Aconitase (PDB ID:6ACN) from Sus scrofa
Created by: Candice Kremer
Aconitase (pdb: 6ACN) from Sus scrofa is a member of the aconitase superfamily and is classified as a dehydratase. It is also known as aconitate hydratase or citrate hydrolyase. Aconitase plays a significant role in the tricarboxylic acid cycle portion of cellular respiration (aka TCA, Krebs cycle, or citric acid cycle). Aerobic organisms use this cycle to oxidize acetyl-coA into carbon dioxide and generate energy in the form of ATP, NADH, and FAHD2 (Figure 1). Aconitase catalyzes the second step of the cycle, where citrate is converted to isocitrate via a cis-aconitate intermediate (Figure 2). Aconitase is also capable of the reverse reaction, converting isocitrate to citrate. This conversion step is critical in the TCA cycle, as citrate is a tertiary alcohol, and therefore cannot easily be oxidized. Isocitrate, however, is a secondary alcohol, which can be oxidized readily to continue the TCA cycle (2).
The structure of aconitase was resolved by crystallizing the protein in a buffer solution of 15 mM tricarballylate, 60 mM tris HCL (at pH 7.8), 2 mM sodium dithionite, and 2 mM ferrous ammonium sulfate (7). This crystallized form of aconitase has sulfate bound to the active site in lieu of citrate or isocitrate. Due to structural similarities however, this sulfate bound form of the enzyme can be considered equivalent to the substrate-free enzyme (9).
Aconitase is a monomer globular protein. Inactive aconitase has a [3Fe-4S] cluster at its active site, while active aconitase has a [4Fe-4S] cluster. The protein consists of 754 amino acids, with a total molecular weight of 82693.1 Da and a theoretical isoelectric point of 7.2 (3). Aconitase has a secondary structure composition of 34% helical (31 helices made of 263 total residues) and 20% beta sheets (52 strands made of 154 total residues) (6).
Aconitase consists of four domains: domain one contains residues 1-200, domain 2 contains residues 201-319, domain 3 contains residues 320-512, and domain 4 contains residues 537-754 (10). Another segment of the protein, the hinge-linker, contains residues 513-536. The N-terminus domain and the next two domains are tightly associated due to the presence of antiparallel beta strands, alpha helices, and parallel beta sheets. These three domains come together and their surface forms a shallow depression near the center of the molecule. This depression is the location of the catalytic iron-sulfur cluster. The C-terminus domain is tethered to the first three by a polypeptide chain segment termed the hinge-linker. This domain is complementary to the surface formed by the first three domains and lies atop the other three domains (9). The area between domains 1-3 and domain 4 forms a cleft that reaches from the surface of the protein directly to the iron-sulfur cluster. The presence of the hinge-linker suggests that the protein is capable of moving in a manner that allows substrates to diffuse in and out of the active site (14).
The active site of aconitase (illustrated with the binding pocket) is located at the end of this cleft. It consists of 21 amino acids and the iron-sulfur cluster. The Fe-S cluster is bound to aconitase via three cysteines from domain 3: cys358, cys421, and cys 424 (11). In its inactive form, the cluster is a [3Fe-4S] cluster, with each iron molecule ligated to cysteine. The cluster forms a simple cubane structure, with one corner missing. To become active, an additional iron molecule must ligate to the cluster, creating a [4Fe-4S]2+ cluster. Interestingly, the fourth iron molecule adds to the vacant corner of the cubane structure without causing any significant change to the conformation of the protein (9). In lieu of ligating to a cysteine amino acid residue, the fourth iron molecule ligates to a free hydroxide (4,9).
The 21 amino acids of the active site come from all four domains: 7 from domain one, 2 from domain two, 7 from domain three, and 5 from domain four. These amino acids are: gln72, asp100, his101, his147, asp165, ser166, his167, glu262, asn258, cys358, cys421, cys424, ile425, asn446, arg447, arg452, asp568, arg580, ser642, ser64, and arg644. The amino acid residues that contribute most significantly to the catalytic activity of aconitase are the three cysteines ligated to the iron-sulfur cluster, ser642, and his101. The remaining 16 amino acids contribute to hydrogen bonding, either between amino acid residues or to the substrate (4). The amino acid composition of the active site explains why sulfate is bound to the crystallized form of aconitase: the active site has a net positive charge, so negatively charged sulfate from the buffer solution is attracted to it. Specifically, the four sulfate oxygens hydrogen bind to gln72, arg580, ser643, and arg644 (4,9).
This enzyme catalyzes the interconversion of citrate and isocitrate by a dehydration-rehydration mechanism. When either citrate or isocitrate bind to the iron-sulfur cluster, the fourth iron molecule expands its coordination from 4 to 6. When citrate acts as the substrate, the iron molecule binds to the hydroxyl group and the carboxyl oxygen attached to Cβ. When isocitrate acts as the substrate, the iron molecule binds to the hydroxyl group and the carboxyl oxygen of the Cα. Despite the differences in substrate binding, the conformation of aconitase is the same in both situations (8).
In the conversion of citrate to isocitrate, the first step in the reaction mechanism is the protonation of the hydroxyl on Cβ by his101. Next, ser642 abstracts a proton from Cα, which causes the formation of a double bond between Cα and Cβ as the protonated hydroxyl group leaves the molecule. This step forms cis-aconitate. To form isocitrate, the molecule must flip orientations and then be rehydrated. The mechanism for this flip is unknown, but it is postulated that cis-aconitate actually diffuses away from the enzyme and a new molecule of cis-aconitate diffuses in and binds in the flipped orientation (4). The rehydration mechanism proceeds in the reverse manner as the hydration mechanism.
Aconitase is a participant in a crucial biochemical reaction, cellular respiration. As such, one can expect that aconitase would be highly conserved across species. Many molecules of the aconitase family achieved high Z-scores through DALI analysis and low E values through PSI-BLAST, indicating similarities in tertiary structure and primary sequence respectively. One such molecule is aconitase from Bos taurus (pdb: 1ACO), which has a Z-score of 66.3 and E value of 0.0 when compared with aconitase from Sus scrofa. Aconitase from Homo sapiens (pdb: 2B3Y) has a Z-score of 38 and E value of 0.0 (1,5). The superpositioning of aconitase from Sus scrofa and aconitase from Homo sapiens demonstrates the high level of similarity between the two proteins.
Another thing of note about aconitase is that there are two variants of the protein, known as m-aconitase and c-aconitase. 6ACN is an m-aconitase protein, the mitochondrial form. C-aconitase, the cytoplasmic enzyme, differs from m-aconitase in one major way. In addition to converting isocitrate and citrate, c-aconitase can also function as an iron regulatory protein. In the absence of iron, this enzyme binds to iron-responsive elements (found at specific regions of certain mRNAs) to either promote or block translation of that mRNA. C-aconitase has a molecular weight of 98,400 and is made of 889 residues (4).