Solanum lycopersicum ACC
Amanda He '16 and Andrew Pearlman '15
1-aminoacyclopropane 1-carboxylate synthase (ACC synthase) is an
enzyme that plays a significant role in synthesizing ethylene, a plant
hormone produced in a variety of higher plants .
Ethylene is an important hormone for ripening and germination
biosynthesis consists of three main steps. In the second step,
ACC synthase catalyzes the cyclization of the
(SAM), which produces 5'-methylthioadenosine and ACC. This step is the
rate-limiting step in ethylene synthesis .
In order for ACC synthase to function, it requires the cofactor,
pyridoxal-5'-phosphate (PLP). PLP has been found to activate catalysis
in enzymes, including ACC synthase, by binding to the substrate active
Better understanding of
II. General Structure
The tomato ACC synthase monomer has two
, an alpha-beta-alpha sandwich
domain and an alpha-beta domain.
The alpha-beta-alpha sandwich domain has a central seven-strand
beta-sheet that is flanked between nine alpha-helices [button
of beta sheet]. The alpha-beta domain has five
beta-strands and five alpha-helices
ACC synthase typically appears in a dimeric form as do most
PLP-dependent enzymes. However, there is not a substantial
difference between the functionality of the monomer and dimer forms
because there were not a significant difference in conformational
change between the two when the inhibitor, AVG, binded .
CAP is a dimer of 22, 500 molecular weight,
composed of two chemically identical polypeptide chains each 209
amino acids in length.
The overall structure of the dimer is assymetric; one subunit adopts a
"closed" conformation in which the
amino- and carboxy-termini are closer together than in the more "open"
subunit. Each subunit is composed of two distinct domains
connected by a hinge region.
The N-terminal domain is responsible for
dimerization and cAMP
binding. The carboxy-terminal
domain contains a helix-turn helix DNA
and is also responsible for DNA bending.
III. Cofactor PLP Binding
ACC synthase forms a complex with cofactor,
pyridoxal-5'-phosphate (PLP) in order to be able to catalysis
its substrate, S-adenosylmethionine (SAM). PLP
is an active form of vitamin B6 that is able to
catalyze reactions by forming a covalent bond. In the case of
ACC synthase, PLP forms a covalent bond to [Lys 278]
In ACC synthase's
dimeric form, the active site for substrate binding is dually
occupied by PLP and the substrate, SAM.
The nitrogen atom of PLP forms a [hydrogen bond]
with Asp 237 of the ACC synthase. Since aspartate is
negatively charged, it is able to stabilize the protonation
state of the pyridine nitrogen .
An important recognition site for cAMP within CAP is the
ionic bond formed between the side chain of Arg-82
and the negatively charged phosphate group
of cAMP. In the crystal structure, the two cAMP molecules are
buried deep within the beta roll
and the C-helix.
It is unclear how cAMP enters or leaves the binding site, but this
probably requires the separation of the two subunits of the dimer,
or the movement of the beta roll and the C helix away from each
other. Other side-chain interactions between the protein and cAMP
are hydrogen bonds occuring at Thr-127,
Ser-128, Ser-83, and Glu-72.
Additional hydrogen bonding between is seen between cAMP and the
polypeptide backbone at residues 83
IV. Inhibitor AVG Binding
Workers in the agricultural industry are familiarized
with working with aminoethoxyvinylglycine (AVG), a naturally
produced amino acid that has a role in inhibition of ethylene
biosynthesis . AVG
is a competitive inhibitor of ACC synthase.
AVG binds to a location neighboring the cofactor, PLP. The
alpha-carboxylate group of AVG forms three hydrogen bonds with PLP
at [Ala 54], [Arg 412], and water. Additionally, AVG forms a van
der Waals contact with [Tyr 152]. All these sites are highly
conserved because they are also the sites at which the substrate,
SAM would interact with to bind.
Once CAP has bound cAMP, it is ready to bind to the DNA.
Binding occurs at the conserved sequence of
Hydrogen bonds between the protein and the DNA phsophates occur at the
backbone amide of residue
139, and the side chains of Thr-140,
Ser-179, and Thr-182
In addition to these phosphate interactions, the side chains of Glu-181
and Arg-185, both emanating from
directly contact the bases within the major groove of the DNA. Because
of the way that the protein binds to the DNA, a kink of ~40
degrees occurs between nucleotide base pairs six
and seven on each side of the dyad
This sequence has been shown to favor DNA flexibility and bending in
other systems as well. Because of this kink, an additional five
ionic interactions and four hydrogen bonds are able to occur
between the protein and the DNA strand. Examples of these new
interactions occur between Lys-26,
Lys-166, His-199 and the DNA sugar-phosphate backbone
The DNA bend is integral to the mechanism of transcription activation.
Not only does it place CAP in the proper orientation for
interaction with RNA polymerase, but wrapping the DNA around the
protein may result in direct contacts between upstream DNA and
V. Activating Regions
Transcription activation by CAP requires more than merely
the binding of cAMP and binding and bending of DNA. CAP contains
an "activating region" that has been proposed to participate in
direct protein-protein interactions with RNA polymerase and/or
other basal transcription factors. Specifically, amino acids 156,
have been proposed to be critical for transcription activation by CAP.
These amino acids are part of a surface loop composed of
Researchers have concluded that the third and final step in
transcription activation is this direct protein-protein contact
between amino acids 156-162 of CAP, and RNA polymerase.
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