteine is the initial step of
many biosynthetic pathways that supply the cell with
biomolecules such as Fe–S clusters, modified tRNAs
(thiouridine), thiamine, biotin, glutathione, trypanothione
and mycothiol (Beinert, 2000;Kessler, 2006). Mammals rely
on sulphated amino acids, mostly the essential amino acid
methionine, for their sulphur supplies whereas most bacteria,
protists and plants assimilate sulphur into cysteine through
the reductive sulphate assimilation pathway (RSAP). Apart
from functioning as a protein building block and as a
component of important biomolecules, cysteine participates
directly, or as a precursor of reducing agents, in the
maintenance of the redox state of the cell. This function is of
special interest to microorganisms that spend part of their
life cycle in highly oxidizing environments, e.g., when
establishing an infection in the human host or inside human
macrophages (Mozzarelli et al., 2011).
The ability of pathogens to counteract the oxidative
defences of a host is critical for the survival of these
*Address correspondence to this author at the School of Life Sciences,
Jawaharlal Nehru University, New Delhi 110 067, India;
Tel: ???????????????; Fax: ????????????????;
E-mail: samudralag@yahoo.com
anaerobic pathogens such as Entamoeba histolytica and
pathogens such as Leishmania donovani, Trichomonas
vaginalis, and Salmonella typhimurium poses a severe threat
to health. Due to the increase in drug resistance, treatment of
such diseases have become complicated and hence life
threating. All of these organisms rely on the de novo cysteine
biosynthetic pathway to assimilate sulphur and maintain a
ready supply of cysteine (Campanini et al., 2015). The de
novo cysteine biosynthetic pathway, on account of its being
important for the pathogen and at the same time being absent
in mammals, is an important drug target. An infection by
these pathogens cause diseases such as amebiasis,
leishmaniosis and tuberculosis where the inhibitors of
cysteine biosynthetic pathway could result in better
treatment then the presently available antibiotics.
In cysteine biosynthesis, the first reaction is catalysed by
the enzyme serine acetyltransferase (SAT, EC 2.3.1.30),
which generates the activated sulphide acceptor O-
acetylserine (OAS) from serine and acetyl CoA. In the sec-
ond step, O-acetylserine sulfhydrylase (OASS, EC 2.5.1.47)
catalyzes O-acetylserine where sulphide is inserted into O-
acetylserine in a β replacement reaction catalyzed by the
cofactor pyridoxal phosphate (PLP) to yield cysteine and
acetate (Fig. 1). The enzymatic pathway of cysteine
synthesis was characterized by the pioneering work of

1568-0266/16 $58.00+.00 © 2016 Bentham Science Publishers

2 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 Mazumder and Gourinath

Fig. (1). Cysteine biosynthetic pathway. A) Cysteine synthesis occurs in two steps, the first one catalyzed by serine acetyltransferase (SAT),
which results in acetylation of serine, and the second catalyzed by OASS, which substitutes acetate with sulfide. B) Upon the association of
OASS and SAT forms cysteine synthase complex that is favored under excess sulfur concentrations. In the cysteine synthase complex, the C-
terminal end of SAT binds the active site of OASS and decreases its activity while SAT activity increases resulting in excessive production of
O-acetylserine (OAS), which induces the dissociation of the cysteine synthase complex and downregulates the SAT activity. The free OASS
catalyzes cysteine formation and reduces OAS levels, which in turn allows re-association of SAT with OASS.

Kredich and colleagues (Becker et al., 1969); they also
provided the first enzymatic data for SAT and OASS as free
homomers and when bound to the hetero-oligomeric cysteine
synthase complex.
The second enzyme in the cysteine biosynthetic pathway,
OASS, belongs to the family of tryptophan synthase beta
subunit-like PLP-dependent enzymes and is also the last
enzyme in the RSAP. Together with ATP sulfurylase
(ATPS), OASS forms a class of energy-transfer systems
where an ATPase-driven energy pump can be constructed
using the allosteric interactions between the RSAP and
cysteine biosynthetic enzymes. The multifunctional protein
complex involving ATPS and OASS allows the efficient
synthesis of Adenosine 5'-phosphosulfate (APS) and thereby
drives the sulphate assimilation process in many
microorganisms (Wei et al., 2002) and in plants
(Hawkesford and De Kok, 2006). As this process is crucial
for the survival of these pathogens, these enzymes especially
OASS, which is the last enzyme of the RSAP could be
crucial targets for inhibitor development and possible drug
development.
2. STRUCTURAL CHARACTERIZATION OF OASS
OASS belongs to fold type II of PLP dependent enzymes,
where the internal aldimine is formed at lysine residues of an
α-helix on the N-terminal domain, and the apo enzyme is
stabilized by interactions of its serine and threonine residues
with a pyridine ring of PLP (Percudani and Peracchi, 2003).
This family is represented by tryptophan synthase. The first
structure of OASS to be solved was that from S.
typhimurium in 1998 by Burkhard et al. (Burkhard et al.,
1998). Since then, a number of OASS structures from
various plants, bacteria and protozoans have been
determined (Bonner et al., 2005;Huang et al., 2005;Krishna
et al., 2007;Schnell et al., 2007;Chinthalapudi et al., 2008).
All OASSs in solution are homodimers of around 75 kDa,
and the crystal structures reveal each monomer to be
composed of two domains, each domain having a α/β−fold
(Fig. 2B) typical of the fold type II PLP enzymes. The N-
terminal domain includes a central four-stranded parallel β
sheet, which is flanked by three α helices on one side and
one α helix on the other side.
The C-terminal domain folds into a six-stranded mixed β
sheet sandwiched by four α helices. The community network
analysis, which identifies communities (clusters of highly
connected residues) based on residue-by-residue correlation
and proximity, is shown in Fig. (2C and D). As seen in these
figures, the ligand binding sites are present in cluster 4,
which is strongly connected to cluster 11. The residues
responsible for the opening and closing of the active site are
present in cluster 11. The figure demonstrates the positive
correlation of the motion of cluster 11 with that of cluster 4,
which triggers the closing and opening of the active site
upon ligand binding.
The overall structures of OASSs are very similar. For
example, E. histolytica OASS (EhOASS) (Krishna et al.,
2007), L. donovani OASS (LdOASS) (Raj et al., 2012), S.
typhimurium OASS (StOASS) (Burkhard et al., 1998),
Arabidopsis thaliana OASS (AtOASS) (Francois et al., 2006),
Escherichia coli OASS (EcOASS) (Claus et al., 2005),
Haemophilus influenzae OASS (HiOASS) (Salsi et al.,
2010b), and Mycobacterium tuberculosis OASS (MtbOASS)
(Schnell et al., 2007) all display 40-50 % sequence identity
with each other, and their three-dimensional structures differ
from one another by RMSDs of less than 1.5 Å (Fig. 2A).
The StOASS, AtOASS, HiOASS, MtbOASS and
EhOASS structures have been solved in various
conformations. These have included inhibitor-bound open
conformations for MtbOASS (PDB ID 2Q3C), AtOASS
(PDB ID 2ISQ), and HiOASS (PDB IDs 1Y7L, 3IQG, 3IQH
and 3IQI) (Huang et al., 2005;Francois et al., 2006;Schnell
et al., 2007;Salsi et al., 2010a). They have also included
closed conformations when (a) forming an external aldimine
for AtOASS (PDB ID 1Z7Y) (Bonner et al., 2005), and for
StOASS (PDB ID 1D6S) (Burkhard et al., 1999) or (b) α-
aminoacrylate for MtbOASS (PDB ID 2Q3D) (Schnell et al.,
2007) as well as when (c) Cys bound for EhOASS (PDB ID
3BM5) (Chinthalapudi et al., 2008), (D) Met bound for
EhOASS (PDB ID 4JBL) and (E) Ile bound for EhOASS
(PDB ID 4IL5) in (Fig. 2B) (Raj et al., 2013a).
Inhibitors of O-Acetyl Serine Sulfhydrylase Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 3

Fig. (2). Structural characterizations of OASS. A) Structure-based sequence alignment of Entamoeba histolytica OASS (EhOASS),
Leishmania donovani OASS (LdOASS), Haemophilus influenzae OASS (HiOASS), Salmonella typhimurium OASS (StOASS),
Mycobacterium tuberculosis OASS (MtbOASS), E. coli OASS (EcOASS) and Arabidopsis thaliana OASS (AtOASS). The secondary
structures were plotted using the EhOASS structure. The alignment shows the highly conserved OASS’s from various organisms. B)
Structural superposition of OASS from various organisms (mentioned above) with open, closed and intermediate conformations. The
superposition of all the OASSs suggests that the overall structures of OASS’s are very similar. C &D) shows the Community network graph.
Plot of the complete all-residue network and of the coarse-grained community network of EhOASS. The network was plotted using the
Bio3D package (Grant et al., 2006). The network was generated using normal mode analysis of the EhOASS structure showing C) highly
intra-connected and loosely inter-connected nodes and a D) simplified coarse-grained representation. The plots show the conformational
arrangement of EhOASS and the radius of the circle indicates the number of residues in the community. The images were created using
PyMol (DeLano, 2002) and Bio3D software.

3. OASSS FROM DIFFERENT ORGANISMS
3.1. OASS in Plants
Like in bacteria, cysteine biosynthesis in plants is a two-
step process carried out by SAT and OASS (Kumaran et al.,
2009). In plants, cysteine biosynthesis plays a central role in
fixing inorganic sulfur from the environment and provides
the only metabolic sulfide donor for the generation of
methionine, glutathione, phytochelatins, iron-sulfur clusters,
vitamin cofactors, and multiple secondary metabolites. As
OASS has a central position between assimilatory sulphate
reduction and the branching point of pathways that lead to
the array of organic compounds containing reduced sulphur
(Leustek et al., 2000;Hell et al., 2002;Droux, 2004), it plays
an integral role in the regulation of the rate of cysteine
synthesis (Wirtz et al., 2004). Most plants have several
nuclear genes that encode for different isoforms of SAT and
OASS for each of the three compartments, namely the
cytosol, plastids and mitochondria (Hell et al., 2002). The
isoforms present in the cytoplasm, chloroplasts, and
mitochondria are named OASS-A, OASS-B, and OASS-C,
respectively. Its importance is highlighted by the
observations that each subcellular compartment with the
capability of protein biosynthesis carries out cysteine
synthesis (Lunn et al., 1990).
The location of these isoforms were determined by
molecular cloning of the corresponding genes (Saito et al.,
1992;Saito et al., 1993;Saito et al., 1994) as well as physical
studies of organelle separation (Fankhauser et al.,
1976;Rolland et al., 1992). The OASS from different plant
organelles share approximately 40% amino acid sequence
identity with the bacterial enzymes (Bonner et al., 2005).
SAT and OASS in plants form a hetero-oligomeric and
regulatory cysteine synthase complex (CSC) where SAT
activity increases and OASS activity decreases (Bogdanova
4 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9
and Hell, 1997). The function of the complex is not
metabolic channelling, since O-acetylserine freely diffuses
out of the complex (Kredich et al., 1969;Cook and Wedding,
1976;Droux et al., 1998). Instead, interaction of SAT and
OASS coordinates sulfate assimilation and modulates cys-
teine synthesis at the cellular level in plants (Hell and
Hillebrand, 2001). Plant OASSs were also shown to catalyse
the production of beta-substituted alanines as well as
cysteine, and are considered to be involved, at least partly, in
detoxifying toxins byproducts in plant metabolism such as
cyanide, pyrazole, and 3,4-dihydroxypyridine (Murakoshi,
1994).
3.2. Bacterial OASSs
Cysteine biosynthesis in bacteria follows the same general
scheme as depicted in Fig. (1). In enteric bacteria such as S.
typhimurium and E. coli, the genes cysK and cysM code for
two isozymes of OASS, OASS-A and OASS-B, respectively
(Chattopadhyay et al., 2007;Zocher et al., 2007). Both of
these OASSs use sulfide as a nucleophile, but OASS-B has
the added ability to use thiosulfate in the place of H2S,
leading to the production of S-sulfocysteine. OASS-B can
also accept other bigger substrates (Tai et al.,
1993;Chattopadhyay et al., 2007). Of the two proteins,
OASS-A is the best characterized (Becker et al., 1969;Cook
and Wedding, 1976; 1977;Tai and Cook, 2000) and is highly
expressed throughout the life cycle (Kredich, 1971). The
exact role and function of OASS-B is still not clear: at any
time, its activity accounts for a maximum of 20% of total
cysteine biosynthesis of the cell (Nakamura et al., 1983). The
two OASS isozymes share 43% sequence identity and have
almost superimposable three-dimensional structures.
Although it has been reported that OASS-B is mainly
expressed under anaerobic growth conditions (Claus et al.,
2005;Chattopadhyay et al., 2007), the distinct roles of the two
enzymes in infection and long-term survival of the pathogen
have not yet been ascertained (Mozzarelli et al., 2011);
however the redundancy in enzymatic function emphasises
the central role played by the cysteine biosynthetic pathway,
especially in parasitic pathogens that face highly oxidizing
environments during the persistent phase of their life cycle
(Schelle and Bertozzi, 2006;Agren et al., 2009). It is well
documented that SAT and OASS interact to form a regulatory
CSC (Campanini et al., 2005;Salsi et al., 2010b). OASS-B is
unable to interact with SAT and form the CSC; only OASS-A
has this ability. The cysteine biosynthetic pathway in this case
becomes essential for the persistent phase of infection and is
important for long–term survival of the parasites. Since
OASS catalyses the essential final step of the cysteine
biosynthetic pathway, it is at the centre of the regulatory
mechanism of the RSAP. The importance of OASS in
pathogenic bacteria is highlighted by the observation that
CysM is essential for the survival of M. tuberculosis in
macrophages (Sassetti and Rubin, 2003;Rengarajan et al.,
2005) and CysK/CysM knockout strains of S. typhimurium
are deficient in their ability to develop antibiotic resistance
(Turnbull and Surette, 2008).
3.2.1. Inhibitors of Bacterial OASS
The inhibitors of OASS were developed based on
analysis of the SAT-OASS interactions, where the C-
Mazumder and Gourinath
terminal end of SAT binds in the active site of OASS, with
the last amino acid, isoleucine, playing a pivotal role in the
binding. Based on analysis of the crystal structures HiOASS,
MtbOASS and StOASS, different combinations of
pentapeptides were tested for the inhibition of OASS (Salsi
et al., 2010a). The inhibition of OASS by the peptide
inspired the search for isoleucine-like inhibitors in HiOASS.
A large-scale interaction study was carried out by the A.
Mozzarelli group using various combinations of C-terminal
SAT peptides (Salsi et al., 2010a). This study was useful for
further development of pharmacophores and peptide-based
inhibitors. In another study, 2-substituted-cyclopropane-1-
carboxylic acids (Amori et al., 2012) were used for the
scaffold and several derivate were used to calculate the
dissociation constant, with the best binding constant value
being 1.5 µM. Very recently, the Kumaran group determined
a few structures (PDB ID’s: 4ORE, 4NU8, 4MH9) from co-
crystals of HI-OASS with different peptides and substrates,
which showed open, intermediate and closed conformations.
The structures showing the binding of the peptide inhibitors
in open and closed conformations are shown in Fig. (3A).
The availability of the intermediate state as well as the
open and closed conformations can be good starting points
for virtual screening, which may lead to structure-based drug
development (Fig. 3). In one of the approaches using virtual
high-throughput screening, 30 compounds were synthesized
and tested for inhibition and cytotoxicity on MtbOASS (Jean
Kumar et al., 2013). Similarly, in this approach, an SAT-
binding peptide was used as a template for docking studies.
The best-scoring peptide was then optimized (8-nitro-4-(2-
(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido[5,4-
e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione), which yielded
an IC 50 of 17.7 µM for purified OASS and an MIC of 7.6
µM (Jean Kumar et al., 2013). In another study, the structure
of MtbOASS was used to build a pharmacophore (Poyraz et
al., 2013). The pharmacophore was then screened using a
commercial database (Asinex) to identify thiazolidine
inhibitors, with the most potent molecule (3-((Z)-((Z)-5-(4-
fluorobenzylidene)-3-methyl-4-oxo-thiazolidin-2-ylidene)
amino) benzoic acid) having an IC50 value of 19 nM (Poyraz
et al., 2013). Analysis of the pharmacophore developed
molecule and its co-crystal structure of MtbOASS and the
structure of OASS with SAT C-terminal peptide suggested
that the peptide and the inhibitor bind in the same active site
and interact with similar residues (Fig. 4).
The OASS A and OASS B isoforms of S. typhimurium
were used for insilico screening using the ZINC Database
(Spyrakis et al., 2013). The best hits in terms of their
binding/ dissociation constants were in the range of 3-34
µM. Due to the similarity of their active site regions, the
small molecules identified could bind to both enzymes,
albeit with different dissociation constants.
3.3. Protozoan OASS
Protozoa are single-celled eukaryotic organisms and are
the cause of more sickness, death, mutilation, and
debilitation in the world than any other group of disease-
causing organisms. Malaria, which is caused by the genus
Plasmodium, diseases including leishmaniasis and
trypanosomiasis, which are caused by members of the order
Inhibitors of O-Acetyl Serine Sulfhydrylase Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 5

Fig. (3). Bacterial OASS. The crystal structures of A) Haemophilus influenza OASS B) Salmonella typhimurium OASS and C)
Mycobacterium tuberculosis OASS in open, intermediate, and peptide/inhibitor-bound closed conformations. The open conformations are
shown in yellow color and the closed conformations are shown in red color scale.

Kinetoplastida, and amebiasis, which is caused by E.
histolytica, are the three major groups of serious diseases
afflicting very large populations in developing countries
(Turrens, 2004). Most eukaryotes have glutathione as a key
redox buffer and antioxidant, but amitochondriate protists
such as E. histolytica and Trichomonas vaginalis lack this
and related thiols (Ellis et al., 1994;Mehlotra, 1996). In both
of these organisms, cysteine is the major cellular reducing
agent and antioxidant (Fahey et al., 1984;Muller et al.,
2003). In trypanosomatids such as Leishmania and
Trypanosoma brucei, the low molecular weight thiol
trypanothione, a conjugate of glutathione (GSH) and
spermidine, plays a pivotal role in maintaining intracellular
redox homeostasis and providing defence against oxidative
stress (Krauth-Siegel and Comini, 2008). The synthesis of
glutathione and hence trypanothione depends on the
availability of cysteine.
This sulfur-containing amino acid is an essential growth
factor for the trypanosomatid T. brucei also (Duszenko et al.,
1992), whereas T. cruzi can generate it from homocysteine
(Nozaki et al., 2001). Cysteine is essential for a variety of
biological activities, including protein synthesis,
methylation, polyamine synthesis, coenzyme A production,
cysteamine production, taurine production, iron-sulfur
cluster biosynthesis, and antioxidative stress defence
(Nozaki et al., 2005). It was also previously shown that a
high concentration of extracellular cysteine is required for
the growth, attachment, and survival of E. histolytica, T.
vaginalis and G. intestinalis under oxidative stress (Gillin
and Diamond, 1980a;b; 1981a;b;Gillin et al., 1984;Abe et
al., 1999). Some protozoans use different cysteine
biosynthetic pathways than do other protozoans. In
Entamoeba, the typical two-step de novo cysteine
biosynthetic pathway catalyzed by SAT and OASS is
6 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 Mazumder and Gourinath

Fig. (4). The binding mode of peptide and inhibitor in MtbOASS. A) The conformational space occupied by the inhibitor 3-[(Z)-[(5Z)-5-
[[2-(2-hydroxy-2-oxoethyloxy)phenyl]methylidene]- 3-methyl-4-oxidanylidene-1,3-thiazolidin- 2-ylidene]amino]benzoic acid (AWH) and
the ligand DFSI in the superimposed structures of MtbOASS. B) Expanded view of the active site, including those active site residues
involved in inhibitor binding. C) Zoomed in view of the active site residues involved in the peptide binding. The residues of the peptide and
inhibitor are shown in red color.

operational; in contrast, Trichomonas does not have SAT
and instead utilizes O-phosphoserine (OPS) for cysteine
biosynthesis. In both of these organisms, part of reverse
trans-sulfuration is absent, but in Leishmania both reverse
trans-sulfuration and de novo cysteine biosynthetic pathway
are present (Williams et al., 2009).
E. histolytica has been shown to possess three SATs and
three allelic isotypes of OASS (Nozaki et al., 1998;Loftus et
al., 2005). There are several unique features of the amoebic
SAT and OASS which are remarkably different from those
of other organisms. It has been shown that SAT1 is a key
regulated enzyme of the cysteine biosynthetic pathway in E.
histolytica (Nozaki et al., 1998). SAT1 is regulated by
allosteric negative feedback of cysteine, which is similar to
that for plant SATs. Interestingly, SAT3 does not have
feedback inhibition with cysteine (Kumar et al., 2013).
Another unique aspect of the amoebic SAT1 is a lack of
protein-protein interactions including OASS (Kumar et al.,
2011;Raj et al., 2015). OASS1 (and OASS2) and SAT1 form
homodimers and homotrimers, respectively, but they do not
interact with each other under physiological conditions
(Kumar et al., 2011). Among the three OASS isotypes,
OASS1 and OASS2 are identical except for two conservative
amino acid residue changes (Nozaki et al., 1998). OASS3
has diverged from the other two isotypes, and shares only
83% amino acid sequence identity with them (Ali and
Nozaki, 2007). Entamoeba dispar also has two OASS
isotypes, named OASS1 and OASS2, which share 82 to 83%
sequence identity (Nozaki et al., 2000). E. dispar OASS1
and OASS2 correspond to E. histolytica OASS 1 and 2,
respectively. All of these OASS isotypes lack signal
sequences or organelle-targeting sequences, suggesting a
cytosolic location. The presence of multiple cytosolic OASS
isotypes in the non-pathogenic E. dispar species suggests
that this enzyme is not directly associated with the
pathogenicity of the amoeba but that it plays an important
housekeeping role in Entamoeba. Sulfur-assimilatory
cysteine biosynthesis is also characterized in T. vaginalis
(Westrop et al., 2006). The genomic analysis of this
organism revealed six copies of OASS but no SAT.
Inhibitors of O-Acetyl Serine Sulfhydrylase
Enzymological characterization of OASS indicates that T.
vaginalis OASS is able to utilize O-phosphoserine as well as
O-acetylserine as an alanyl donor. Although T. vaginalis
lacks SAT and is thus unable to produce O-acetylserine, it
does possess three copies each of 3-phosphoglycerate
dehydrogenase and phosphoserine aminotransferase and is
presumably able to form O-phosphoserine from 3-
phosphoglycerate derived from glycolysis. Examination and
analysis of Leishmania genomes suggest that L. major, L.
donovani, L. infantum and L. braziliensis all have enzymes
comprising two cysteine biosynthetic routes: the de novo
biosynthesis and the reverse trans-sulfuration (RTS)
pathways. As determined by Southern blot analysis, the L.
major genome contains single-copy genes for cystathionine
β-synthase and cystathionine γ lyase of the RTS pathway,
and OASS and SAT of the de novo biosynthesis pathway
(Westrop et al., 2006). The OASS from Leishmania has high
sequence identities with OASSs of other protozoa — 72%
for T. cruzi, 42% for E. histolytica and 34-38% for T.
vaginalis. It has been observed that SAT and OASS interact
in L. major in a manner similar to that in bacteria and plants,
i.e., they form a regulatory complex in which SAT activity is
enhanced but OASS activity is inhibited (Williams et al.,
2009).
3.3.1. Inhibitors of Protozoan OASS
The C-terminus of E. histolytica SAT1 (EhSAT1) was
expected to bind the OASS active site, since this SAT1 C-
terminus has a sequence (SPSI) that includes an Ile (Kumar
et al., 2011), similar to SATs from E. coli, A. thaliana, H.
influenza and M. tuberculosis (Fig. 5). EhSAT1 and
EhOASS, however, have been shown not to interact with
each other. To explain why EhSAT1 does not interact with
EhOASS, a library of peptides that mimic the C-terminal
region of SAT was generated to study the binding affinity of
these peptides with EhOASS and to determine their
inhibition activities (Kumar et al., 2011). Specifically, a
library of different combinations of tetrapeptides was
generated, keeping Ile at the C-terminal end for docking
studies. These four-residue peptides were docked into the
active site of EhOASS. According to energy parameters
calculated by GLIDE docking software (Friesner et al.,
2004), three peptides, DFSI, DWSI and DYSI, showed better
inhibition against and binding affinity for EhOASS than did
SPSI, which is the native SAT C-terminus. It may be noted
that DFSI is the C-terminal sequence of M. tuberculosis
SAT, where it interacts with OASS to form a cysteine
synthase complex (Schnell et al., 2007). ITC studies
reaffirmed that DFSI is a better binder of EhOASS than is
the EhSAT1-derived SPSI peptide. The docking studies
suggest that aspartate and aromatic amino acids like
phenylalanine and tryptophan make several interactions with
the EhOASS active site cavity. It appears that if the C-
terminal end of EhSAT1 had any of the peptide sequences
DFSI, DWSI, or DYSI, then EhSAT1 would have interacted
with EhOASS to form a multi-enzyme complex. But this
multi-enzyme complex would not be a decamer as observed
in other CS complexes, because SAT in other organisms is a
hexamer and EhSAT1 is a trimer (Raj et al., 2015). By slight
modification of the EhSAT1 C-terminal peptide, E.
histolytica escapes inhibition of EhOASS in the cysteine
biosynthesis regulation process to ensure ready availability
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 7
of cysteine at all times. The closed, intermediate and open
structures of this enzyme in different protozoans are shown
in Fig. (5).
In another investigation, a similar kind of peptide library
was used to study the interactions of the peptides with
LdOASS and it was reported that all kinds of peptides
containing small amino acids can bind and interact with this
enzyme (Raj et al., 2012), although peptides containing an
aromatic residue at the third position from the C-terminus
have higher binding affinities than do the others. While these
SAT C-terminal-mimicking peptides may be considered as
potential inhibitors of OASS, their affinities for OASS are
only in the millimolar to micromolar range.
In the quest for promising inhibitors, the ZINC database
was also used for insilico screening of natural compounds
against EhOASS (Nagpal et al., 2012). Four of the
shortlisted natural inhibitor compounds derived from this
screening were tested for their effects on the enzyme active
site. Out of the selected molecules, the highest ranked
compound (4-hydroxy-2-[2-(1H-indol-3-yl)-2-
oxoethyl]sulfanyl-1H-pyrimidin-6-one) and the sixth highest
ranked compound (3-[3 3-acetyl-4-hydroxy-2-(4-metho
oxyphenyl)-5-oxo-2,5-dihydro-1H- -pyrrol-1-yl]propanoic
acid) caused appreciable decreases in the enzymatic activity.
The binding affinity of 4-hydroxy-2-[2-(1H-indol-3-yl)-2-
oxoethyl]sulfanyl-1H-pyrimidin-6-one to EhOASS was
calculated to be approximately 8 µM whereas that for 3-[3 3-
acetyl-4-hydroxy-2-(4-metho oxyphenyl)-5-oxo-2,5-dihydro-
1H- -pyrrol-1-yl]propanoic acid was 0.18 µM. The
compound 3-[3 3-acetyl-4-hydroxy-2-(4-metho oxyphenyl)-
5-oxo-2, 5-dihydro-1H- pyrrol-1-yl] propionic acid showed
better binding affinity but less inhibition. This difference is
not an aberration as compounds showing better Kd do not
necessarily have better efficacy (Copeland et al., 2006).
Hence, though 4-hydroxy-2-[2-(1H-indol-3-yl)-2-oxoethyl]
sulfanyl-1H-pyrimidin-6-one has a lower binding affinity, it
has a higher efficacy at inhibiting the enzyme. This study
concluded that the insilico screening of natural compounds
resulted in the identification of a few lead-like compounds
that could inhibit EhOASS activity (Nagpal et al., 2012).
The top ranked compound, which inhibited the activity of the
enzyme by almost 70% at a concentration of 100 µM and
with a Kd of 0.18 µM, may be a good starting point for
further optimization.
4. MECHANISM OF THE OPENING AND CLOSING
OF ACTIVE SITE IN OASS
The binding of a substrate at the OASS active site results
in an external aldimine, leading to both local conformational
changes at the active site and global conformational changes
throughout the protein (Fig. 6A). In S. typhimurium, the
formation of the external aldimine with methionine causes
the cofactor to rotate by about 13° about the axis defined by
the line between C20 and the phosphate (Burkhard et al.,
1999). This rotation moves the pyridinium nitrogen
“inwards” towards the protein interior and the aldimine
linkage “outwards” toward the active site entrance. The
cofactor also rotates by about 6°, about an axis perpendicular
to the cofactor ring plane, so as to bring the α-carboxylate
group of the inhibitor close to the N terminus of α-helix 2,
8 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 Mazumder and Gourinath

Fig. (5). Protozoan OASS. A) Entamoeba histolytica OASS and B) Leishmania donovani OASS in open, intermediate, peptide / inhibitor
bound and closed conformations. The open conformations are shown in yellow color.

Fig. (6). Conformational changes induced by ligand binding in OASS. A) Superimposition of the open and closed conformations of
StOASS, EhOASS, HiOASS and MtbOASS. The closure of the active site is located at the middle of the β-sheets in the movable domain,
where the twisting of β-sheets is centred. B) Superimposition of intermediates (Cys and Met in yellow and orange) with the cofactor PLP at
the active site of the above-mentioned OASS structures. C) and D) show the active sites of StOASS and EhOASS in closed and open
conformations and their corresponding interacting residues are shown in stick representation.
Inhibitors of O-Acetyl Serine Sulfhydrylase
and thus more under the influence of the positive electric
field of its dipole (Fig. 6B). The α-carboxylate group of
methionine participates in a strong hydrogen-bonding
network with the residues of the asparagine loop, i.e., 68-
TNGNT-72 (Fig. 6C). Of special interest is the side-chain
amide nitrogen N δ2 of Asn69, which makes one hydrogen
bond each to the inhibitor carboxylate, as well as to the O 3’
oxygen of the cofactor. Asn69 has a conformation
completely different from that in the wild-type enzyme; its
side-chain amide nitrogen moves more than 7 Å to make the
two hydrogen bonds (Fig. 6A). Together with Asn69, all
residues of the asparagine loop undergo a large
rearrangement upon substrate binding (Fig. 6C), resulting in
the above-mentioned interactions with the α-carboxylate
moiety of the substrate. The binding of the α-carboxylate to
the N terminus of α-helix 2 and the rearrangement of Asn69
also has a dramatic influence on the overall topology of the
protein. The movement of the side chain of Asn69 leaves a
hole within the protein structure, which is filled by the
protein itself. Met95, adjacent to Asn69 in the native
structure, follows the side-chain movement of Asn69. The
methionine side-chain sulfur atom shifts over 6.5 Å. Met95
is located in a subdomain (residues 87-131) of the N-
terminal domain (Burkhard et al., 1998). This subdomain,
comprising β-strand 4, α−helix 3, β-strand 5 and α-helix 4,
rotates as a rigid body by about 13°. The subdomain
movement does not disrupt the overall topology of the N-
terminal domain: the RMSD of the Cα positions of the
subdomain (residues 87-131) between the open and the
closed structures is only 0.56 Å. The RMSD is 0.16 Å for the
C-terminal domain (residues 16-31 and 147-298) and 1.56 Å
for the whole molecule (residues 16-298). Thus, the
conformational change in the protein, with the exception of
the local rearrangement in the asparagine loop, basically
consists of a rigid body motion of a subdomain of the N-
terminal domain, while no local conformational changes in
this subdomain or in the C-terminal domain occur. The
movement of the subdomain (residues 87 -131) brings
residues that are far apart in the open conformation into close
proximity, resulting in burying the substrate within the
protein and making the active site cleft no longer accessible
to bulk solvent (Fig. 6C).
Similar changes in conformation were observed in cys-
teine and methionine bound EhOASS structures
(Chinthalapudi et al., 2008;Raj et al., 2013a), where part of
the N-terminal domain (64 to 165) is twisted by about
15°relative to C-terminal domain, which causes closure of
the active site (Fig. 6D). These residues are well conserved
among members of the cysteine synthase family. The
proximity of the amino group of Cys to the C4A atom of
PLP makes the conditions potentially favourable for forming
a covalent bond between them; this proximity may
alternatively be interpreted as as a snapshot depicting the
beginning of the dissociation between the product (Cys) and
cofactor (PLP).
4.1. Targeting the Closed or the Intermediate
Conformation
A recent paper (Raj et al., 2013b) from our laboratory
regarding the binding free energies in both complexes, i.e.,
the methionine-bound closed conformation and the
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 9
isoleucine-containing peptide-bound open conformation,
revealed that binding of methionine to EhOASS is more than
twice as favourable as the binding of isoleucine, suggesting
methionine to be more stable at the active site than
isoleucine. The approach to screen and design molecules that
can mimic methionine binding can yield small-molecule
drugs capable of disrupting the activity of the enzyme. Any
such inhibitor would act by closing the active site cleft and
maintaining it in this conformation, making the active site
inaccessible to substrate. So far, the strategy of such work
has involved using isoleucine as a basis for modeling
inhibitors (Salsi et al., 2010a;Amori et al., 2012;Nagpal et
al., 2012), since isoleucine itself is a known inhibitor.
Methionine, however, is shown here to be the stronger
binding ligand, since it is a substrate analog that stops the
cysteine biosynthetic reaction at the sulfide addition step; it
can thus also act as an inhibitor. Hence, the design of an
inhibitor using the methionine-complexed closed cleft
conformation structure as a template should lead to a more
energetically favourable complex compared to isoleucine-
based inhibitor molecules, which all have binding affinities
in the micromolar range.
CONCLUSION
Over the recent few years, the sulphur metabolism
pathway and the enzymes involved in it have been targeted
as the prospective source for drug targets due to the
importance of this pathway in the life cycles of many
microorganisms. This is due to the fact that these microbes
live in very challenging conditions and require certain
metabolites for their survival and growth. The identification
of potential drug targets for mutating and evolving microbes
is a very difficult task, with the success rate of such
endeavours being very low. The failures are due to the
limited understanding of the complex mechanism exhibited
by these microorganisms. Targeting such a pathway for
which there is complete knowledge of the mechanism of the
responsible enzyme would definitely increase the chances of
discovering an effective drug. Molecules developed to target
enzymes such as OASS, whose mechanism of binding is
known, can be used to develop effective inhibitors. The
growing availability of crystal structures of the intermediate
and closed conformations of OASS from various organisms
suggests that these conformations are preferred in this
enzyme. So far, all the inhibitors developed against any
OASS are against the open state conformation, and most of
the inhibitors bind to similar residues located close to the
PLP. We therefore predict that designing drugs based on the
more stable methionine-bound OASS structure should yield
higher-binding and more effective inhibitors than those
currently available.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
We thank Indian Council of Medical Research and the
Department of Biotechnology (COE), Government of India
for funding. We thank DST-PURSE for extending
10 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9
institutional funding. We would also like to thank Dr. Isha
Raj for the valuable inputs from her thesis work.
REFERENCES
[1] Abe, N., Nishikawa, Y., Yasukawa, A., and Haruki, K. (1999).
Entamoeba histolytica outbreaks in institutions for the mentally
retarded. Jpn J Infect Dis 52, 135-136.
[2] Agren, D., Schnell, R., and Schneider, G. (2009). The C-terminal
of CysM from Mycobacterium tuberculosis protects the
aminoacrylate intermediate and is involved in sulfur donor
selectivity. FEBS Lett 583, 330-336. doi:
10.1016/j.febslet.2008.12.019.
[3] Ali, V., and Nozaki, T. (2007). Current therapeutics, their
problems, and sulfur-containing-amino-acid metabolism as a novel
target against infections by "amitochondriate" protozoan parasites.
Clin Microbiol Rev 20, 164-187. doi: 10.1128/CMR.00019-06.
[4] Amori, L., Katkevica, S., Bruno, A., Campanini, B., Felici, P.,
Mozzarelli, A., and Costantino, G. (2012). Design and synthesis of
trans-2-substituted-cyclopropane-1-carboxylic acids as the first
non-natural small molecule inhibitors of O-acetylserine
sulfhydrylase. MedChemComm 3, 1111-1116. doi:
10.1039/C2MD20100C.
[5] Becker, M.A., Kredich, N.M., and Tomkins, G.M. (1969). The
purification and characterization of O-acetylserine sulfhydrylase-A
from Salmonella typhimurium. J Biol Chem 244, 2418-2427.
[6] Beinert, H. (2000). A tribute to sulfur. Eur J Biochem 267, 5657-
5664.
[7] Bogdanova, N., and Hell, R. (1997). Cysteine synthesis in plants:
protein-protein interactions of serine acetyltransferase from
Arabidopsis thaliana. Plant J 11, 251-262.
[8] Bonner, E.R., Cahoon, R.E., Knapke, S.M., and Jez, J.M. (2005).
Molecular basis of cysteine biosynthesis in plants: structural and
functional analysis of O-acetylserine sulfhydrylase from
Arabidopsis thaliana. J Biol Chem 280, 38803-38813. doi:
M505313200 [pii]10.1074/jbc.M505313200.
[9] Burkhard, P., Rao, G.S., Hohenester, E., Schnackerz, K.D., Cook,
P.F., and Jansonius, J.N. (1998). Three-dimensional structure of O-
acetylserine sulfhydrylase from Salmonella typhimurium. J Mol
Biol 283, 121-133. doi: S002228369892037X [pii].
[10] Burkhard, P., Tai, C.H., Ristroph, C.M., Cook, P.F., and Jansonius,
J.N. (1999). Ligand binding induces a large conformational change
in O-acetylserine sulfhydrylase from Salmonella typhimurium. J
Mol Biol 291, 941-953. doi: 10.1006/jmbi.1999.3002S0022-
2836(99)93002-4 [pii].
[11] Campanini, B., Pieroni, M., Raboni, S., Bettati, S., Benoni, R.,
Pecchini, C., Costantino, G., and Mozzarelli, A. (2015). Inhibitors
of the sulfur assimilation pathway in bacterial pathogens as
enhancers of antibiotic therapy. Curr Med Chem 22, 187-213.
[12] Campanini, B., Speroni, F., Salsi, E., Cook, P.F., Roderick, S.L.,
Huang, B., Bettati, S., and Mozzarelli, A. (2005). Interaction of
serine acetyltransferase with O-acetylserine sulfhydrylase active
site: evidence from fluorescence spectroscopy. Protein Sci 14,
2115-2124. doi: ps.051492805 [pii]10.1110/ps.051492805.
[13] Chattopadhyay, A., Meier, M., Ivaninskii, S., Burkhard, P.,
Speroni, F., Campanini, B., Bettati, S., Mozzarelli, A., Rabeh,
W.M., Li, L., and Cook, P.F. (2007). Structure, mechanism, and
conformational dynamics of O-acetylserine sulfhydrylase from
Salmonella typhimurium: comparison of A and B isozymes.
Biochemistry 46, 8315-8330. doi: 10.1021/bi602603c.
[14] Chinthalapudi, K., Kumar, M., Kumar, S., Jain, S., Alam, N., and
Gourinath, S. (2008). Crystal structure of native O-acetyl-serine
sulfhydrylase from Entamoeba histolytica and its complex with
cysteine: structural evidence for cysteine binding and lack of
interactions with serine acetyl transferase. Proteins 72, 1222-1232.
doi: 10.1002/prot.22013.
[15] Claus, M.T., Zocher, G.E., Maier, T.H., and Schulz, G.E. (2005).
Structure of the O-acetylserine sulfhydrylase isoenzyme CysM
from Escherichia coli. Biochemistry 44, 8620-8626. doi:
10.1021/bi050485+.
[16] Cook, P.F., and Wedding, R.T. (1976). A reaction mechanism from
steady state kinetic studies for O-acetylserine sulfhydrylase from
Salmonella typhimurium LT-2. J Biol Chem 251, 2023-2029.
[17] Cook, P.F., and Wedding, R.T. (1977). Overall mechanism and rate
equation for O-acetylserine sulfhydrylase. J Biol Chem 252, 3459.
Mazumder and Gourinath
[18] Delano, W. (2002). Pymol. DeLano Scientific, South San
Francisco, CA. .
[19] Droux, M. (2004). Sulfur assimilation and the role of sulfur in plant
metabolism: a survey. Photosynth Res 79, 331-348. doi:
10.1023/B:PRES.0000017196.95499.11.
[20] Droux, M., Ruffet, M.L., Douce, R., and Job, D. (1998).
Interactions between serine acetyltransferase and O-acetylserine
(thiol) lyase in higher plants--structural and kinetic properties of
the free and bound enzymes. Eur J Biochem 255, 235-245.
[21] Duszenko, M., Muhlstadt, K., and Broder, A. (1992). Cysteine is an
essential growth factor for Trypanosoma brucei bloodstream forms.
Mol Biochem Parasitol 50, 269-273.
[22] Ellis, J.E., Yarlett, N., Cole, D., Humphreys, M.J., and Lloyd, D.
(1994). Antioxidant defences in the microaerophilic protozoan
Trichomonas vaginalis: comparison of metronidazole-resistant and
sensitive strains. Microbiology 140 ( Pt 9), 2489-2494.
[23] Fahey, R.C., Newton, G.L., Arrick, B., Overdank-Bogart, T., and
Aley, S.B. (1984). Entamoeba histolytica: a eukaryote without
glutathione metabolism. Science 224, 70-72.
[24] Fankhauser, H., Brunold, C., and Erismann, K.H. (1976).
Subcellular localization of O-acetylserine sulfhydrylase in spinach
leaves. Experientia 32, 1494-1497.
[25] Francois, J.A., Kumaran, S., and Jez, J.M. (2006). Structural basis
for interaction of O-acetylserine sulfhydrylase and serine
acetyltransferase in the Arabidopsis cysteine synthase complex.
Plant Cell 18, 3647-3655. doi: tpc.106.047316
[pii]10.1105/tpc.106.047316.
[26] Friesner, R.A., Banks, J.L., Murphy, R.B., Halgren, T.A., Klicic,
J.J., Mainz, D.T., Repasky, M.P., Knoll, E.H., Shelley, M., Perry,
J.K., Shaw, D.E., Francis, P., and Shenkin, P.S. (2004). Glide: a
new approach for rapid, accurate docking and scoring. 1. Method
and assessment of docking accuracy. J Med Chem 47, 1739-1749.
doi: 10.1021/jm0306430.
[27] Gillin, F.D., and Diamond, L.S. (1980a). Attachment and short-
term maintenance of motility and viability of Entamoeba histolytica
in a defined medium. J Protozool 27, 220-225.
[28] Gillin, F.D., and Diamond, L.S. (1980b). Attachment of Entamoeba
histolytica to glass in a defined maintenance medium: specific
requirement for cysteine and ascorbic acid. J Protozool 27, 474-
478.
[29] Gillin, F.D., and Diamond, L.S. (1981a). Entamoeba histolytica and
Giardia lamblia: effects of cysteine and oxygen tension on
trophozoite attachment to glass and survival in culture media. Exp
Parasitol 52, 9-17. doi: 0014-4894(81)90055-2 [pii].
[30] Gillin, F.D., and Diamond, L.S. (1981b). Entamoeba histolytica
and Giardia lamblia: growth responses to reducing agents. Exp
Parasitol 51, 382-391.
[31] Gillin, F.D., Reiner, D.S., Levy, R.B., and Henkart, P.A. (1984).
Thiol groups on the surface of anaerobic parasitic protozoa. Mol
Biochem Parasitol 13, 1-12.
[32] Grant, B.J., Rodrigues, A.P., Elsawy, K.M., Mccammon, J.A., and
Caves, L.S. (2006). Bio3d: an R package for the comparative
analysis of protein structures. Bioinformatics 22, 2695-2696. doi:
10.1093/bioinformatics/btl461.
[33] Hawkesford, M.J., and De Kok, L.J. (2006). Managing sulphur
metabolism in plants. Plant Cell Environ 29, 382-395.
[34] Hell, R., and Hillebrand, H. (2001). Plant concepts for mineral
acquisition and allocation. Curr Opin Biotechnol 12, 161-168.
[35] Hell, R., Jost, R., Berkowitz, O., and Wirtz, M. (2002). Molecular
and biochemical analysis of the enzymes of cysteine biosynthesis
in the plant Arabidopsis thaliana. Amino Acids 22, 245-257.
[36] Huang, B., Vetting, M.W., and Roderick, S.L. (2005). The active
site of O-acetylserine sulfhydrylase is the anchor point for
bienzyme complex formation with serine acetyltransferase. J
Bacteriol 187, 3201-3205. doi: 187/9/3201
[pii]10.1128/JB.187.9.3201-3205.2005.
[37] Jean Kumar, V.U., Poyraz, O., Saxena, S., Schnell, R., Yogeeswari,
P., Schneider, G., and Sriram, D. (2013). Discovery of novel
inhibitors targeting the Mycobacterium tuberculosis O-acetylserine
sulfhydrylase (CysK1) using virtual high-throughput screening.
Bioorg Med Chem Lett 23, 1182-1186. doi:
10.1016/j.bmcl.2013.01.031.
[38] Kessler, D. (2006). Enzymatic activation of sulfur for incorporation
into biomolecules in prokaryotes. FEMS Microbiol Rev 30, 825-
840. doi: 10.1111/j.1574-6976.2006.00036.x.
Inhibitors of O-Acetyl Serine Sulfhydrylase
[39] Krauth-Siegel, R.L., and Comini, M.A. (2008). Redox control in
trypanosomatids, parasitic protozoa with trypanothione-based thiol
metabolism. Biochim Biophys Acta 1780, 1236-1248. doi:
10.1016/j.bbagen.2008.03.006.
[40] Kredich, N.M. (1971). Regulation of L-cysteine biosynthesis in
Salmonella typhimurium. I. Effects of growth of varying sulfur
sources and O-acetyl-L-serine on gene expression. J Biol Chem
246, 3474-3484.
[41] Kredich, N.M., Becker, M.A., and Tomkins, G.M. (1969).
Purification and characterization of cysteine synthetase, a
bifunctional protein complex, from Salmonella typhimurium. J Biol
Chem 244, 2428-2439.
[42] Krishna, C., Jain, R., Kashav, T., Wadhwa, D., Alam, N., and
Gourinath, S. (2007). Crystallization and preliminary
crystallographic analysis of cysteine synthase from Entamoeba
histolytica. Acta Crystallogr Sect F Struct Biol Cryst Commun 63,
512-515. doi: S1744309107022154
[pii]10.1107/S1744309107022154.
[43] Kumar, S., Mazumder, M., Dharavath, S., and Gourinath, S.
(2013). Single residue mutation in active site of serine
acetyltransferase isoform 3 from Entamoeba histolytica assists in
partial regaining of feedback inhibition by cysteine. PLoS One 8,
e55932. doi: 10.1371/journal.pone.0055932.
[44] Kumar, S., Raj, I., Nagpal, I., Subbarao, N., and Gourinath, S.
(2011). Structural and biochemical studies of serine
acetyltransferase reveal why the parasite Entamoeba histolytica
cannot form a cysteine synthase complex. J Biol Chem 286, 12533-
12541. doi: M110.197376 [pii]10.1074/jbc.M110.197376.
[45] Kumaran, S., Yi, H., Krishnan, H.B., and Jez, J.M. (2009).
Assembly of the cysteine synthase complex and the regulatory role
of protein-protein interactions. J Biol Chem 284, 10268-10275. doi:
10.1074/jbc.M900154200.
[46] Leustek, T., Martin, M.N., Bick, J.A., and Davies, J.P. (2000).
Pathways and Regulation of Sulfur Metabolism Revealed through
Molecular and Genetic Studies. Annu Rev Plant Physiol Plant Mol
Biol 51, 141-165. doi: 10.1146/annurev.arplant.51.1.141.
[47] Loftus, B., Anderson, I., Davies, R., Alsmark, U.C., Samuelson, J.,
Amedeo, P., Roncaglia, P., Berriman, M., Hirt, R.P., Mann, B.J.,
Nozaki, T., Suh, B., Pop, M., Duchene, M., Ackers, J., Tannich, E.,
Leippe, M., Hofer, M., Bruchhaus, I., Willhoeft, U., Bhattacharya,
A., Chillingworth, T., Churcher, C., Hance, Z., Harris, B., Harris,
D., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares, R.,
Whitehead, S., Quail, M.A., Rabbinowitsch, E., Norbertczak, H.,
Price, C., Wang, Z., Guillen, N., Gilchrist, C., Stroup, S.E.,
Bhattacharya, S., Lohia, A., Foster, P.G., Sicheritz-Ponten, T.,
Weber, C., Singh, U., Mukherjee, C., El-Sayed, N.M., Petri, W.A.,
Jr., Clark, C.G., Embley, T.M., Barrell, B., Fraser, C.M., and Hall,
N. (2005). The genome of the protist parasite Entamoeba
histolytica. Nature 433, 865-868. doi: 10.1038/nature03291.
[48] Lunn, J.E., Droux, M., Martin, J., and Douce, R. (1990).
Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in
Spinach Leaves. Plant Physiol 94, 1345-1352.
[49] Mehlotra, R.K. (1996). Antioxidant defense mechanisms in
parasitic protozoa. Crit Rev Microbiol 22, 295-314. doi:
10.3109/10408419609105484.
[50] Mozzarelli, A., Bettati, S., Campanini, B., Salsi, E., Raboni, S.,
Singh, R., Spyrakis, F., Kumar, V.P., and Cook, P.F. (2011). The
multifaceted pyridoxal 5'-phosphate-dependent O-acetylserine
sulfhydrylase. Biochim Biophys Acta 1814, 1497-1510. doi:
10.1016/j.bbapap.2011.04.011.
[51] Muller, S., Liebau, E., Walter, R.D., and Krauth-Siegel, R.L.
(2003). Thiol-based redox metabolism of protozoan parasites.
Trends Parasitol 19, 320-328.
[52] Murakoshi, F.I.I. (1994). Enzymic synthesis of non-protein β-
substitutedalanines and some higher homologues in plants.
Phytochemistry 35, 1089–1104.
[53] Nagpal, I., Raj, I., Subbarao, N., and Gourinath, S. (2012). Virtual
screening, identification and in vitro testing of novel inhibitors of
O-acetyl-L-serine sulfhydrylase of Entamoeba histolytica. PLoS
One 7, e30305. doi: 10.1371/journal.pone.0030305PONE-D-11-
20729 [pii].
[54] Nakamura, T., Kon, Y., Iwahashi, H., and Eguchi, Y. (1983).
Evidence that thiosulfate assimilation by Salmonella typhimurium
is catalyzed by cysteine synthase B. J Bacteriol 156, 656-662.
Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9 11
[55] Nozaki, T., Ali, V., and Tokoro, M. (2005). Sulfur-containing
amino acid metabolism in parasitic protozoa. Adv Parasitol 60, 1-
99. doi: 10.1016/S0065-308X(05)60001-2.
[56] Nozaki, T., Asai, T., Kobayashi, S., Ikegami, F., Noji, M., Saito,
K., and Takeuchi, T. (1998). Molecular cloning and
characterization of the genes encoding two isoforms of cysteine
synthase in the enteric protozoan parasite Entamoeba histolytica.
Mol Biochem Parasitol 97, 33-44. doi: S0166-6851(98)00129-7
[pii].
[57] Nozaki, T., Shigeta, Y., Saito-Nakano, Y., Imada, M., and Kruger,
W.D. (2001). Characterization of transsulfuration and cysteine
biosynthetic pathways in the protozoan hemoflagellate,
Trypanosoma cruzi. Isolation and molecular characterization of
cystathionine beta-synthase and serine acetyltransferase from
Trypanosoma. J Biol Chem 276, 6516-6523. doi:
10.1074/jbc.M009774200.
[58] Nozaki, T., Tokoro, M., Imada, M., Saito, Y., Abe, Y., Shigeta, Y.,
and Takeuchi, T. (2000). Cloning and biochemical characterization
of genes encoding two isozymes of cysteine synthase from
Entamoeba dispar. Mol Biochem Parasitol 107, 129-133.
[59] Percudani, R., and Peracchi, A. (2003). A genomic overview of
pyridoxal-phosphate-dependent enzymes. EMBO Rep 4, 850-854.
doi: 10.1038/sj.embor.embor914.
[60] Poyraz, Ö., Jeankumar, V.U., Saxena, S., Schnell, R., Haraldsson,
M., Yogeeswari, P., Sriram, D., and Schneider, G. (2013).
Structure-Guided Design of Novel Thiazolidine Inhibitors of O-
Acetyl Serine Sulfhydrylase from Mycobacterium tuberculosis.
Journal of Medicinal Chemistry 56, 6457-6466. doi:
10.1021/jm400710k.
[61] Raj, I., Kumar, S., and Gourinath, S. (2012). The narrow active-site
cleft of O-acetylserine sulfhydrylase from Leishmania donovani
allows complex formation with serine acetyltransferases with a
range of C-terminal sequences. Acta Crystallographica Section D
Biological Crystallography D68, 909-919.
[62] Raj, I., Kumar, S., Mazumder, M., and Gourinath, S. (2015).
"Structural Biology of Cysteine Biosynthetic Pathway Enzymes,"
in Amebiasis, eds. T. Nozaki & A. Bhattacharya. Springer Japan),
373-391.
[63] Raj, I., Mazumder, M., and Gourinath, S. (2013a). Molecular basis
of ligand recognition by OASS from E. histolytica: Insights from
structural and molecular dynamics simulation studies. Biochimica
et Biophysica Acta (BBA) - General Subjects 1830, 4573-4583. doi:
http://dx.doi.org/10.1016/j.bbagen.2013.05.041.
[64] Raj, I., Mazumder, M., and Gourinath, S. (2013b). Molecular basis
of ligand recognition by OASS from E. histolytica: insights from
structural and molecular dynamics simulation studies. Biochim
Biophys Acta 1830, 4573-4583. doi: 10.1016/j.bbagen.2013.05.041.
[65] Rengarajan, J., Bloom, B.R., and Rubin, E.J. (2005). Genome-wide
requirements for Mycobacterium tuberculosis adaptation and
survival in macrophages. Proc Natl Acad Sci U S A 102, 8327-
8332. doi: 10.1073/pnas.0503272102.
[66] Rolland, N., Droux, M., and Douce, R. (1992). Subcellular
Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica
oleracea L.) Inflorescence. Plant Physiol 98, 927-935.
[67] Saito, K., Miura, N., Yamazaki, M., Hirano, H., and Murakoshi, I.
(1992). Molecular cloning and bacterial expression of cDNA
encoding a plant cysteine synthase. Proc Natl Acad Sci U S A 89,
8078-8082.
[68] Saito, K., Tatsuguchi, K., Murakoshi, I., and Hirano, H. (1993).
cDNA cloning and expression of cysteine synthase B localized in
chloroplasts of Spinacia oleracea. FEBS Lett 324, 247-252.
[69] Saito, K., Tatsuguchi, K., Takagi, Y., and Murakoshi, I. (1994).
Isolation and characterization of cDNA that encodes a putative
mitochondrion-localizing isoform of cysteine synthase (O-
acetylserine(thiol)-lyase) from Spinacia oleracea. J Biol Chem 269,
28187-28192.
[70] Salsi, E., Bayden, A.S., Spyrakis, F., Amadasi, A., Campanini, B.,
Bettati, S., Dodatko, T., Cozzini, P., Kellogg, G.E., Cook, P.F.,
Roderick, S.L., and Mozzarelli, A. (2010a). Design of O-
acetylserine sulfhydrylase inhibitors by mimicking nature. J Med
Chem 53, 345-356. doi: 10.1021/jm901325e.
[71] Salsi, E., Campanini, B., Bettati, S., Raboni, S., Roderick, S.L.,
Cook, P.F., and Mozzarelli, A. (2010b). A two-step process
controls the formation of the bienzyme cysteine synthase complex.
J Biol Chem 285, 12813-12822. doi: M109.075762
[pii]10.1074/jbc.M109.075762.
 12 Current Topics in Medicinal Chemistry, 2016, Vol. 16, No. 9
[72] Sassetti, C.M., and Rubin, E.J. (2003). Genetic requirements for
mycobacterial survival during infection. Proc Natl Acad Sci U S A
100, 12989-12994. doi: 10.1073/pnas.2134250100.
[73] Schelle, M.W., and Bertozzi, C.R. (2006). Sulfate metabolism in
mycobacteria. Chembiochem 7, 1516-1524. doi:
10.1002/cbic.200600224.
[74] Schnell, R., Oehlmann, W., Singh, M., and Schneider, G. (2007).
Structural insights into catalysis and inhibition of O-acetylserine
sulfhydrylase from Mycobacterium tuberculosis. Crystal structures
of the enzyme alpha-aminoacrylate intermediate and an enzyme-
inhibitor complex. J Biol Chem 282, 23473-23481. doi:
M703518200 [pii]10.1074/jbc.M703518200.
[75] Spyrakis, F., Singh, R., Cozzini, P., Campanini, B., Salsi, E., Felici,
P., Raboni, S., Benedetti, P., Cruciani, G., Kellogg, G.E., Cook,
P.F., and Mozzarelli, A. (2013). Isozyme-specific ligands for O-
acetylserine sulfhydrylase, a novel antibiotic target. PLoS One 8,
e77558. doi: 10.1371/journal.pone.0077558.
[76] Tai, C.H., and Cook, P.F. (2000). O-acetylserine sulfhydrylase. Adv
Enzymol Relat Areas Mol Biol 74, 185-234.
[77] Tai, C.H., Nalabolu, S.R., Jacobson, T.M., Minter, D.E., and Cook,
P.F. (1993). Kinetic mechanisms of the A and B isozymes of O-
acetylserine sulfhydrylase from Salmonella typhimurium LT-2
using the natural and alternative reactants. Biochemistry 32, 6433-
6442.
Received: ???????????????? Revised: ???????????????? Accepted: ????????????????
View publication stats
Mazumder and Gourinath
[78] Turnbull, A.L., and Surette, M.G. (2008). L-Cysteine is required
for induced antibiotic resistance in actively swarming Salmonella
enterica serovar Typhimurium. Microbiology 154, 3410-3419. doi:
10.1099/mic.0.2008/020347-0.
[79] Turrens, J.F. (2004). Oxidative stress and antioxidant defenses: a
target for the treatment of diseases caused by parasitic protozoa.
Mol Aspects Med 25, 211-220. doi: 10.1016/j.mam.2004.02.021.
[80] Wei, J., Tang, Q.X., Varlamova, O., Roche, C., Lee, R., and Leyh,
T.S. (2002). Cysteine biosynthetic enzymes are the pieces of a
metabolic energy pump. Biochemistry 41, 8493-8498.
[81] Westrop, G.D., Goodall, G., Mottram, J.C., and Coombs, G.H.
(2006). Cysteine biosynthesis in Trichomonas vaginalis involves
cysteine synthase utilizing O-phosphoserine. J Biol Chem 281,
25062-25075. doi: 10.1074/jbc.M600688200.
[82] Williams, R.A., Westrop, G.D., and Coombs, G.H. (2009). Two
pathways for cysteine biosynthesis in Leishmania major. Biochem J
420, 451-462. doi: 10.1042/BJ20082441.
[83] Wirtz, M., Droux, M., and Hell, R. (2004). O-acetylserine (thiol)
lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited
in Arabidopsis thaliana. J Exp Bot 55, 1785-1798. doi:
10.1093/jxb/erh201.
[84] Zocher, G., Wiesand, U., and Schulz, G.E. (2007). High resolution
structure and catalysis of O-acetylserine sulfhydrylase isozyme B
from Escherichia coli. FEBS J 274, 5382-5389. doi:
10.1111/j.1742-4658.2007.06063.x.