The metabolic flux is controlled by substrate availability, so controlling the levels of acetyl-CoA and oxaloacetate in the mitochondria controls the rate of reaction. Furthermore, citrate synthase is inhibited by NADH, which competes with oxaloacetate , and succinyl-CoA an example of competitive feedback inhibition [8]. In many plants, bacteria and fungi, such as the peroxisomes of baker's yeast, citrate synthase plays a role in the glyoxylate cycle [9] [10] [11].
Citrate Synthase 3D structures. Citrate Synthase From Proteopedia. Jump to: navigation , search. Show: Asymmetric Unit Biological Assembly. Export Animated Image.
The large and small domains are distinguished magenta and green colors, respectively. Residues involved in the formation of oxaloacetate binding pocket are shown as a stick model with appropriate labels. The oxaloacetate substrate bound in Ms CS is shown as a yellow colored stick. The carbon numbers of oxaloacetate substrate are labeled with white.
Red dotted lines indicate hydrogen bonds contributing to oxaloacetate substrate binding. In order to reveal oxaloacetate binding mode of Ms CS, high concentration of oxaloacetate was added to the purified Ms CS protein at the crystallization step.
Ms CS crystals in complex with oxaloacetate were successfully obtained, and we determined its crystal structure at 2. The structural examination revealed two Ms CS molecules and two glycerol molecules in the asymmetric unit.
In the large domain, Asn interacts with the C2 and C3 carboxyl-groups and Arg stabilizes the C3 carboxyl-group of oxaloacetate via a salt bridge. In addition, His from the small domain interacts with the C3 carbonyl-group and Arg is associated with the stabilization of C3 carbonyl- and carboxyl-groups of oxaloacetate through hydrogen bonds Fig 2D. Interestingly, Asn is not conserved in other CS proteins, and some other CS structures contain Pro at the corresponding position Fig 1B , indicating that stabilization mode of oxaloacetate might be somewhat different among CSs.
CSs undergo domain movement to the closed conformation upon binding of the oxaloacetate substrate, which facilities the formation of an optimal binding pocket for acetyl-CoA [ 8 , 20 , 33 — 35 ].
It has been also known that the citrate product inhibits the enzyme activity by binding to the substrate binding site, and the binding of citrate to the enzymes induces the closed conformation. However, detailed comparison of the structure of Ms CS in complex with oxaloacetate with that in complex with citrate revealed that the conformation of these structures were somewhat different from each other. First the Ms CS structure in complex with the citrate product shows a more closed conformation compared with that in complex with the oxaloacetate substrate Fig 3A.
When we superposed these two structures based on the large domain, the small domain of the structure in complex with the citrate product was positioned closer to the large domain Fig 3A. When we calculate the rotation angle using DynDom [ 36 ], the small domain was rotated by 6. Second, the surrounding regions involved in the binding of the citrate product showed much lower B-factor values than those involved in the binding of the oxaloacetate substrate Fig 3B and 3C , indicating that binding of the citrate product induces a tighter conformation.
Finally, we observed differences in residues involved in the binding of these two compounds. While Arg interacts with the citrate product, this residue does not interact with the oxaloacetate substrate, but rather rotates away from the compound Fig 3E. On the contrary, the Asn residue does not participate in the binding of citrate but is involved in the binding of oxaloacetate Fig 3E.
Based on these observations, we suggest that binding of the citrate product to the enzyme induces a more compact conformation than that of the oxaloacetate substrate, although both compounds induce domain movement and conformational changes towards the closed conformation.
A Structural comparison of citrate and oxaloacetate complex structures. Small domains from citrate and oxaloacetate complex structure are distinguished by magenta and green colors, respectively, and the large domains are shown as a gray color. The left image is a close-up view of the black dotted box to highlight structural movement. E Superposition of the residues involved in the substrate stabilization. Residues from citrate and oxaloacetate complex structures shown as lines with magenta and green colors, respectively.
Citrate and oxaloacetate bound to Ms CS are indicated by magenta and greed colored sticks, respectively. Here, we need to consider that pHs of the crystallization mixtures for oxaloacetate- and citrate-bound forms were 7.
Thus, we measured the Ms CS activity under various pH conditions to compare the activity between these two pHs. Interestingly, the optimal pH was 9. The K m and k cat values of oxaloacetate were 0. When we measured the inhibition kinetics of citrate, the K m values increased while the V max values remained constant, as the concentration of citrate increased Fig 3C , Table 2.
These results indicate that Ms CS is competitively inhibited by the citrate product. As we described above, the citrate product binds tightly to the substrate binding site and its binding induces the compact closed conformation, which is consistent with the inhibition kinetic results.
The inhibition kinetics were measured with both oxaloacetate E and acetyl-CoA F. We also performed inhibition kinetic experiment using NADH. The colored amino acid are indicated to same red , similar green and different blue. In summary, in order to elucidate the molecular mechanism of Ms CS, we determined its crystal structure in complex with oxaloacetate and citrate.
The structural information revealed that Ms CS is inhibited by citrate through conformational change. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
National Center for Biotechnology Information , U. PLoS One. Published online Feb Claudio M. Soares, Editor. Author information Article notes Copyright and License information Disclaimer.
Competing Interests: The authors have declared that no competing interests exist. Received Nov 8; Accepted Feb 8. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Introduction Metallosphaera sedula belongs to the sulfolobaceae family. Open in a separate window. Fig 1. Overall structure of Ms CS. Activity assay of Ms CS The activity of Ms CS was determined by measuring the increase of absorbance at nm extinction coefficient of 1. Results and discussion Overall structure of Ms CS To understand the molecular mechanism of citrate synthase from Metallosphaera sedula Ms CS , we determined its crystal structure at 1.
Table 1 Data collection and refinement statistics. Active site of Ms CS Although we did not add any compound during the purification and crystallization procedure, the citrate product was bound in the structure at pH 6. Fig 2. Active site of Ms CS. Conformation change upon product formation CSs undergo domain movement to the closed conformation upon binding of the oxaloacetate substrate, which facilities the formation of an optimal binding pocket for acetyl-CoA [ 8 , 20 , 33 — 35 ].
Fig 3. Conformation change upon product formation. Fig 4. Inhibition properties analysis of Ms CS. Table 2 Inhibition kinetics of Ms CS. Inhibitor varying substrate Concentration of inhibitor. Fig 5. Data Availability All relevant data are within the manuscript and its Supporting Information files.
References 1. Life in hot acid: pathway analyses in extremely thermoacidophilic archaea. Curr Opin Biotechnol. Physiological versatility of the extremely thermoacidophilic archaeon Metallosphaera sedula supported by transcriptomic analysis of heterotrophic, autotrophic, and mixotrophic growth.
Appl Environ Microbiol. Characterization of a bifunctional archaeal acyl coenzyme A carboxylase. September , David Goodsell. PDB helps teachers, students, and the general public explore the 3D world of proteins and nucleic acids. Learning about their diverse shapes and functions helps to understand all aspects of biomedicine and agriculture, from protein synthesis to health and disease to biological energy.
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Molecule of the Month. Citrate Synthase Citrate synthase opens and closes around its substrates as part of the citric acid cycle Citrate synthase: open form with product top and closed form with substrates bottom. Your body burns up a lot of food every day. However, cells don't burn food like a fireplace. Instead, food molecules are combined with oxygen molecules one-by-one, in many carefully controlled steps.
In this way, the energy that is released can be captured in convenient forms, like ATP or NADH, which are then used elsewhere to power essential cellular functions. Our cells get most of their energy from a long series of reactions that combine oxygen and glucose, forming carbon dioxide and water, and creating lots of ATP and NADH in the process. Citrate synthase is a central enzyme in this process of sugar oxidation.
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