REVIEW ?-replacement enzyme which performs the second half reaction



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(Cys) is a sulfur containing amino acid which plays important structural and
functional roles in proteins and enzymes. Cysteine often participates in the
enzymatic reactions as a nucleophile through its thiol side chain. Cysteine
also plays a major role in the antioxidant defense mechanism in the human
parasite, Entamoeba histolytica; and acts a precursor of methionine,
glutathione, phytochelatins, iron-sulfur clusters, vitamin cofactors and
multiple secondary metabolites. The biosynthesis of cysteine in mammals is
quite different to that of in plants and microorganisms. In plants and
microorganisms, the cysteine biosynthesis is a two-step process and requires
two enzymes: (1) Serine Acetyltransferase (SAT/ CysE) and (2)
O-AcetylserineSulfhydrylase (OASS/ CysK). CysE catalyzes the first half of the
reaction to produce O-acetylserine (OAS) and CysK participates in the second
half of the reaction with OAS and HS (Hydrogen Sulphide) to produce L-cysteine.
The focal point of this paper will however be, CysK, regarding its structure,
mechanism and regulation.

is a pyridoxal-dependent, ?-replacement enzyme which performs the second half
reaction of the cysteine biosynthesis by catalyzing substitution of acetate in
the side-chain of OAS by sulfide to produce L-cysteine. There are many facets
of CysK, some of which will be discussed in this paper. The successfully solved
structures of CysK are found in Salmonella typhimurium, Entamoeba
histolytica and Arabidopsis thaliana with the resolution of 2.2Å
which reveals that it’s a 34.5kDa homodimer protein. Apart from that, many
mutant CysK structures have also been developed in order to study the functions
of significant amino acids in the enzyme. The kinetic mechanism of OASS is Ping
Pong Bi Bi as shown by initial velocity studies in the absence and presence of
products and dead end inhibitors, isotope exchange at equilibrium, and
equilibrium spectral studies. O-acetyl-L-serine binds to the internal aldimine
form of the enzyme, and acetate is released as the first product. Bisulfide
then adds as the second substrate to the ?-aminoacrylate intermediate form of
the enzyme, and L-cysteine is released as the final product. The regulatory
aspect of CysK comes with its complex formation with the preceding enzyme of
the pathway i.e. CysE. This bi-enzyme complex is known as Cysteine Synthase
Complex (CSC). Upon depletion of the substrate for CysK i.e. OAS, CysK forms a
complex with CysE which leads to its inhibition. The inherent inhibition of
CysK comes from its binding to the ?-aminoacrylate intermediate as seen in M.
tuberculosis. But apart from this,
there has been a multitude of researches going on to develop novel inhibitors
of CysK for as therapeutics. For instance, designing the inhibitor peptides
that mimic the terminal peptide sequence of CysE (which directly interacts with
CysK active site), synthetic inhibitors (UPAR40), thiazolidine inhibitors.


Cysteine is a sulfur containing compound which acts
as a metabolic sulfide donor for all cellular components containing reduced
sulfur. In addition to its role in protein structure, cysteine is a precursor
of methionine, glutathione, phytochelatins, iron-sulfur clusters, vitamin
cofactors, and multiple secondary metabolites.

In plants and microorgnisms, the biosynthesis of
cysteine requires primarily two enzymes, serine acetyltransferase (SAT) and
O-Acetylserinesulfhydrylase (OASS). The mechanism involves the transfer of
acetate from acetyl-CoA to serine by serine acetyltransferase generating
O-acetylserine. O-acetylserinesulfhydrylase (Cys K) uses pyridoxal 5′-phosphate
(PLP) as a cofactor to catalyze the formation of cysteine from O-acetylserine
and sulfide.

In Mycobacterium tuberculosis, the mechanism
of formation of O-acetylserine (OAS) by serine acetyltransferase is the same
but there are three genes that had been annotated to be putative O-acetylserine
sulfhydrylase (OASS) encoding genes; Rv2334 (CysK1), Rv0848 (CysK2) and Rv1336
(CysM). There are three de novo cysteine biosynthesis pathways.

CysK1 acts in a PLP dependent manner in which, O-acetylserine forms an
external aldimine with PLP cofactor and forms an intermediate ?-aminoacrylate
in the first step. In the next reaction, the nucleophilic attack of sulfide
onto the intermediate which leads to the formation of cysteine.

The first reaction of cysteine biosynthesis by CysM is same as that of
the CysK1 which yields ?-aminoacrylate intermediate at the end. However, in the
second half reaction, the nucleophilic attack of CysO-SH on the intermediate is
followed by the S,N-acyl shift which results in CysO-cysteine adduct which is
hydrolyzed by Mec+ to release cysteine. Also, CysM prefers O-phosphoserine (OPS)
over O-acetylserine (OAS) as a substrate.

CysK2 uses
O-phosphoserine as an acceptor substrate; however CysK2 does not uses CysO but
sulfide as a sulfur donor to form cysteine.


is a pyridoxal-dependent, ?-replacement enzyme which performs the second half
reaction of the cysteine biosynthesis by catalyzing substitution of acetate in
the side-chain of OAS by sulfide to produce L-cysteine. The crystal structure
of CysK has been solved in Salmonella typhimurium, Entamoeba
histolytica and Arabidopsis thaliana with the resolution of 2.2Å.
From both the cases, it is clear that CysK is a 34.45 kDahomodimer enzyme that
catalyzes the substitution of acetate in the side-chain of OAS by sulfide to
give L-cysteine 3.


has two C- and N-terminal monomers. The N-terminal domain forms an ?/?
structure of 4 ?? units. The helices 1, 2, 3 of N-terminal domain are present
on the face of a four- stranded parallel ?-sheet5, 4, 3, 6; helix 4 is present
on the other face. Similarly in the C-terminal, helices 7, 8, 9 surround
?-sheet strands 9, 8, 7, 10. This similarity raises the possibility of a gene
duplication event in an ancient gene, coding for one-domain ancestor protein.
But still, it has been observed that the c-terminal I bulkier than the
N-terminal and that is because the N-terminus of N-terminal has only 12
residues which can be counted in N-terminal domain. Residues 13-34 belong to
the C-terminal domain. This stretch includes 2 antiparallel ?-sheets which are
attached to the ?-sheet 10 of the central ?-sheet resulting in six ?-sheet
strand in C-terminal 3.

overall topology of A. thaliana resembles that of the S. typhimurium except
for some differences. In AtCysK, there is a three amino acid insertion in
?1A-?2A loop and six amino acid deletion in ?8A-?9A loop as compared to StCysK.
Furthermore, the C-terminus of AtCysK extends along the surface of the adjacent
monomer while the last 20 amino acids were disordered in StCysK 1.


PLP-binding site is buried deeply within the protein and is located at the
interface between N- and C-terminal domains. It forms the internal aldimine
(Schiff-base linkage) with the Lys41. The 5′-phosphate group of PLP is bound to
the N-terminus of helix 7 of CysK. The residues of polypeptide chain between
the strand 7 and helix 7, namely, Gly176, Thr177, Gly178, Thr180 loops around
the phosphate group of PLP making hydrogen bonds to the non-ester phosphate
oxygen atoms. The total of eight hydrogen bonds (six from these four residues
and two from two water molecules) anchor the phosphate group of PLP to the
protein matrix 1.

case of Entamoeba histolytica, the PLP is covalently linked with to the
side-chain of Lys58 instead of Lys41 in C-terminal and the aromatic ring of PLP
is positioned in between three loops from C-terminal domain interacting with
Gly236, Ser280 and Pro307. Also, the phosphate group of PLP forms hydrogen bond
with a glycine rich loop (GTSGT) in between ?7 and ?6 and three structure water
molecules 4.

unprotonated 3′-hydroxyl group of PLP which is in hydrogen bonding with the
side-chain amide nitrogen of the Asn71 (Asn88 in case of E. histolytica),
forms the final attachment point of the coenzyme. The pyridine ring of PLP is
supported at the back by Val40.

active site positions are suspected to be in the vicinity of the coenzyme
binding site. In AtCysK, the ?3B-?2 loop which corresponds to the residues
74-78, forms one side of the active site. The sequence of this loop (TSGNT) is
highly conserved for the plants and microbes except for S. typhimurium where
serine is replaced with the asparagine (TNGNT). In A. thaliana, the
electron density of sulfate ion was near the substrate binding loop in the
active site; specifically, the side-chain hydroxyl group of Thr74, the backbone
nitrogen of Ser75 and the side-chain nitrogen of the Gln147 interact with the
sulfate ion. Whereas in the case of E. histolytica, the loop from Glu83
to Gly 90 is involved in sulfate binding, where, sulfate makes forms two
hydrogen bonds with backbone nitrogen atoms of ser86 and Gly 87 1.


?-amino group of Lys41 which forms the Schiff’s base with PLP in holoenzyme, is
released upon the binding of the substrate OAS. The binding site of OAS is
suspected to include the subsite for the binding of OAS ?-carboxylate. To the
right of the PLP-Lys41 aldimine in the active site, four N-atoms of residues
69, 70, 71, 72 form the wall of a shallow pocket which is structurally suited
to bind to the ?-carboxylate group of OAS.

reported in the case of E.histolytica, the structure of cysteine bound
CysK is similar to that of native CysK. However, there are some key differences
between the two starting from active site region to one end of N-terminal
movable domain. The part of N-terminal domain which contains residues 64-165 is
twisted about 15° causing the site to close. The active site closure is located
at the middle of the ?-sheets in the movable/N-terminal domain.


already discussed, CysK is a ?-replacement enzyme which performs
?,?-elimination reaction to substitute acetate to sulfate in the substrate OAS
to produce L-cysteine. The catalysis reaction of any ?-replacement enzyme
contains two half reactions 5:

The aminoacid substrate binds to the enzyme in a manner to generate substrate
external Schiff base (II) via the intermediacy of geminal di-amines. The
neutral active site of lysine abstracts the ? proton of substrate Schiff base
to produce quinonoid intermediate (III), which then yields ?-aminoacrylate in a
Schiff base likage with PLP (IV).

In the second half, all the reactions of first half go in the reverse manner.
The second substrate adds to give quinonoid intermediate (V), which is then
protonated by lysine to give product external Schiff base and finally the
release of the amino acid product to regenerate the enzyme.


reaction mechanism of CysK starts with the diffusion of OAS into the active
site. The geminal di-amines are formed when the ?-amine of the substrate
attacks C4′ of the internal aldimine between PLP and Lys41. These geminal
di-amines intermediates are converted to an external aldimine intermediate in
which the carboxylate of the substrate interacts with the asparagine-loop
adjacent to the cofactor and the positive end of the ?-helix 2, triggering the
closure of the active site. The closure of the active site serves as an
important phenomenon to expel bulk solvent from it because the ?-aminoacrylate
intermediate that is formed is reactive and to properly position the functional
groups for catalysis. Formation of external aldimine result in the release of
Lys41 which then acts as a general base in the ?,?-elimination of acetate which
then, results in the formation of ?-aminoacrylate intermediate. Once the
acetate has been eliminated, the other substrate sulfide binds and performs a
Michael addition to C3 of ?-aminoacrylate intermediate and generates L-cysteine
with external aldimine. The free enzyme is regenerated via the intermediacy of
geminaldiamine intermediates 6,7.


depletion of the substrate, CysK forms a complex with the preceding the enzyme
of the pathway, CysE and forms Cysteine Synthase Complex. The ratio between the
amount of CysK and CysE is very high and only 5% of CysK binds to the latter and
free CysK acts as an auxiliary enzyme for cysteine synthesis. The complex plays
as a regulator for CysK; however, it has no effect on the activity of CysE. It
is suggested that one of the roles of CysK is to prevent the aggregation of
CysE, therefore, enhancing its stability in the solution 8. It has been
observed in plants and microbes that the activity of CysK in complex with CysE
is reduced with two-fold decrease in kcatand five-fold increase in KM with that
of the free enzyme 9. The stoichiometric titrations of CysK and CysE of H.
influenza suggest that two dimers of CysK form the complex with the hexamer
of CysE 10. The C-terminal region of CysE interacts with the active site of
CysK to convert its open conformation to the closed one thereby, inhibiting it.
This C-terminus is dominated by hexapeptide-repeat domain which is required for
the protein-protein interaction with CysK 11, 12. It is also observed that
aspartic acid and glutamic acid are conserved in the C-terminal domain which
suspects that these amino acids are crucial for the interaction between CysK
and CysE 12.

cysteine synthase complex is dissociated by the accumulation of OAS. Once
dissociated, CysE has the high propensity to form aggregates. However, sulfide
counteracts the action of OAS thereby, strengthening the interaction between
CysE and CysK, keeping the cysteine synthase complex intact.