Succinyl-CoA: acetate
CoA-transferase in complex with CoA (PBD ID: 5DDK) from Acetobacter aceti
Created by: Nikki Aaron
Succinyl-CoA: acetate CoA-transferase in complex with CoA (PBD ID: 5DDK) from Acetobacter aceti is a class I CoA-transferase. It catalyzes the reversible transfer of Coenzyme A (CoA) from
acyl-CoA thioesters to free carboxylates in the citric acid cycle (1). The
citric acid cycle is a series of chemical reactions essential for aerobic
organisms to generate energy through ATP. Since Acetobacter aceti is acetic acid resistant,
succinyl-CoA: acetate CoA-transferase is used instead of the typical enzyme, succinyl-CoA synthetase (PBD ID: 2SCU). Succinyl-CoA: acetate CoA-transferase is
active in the sixth step of the citric acid cycle, which converts succinyl-CoA
and acetate to succinate and acetyl-CoA (2). In this reaction, acylglutamyl
anhydride intermediates are produced. These intermediates undergo attack by the CoA thiolate ligand in complex with succinyl-CoA: acetate CoA-transferase on
either the internal or external carbonyl carbon atoms (3). Exploring the
structure and function of succinyl-CoA: acetate CoA-transferase helps
scientists to understand the reaction of reversible CoA transferase from
acetyl-CoA thioesters in Acetobacter
aceti. Specifically, this crystalized structure of succinyl-CoA: acetate
CoA-transferase is studied in order to determine how one residue, Asn-347,
stabilizes the reaction intermediates (3). Knowledge of the mechanism of succinyl-CoA:
acetate CoA-transferase enables scientists to carry out unfavorable reactions
by using favorable interactions.
Succinyl-CoA: acetate
CoA-transferase was crystalized at 22°C using the hanging drop vapor diffusion
method (3). The crystalized structure of succinyl-CoA: acetate
CoA-transferase includes chloride ions and an imidazole ligand (1). The ligands
were added to help separate the crystalized structure from solution (3). Upon
isolation, structural data was obtained using x-ray diffraction with a
resolution of 2.13 Å (1). 95% of the 514-residue protein was successfully
crystalized (3). Residues 1, 2, and 347 are missing from the final product (1).
Succinyl-CoA: acetate CoA-transferase has a molecular weight of 111,852.80 Daltons
and an isoelectric point at 6.30 (4).
The activity of succinyl-CoA: acetate
CoA-transferase is dependent on its structure. The
enzyme is a homodimer with one unique chain that repeats in subunits A and B. The
active site of subunit A is in the open conformation and binds with the CoA ligand. Subunit B, on the other hand, is in the closed conformation at the
active site (3). The closed active site of subunit B enables succinyl-CoA: acetate
CoA-transferase to engage, immobilize and desolvate acyl-CoA substrates (3).
170 resides comprise 22 alpha helices in the secondary structure of succinyl-CoA: acetate
CoA-transferase (1). Additionally, 106 residues make
up 36 strands of beta sheets (1). The secondary structure is therefore composed
of 33% alpha helical structure and 20% beta sheets. The other 238 residues make
up tight turns, 3/10-helices, beta bridges, and random coils (1).
Coenzyme A in complex with succinyl-CoA: acetate
CoA-transferase is crucial for the protein’s structure and function. CoA is a
coenzyme notable for its role in the synthesis and oxidation of fatty acids and
pyruvate in the citric acid cycle. In complex with succinyl-CoA: acetate
CoA-transferase, CoA acts as a thioester. The
succinyl-CoA binding energy is used to accelerate rate-limiting succinyl
transfers by compressing the substrate thioester tightly against the catalytic glutamate residue of succinyl-CoA:
acetate CoA-transferase. CoA exploits substrate affinity in
order to promote catalysis. Several hydrogen bonds serve to stabilize the interactions
within and between subunits, including a hydrogen bond network between Ser-71, Thr-94, and Arg-228. The CoA ligand is stabilized through hydrogen bonding with Glu-294. The reaction
catalyzed by succinyl-CoA: acetate CoA-transferase, like all class I CoA-
transferases, involves two spatially distinct oxyanion holes. The external
oxyanion is stabilized by hydrogen bonding between CoA and Gly-388, while the
internal oxyanion is stabilized by hydrogen bonding between CoA and Asn-347 (3).
The main residues of interest in the
primary structure of succinyl-CoA:
acetate CoA-transferase are Asn-347 and Glu-294. Asn-347 is purposely excluded from
this structure of succinyl-CoA: acetate CoA-transferase in order to determine its
importance for Glu-294 function and stability of the protein. Glu-294 is an
important nucleophile that enforces rigid structure of oxyanion holes by
confining the thioester oxygen of CoA (3). Through hydrogen bonding, Asn-347 holds Glu-294 at an ideal angle for enzymatic attack as the thioester oxygen is
forced into the oxyanion hole of CoA (5). During this process, Glu-294 in
wild-type succinyl-CoA:
acetate CoA-transferase is nearly immobile. Unexpectedly, in the absence of Asn-347 the succinyl-CoA: acetate CoA-transferase retains significant activity (3). It
is theorized that other polar residues, such as Glu-294, supplement the Asn-347 carboxamide to restore its function (3).
Another
important residue is Val-270, which moves during active site closure. Its
nonpolar side chain is responsible for desolvating and constraining the CoA thioester while its amino group acts as a hydrogen donor to stabilize the CoA thiolate
leaving group from anhydride intermediate formation (3). The amide-thiolate
interaction stabilizes the nucleophile that attacks the anhydride and helps
maintain closed conformation during reactions involving the anhydride adducts (5).
Spatial and sequence conservation of multiple key residues, despite the absence
of Asn-347, indicates that the mechanism of succinyl-CoA: acetate CoA-
transferase can in fact be categorized among other class I CoA-transferases (5).
Many
class I CoA-transferases are responsible for catalyzing reactions in unconventional
citric acid cycles. 4-hydroxybutyrate CoA-transferase (PBD ID: 3GK7) from Clostridium aminobutyricum is
responsible for
binding the succinyl group of succinyl-CoA in succinyl-CoA: 3-oxoacid
CoA-transferase (1). To compare, PSI-BLAST evaluates
the primary structures of 4-hydroxybutyrate
CoA-transferase and succinyl-CoA: acetate
CoA-transferase by assigning gaps between protein sequences. The E-value is 1x10-16, indicating that 4-hydroxybutyrate CoA-transferase
and succinyl-CoA: acetate CoA-transferase have similar primary
structures. An E-value is considered significant if it is less than 0.05 (6). The
Dali server finds similar proteins based on tertiary structure by calculating
differences in the intramolecular distances and using the “sums-of-pairs”
method. It confirms the resemblance between 4-hydroxybutyrate CoA-transferase and
succinyl-CoA: acetate CoA-transferase with a Z-score of 35.4. A Z-score is
considered significant if it is greater than 2 (7).
4-hydroxybutyrate
CoA-transferase is shorter than succinyl-CoA: acetate
CoA-transferase with only 438 residues. The secondary structure of 4-hydroxybutyrate CoA-transferase
is composed of 31% alpha helices and 23% beta sheets (1). This is similar to
the secondary structure composition of succinyl-CoA: acetate
CoA-transferase. 4-hydroxybutyrate
CoA-transferase has a 2-amino-2-hydroxymethyl-propane-1,3-diol ligand added to
help separate the crystalized structure from solution (8). The structure of
4-hydroxybutyrate CoA-transferase also contains two spermidine N-(2-amino-propyl)-1,4-diaminobutane (SPD) ligands (1). These spermidine
residues were added in place of the pantetheine portion of the CoA-thioester.
They are responsible for forming salt bridges with Glu-238 (8).
Like succinyl-CoA: acetate
CoA-transferase, the catalytic activity of 4-hydroxybutyrate CoA-transferase
is identified as its glutamate residue, Glu-238. Though most class I
CoA-transferases contain two subunits, 4-hydroxybutyrate
CoA-transferase consists of only one subunit. However,
4-hydroxybutyrate
CoA-transferase has two domains orthologous to the A and B subunits of succinyl-CoA: acetate CoA-transferase (8). The active sites of 4-hydroxybutyrate
CoA-transferase and succinyl-CoA: acetate CoA-transferase
are both located between the two domains.
4-hydroxybutyrate CoA-transferase has a molecular weight of 97,772.20 Daltons
and an isoelectric point at 5.85 (4).
As
CoA-transferases, 4-hydroxybutyrate CoA-transferase and succinyl-CoA:
acetate CoA-transferase are responsible for catalyzing the activation of
carboxylic acids to CoA-thioesters (5). Both enzymes are categorized as Class I
CoA transferases because they consist of 3-oxoacids, short-chain fatty acids
and glutaconate (8). Most Class I CoA transferases use succinyl-CoA or
acetyl-CoA as CoA donors, unlike 4-hydroxybutyrate
CoA-transferase (8). Additionally, the two enzymes
differ in their cellular respiration processes. 4-hydroxybutyrate CoA-transferase is involved in
fermentation of anaerobic bacterium such as Clostridium
aminobutyricum (8). Succinyl-CoA: acetate
CoA-transferase, on the other hand, is involved in the citric acid cycle of
aerobic respiration (3). Understanding
the structural and functional similarities of 4-hydroxybutyrate CoA-transferase
and succinyl-CoA: acetate CoA-transferase provides insight on class I
CoA-transferases and highlights the importance of an active glutamate residue for the function of this family of enzymes (9).
Since Acetobacter aceti is acetic acid resistant, it requires strong oxygenation for acetate removal. This is especially critical at high acetic acid concentrations and low pH values (2). Acetic acid resistance is improved when strains of succinyl-CoA: acetate CoA-transferase are introduced or overexpressed (2). Succinyl-CoA: acetate CoA-transferase is essential for the organism because it bypasses the usual detoxification pathway required for survival at high acetate concentrations and low pH. Reliance on a complete but unorthodox citric acid cycle that includes succinyl-CoA: acetate CoA-transferase explains the vigorous oxygenation requirements of Acetobacter aceti cultures (3).