Isocitrate_Dehydrogenase

Isocitrate Dehydrogenase (IDH) (PDB ID: 6G3U) is a biomolecule bound to the ligands 2-oxoglutaric acid and NADP. Pseudomonas aeruginosa is a multi-drug resistant bacteria that causes various hospital-acquired infections such as ventilator-associated pneumonia and sepsis syndromes. Infection often occurs during existing diseases or conditions, most notably cystic fibrosis and traumatic burns, leading the bacteria to be described as opportunistic (1). IDH in Pseudomonas aeruginosa is one of three enzymes regulating the citric acid cycle and the glyoxylate shunt to create energy for the cell. IDH is useful to study to develop drugs to combat the multi-drug resistant infections that can occur as a result of contact with Pseudomonas aeruginosa.

The citric acid cycle or tricarboxylic acid cycle (TCA) and the glyoxylate shunt are two separate energy-producing pathways in the cell. The glyoxylate shunt is necessary for bacterial growth since it produces intermediates necessary to synthesize cellular materials, whereas the TCA cycle releases carbons in the form of carbon dioxide, with no net production of intermediates (2). To regulate which cycle is used, different enzymes exist in the cell that respond to various stimuli. In some organisms there are two enzymes, isocitrate dehydrogenase (ICD) (PDB ID: 5M2E), which is the TCA cycle enzyme, and isocitrate lyase (ICL) (PDB ID: 6G1O), which is the glyoxylate shunt enzyme (1). ICD and ICL compete for available substrate, isocitrate. K_M refers to Michaelis constant, which is the concentration at half of the maximum reaction velocity (3). The value of K_M is unique for every protein. Since ICD has a lower K_M than ICL, it requires a smaller amount of substrate to become saturated and therefore reach maximum reaction speed, making it easier for ICD to be used and therefore for TCA to occur. In order for the glyoxylate shunt to occur, ICD must be inactivated. This occurs when a dual function kinase/phosphatase AceK phosphorylates ICD on Ser-113, deactivating ICD and allowing the glyoxylate shunt to occur. However, IDH is a form of isocitrate dehydrogenase which is insensitive to AceK. If IDH is also in an organism, it cannot be phosphorylated to allow ICL to work, and therefore must be regulated in another way.

Most organisms have ICL and either IDH or ICD since IDH and ICD have the same functions but are regulated differently by the cell. Pseudomonas aeruginosa has all three enzymes, IDH, ICL, and ICD, which makes its regulation more complex than other organisms. ICL in Pseudomonas aeruginosa has a higher affinity for isocitrate (K_M = 12 muM) than its competitors (ICD K_M = 26 muM and IDH K_M = 18 muM), making it the favored enzyme in the cell (1). Although Pseudomonas aeruginosa also uses AceK to regulate the activity of its enzymes, it is not the main regulator. Instead, ICL produces two products, glyoxylate, which activates IDH, and succinate, which is an uncompetitive inhibitor of ICL and strong activator of AceK phosphatase activity. Both of these products promote the activity of the enzymes that promote TCA cycle, but the key regulators are oxaloacetate and pyruvate. When these two products are abundant, IDH is activated and ICL is inhibited, promoting the use of TCA cycle. Oxaloacetate and pyruvate also stimulate AceK phosphatase activity, which again stimulates ICD activity. ICD activity is depressed when demand for gluconeogenesis is high since Acetyl-CoA stimulates the kinase activity of AceK, leading to inactivation of ICD. Acetyl-CoA accumulates during growth on fatty acids or acetate when oxaloacetate is in short supply. The differences in the regulation of the two cycles in Pseudomonas aeruginosa is necessary for its function since its demand for energy can quickly increase during oxidative stress or anabolism when infection begins. This may explain why it has evolved to have both ICD and IDH which have the same function but are independently regulated (1).

The Expasy database was used to determine that IDH has a molecular weight of 162275.00 Daltons and an isoelectric point of 5.75 (4). The molecule contains two subunits which are identical in sequence but not in conformation. The molecule is asymmetrical with a small interface between the two monomers, suggesting that the two molecules cannot be considered to be protomers of a functional dimer (1).

The structure of isocitrate dehydrogenase (IDH) from Pseudomonas aeruginosa was solved to a resolution of 2.7 Å. IDH was crystallized using sitting drop vapor diffusion with 17.5 mg/mL of purified protein. IDH crystallized in presence of 200 mM NaH₂PO₄, 21.5% (w/v) PEG 6000, 5% (v/v) glycerol and 150 muM NADP+. The crystals were grown for 2-6 days at 292 K then treated with cryoprotectant composed of 24% (v/v) ethylene glycol and 76% (v/v) mother solution. The activity of IDH and ICD was also measured in the presence of AceK and ATP. Reaction mixtures had a volume of 200 muL and contained 100 mM Tris-HCl (pH 7.0), 1 mM ATP, 2 mM MgCl2, 5 mug purified P. aeruginosa AceK and 10 mug P. aeruginosa ICD or IDH. Reaction mixtures were maintained at 37 °C and aliquots were taken at specified time intervals. The results indicated a decrease in activity of about 80% in ICD and of about 5% in IDH after 60 minutes, showing that ICD is inactivated by AceK while IDH is relatively unaffected (1).

The primary structure of isocitrate dehydrogenase (IDH) contains 737 residues in each identical subunit with hydrophobic, hydrophilic, acidic and basic amino acids. The secondary structure consists of 35 α-helices (48%), 29 β-sheets (16%), and 76 random coils (36%) (5). The helices are particularly important near the active site of IDH where they prevent AceK from accessing the active site serine, effectively preventing regulation (1). The P-loop and AceK recognition segment of ICD are absent from IDH, also making it impossible for AceK to bind. IDH adopts a higher order tertiary structure in solution. In the crystal, subunit A contains one molecule each of NADP⁺ and α-ketoglutarate bound in its active site. Subunit B does not contain any bound small molecules. The two IDH subunits do not show enough interface to be considered a dimer. The binding cleft for α-ketoglutarate is between the two IDH domains and involves side chains from both domains. During the TCA cycle, The ligand 2-oxoglutaric acid is converted into oxaloacetate, which stimulates AceK phosphatase activity and increases the activity of ICD (1). The other ligand, NADP, is an energy rich biomolecule that is used to store and transfer energy. The active site is between the two subunits and is bound to side chains on both domains (1). The region of substrate binding is located between Ser-133 and Arg-140 (5).

Monomeric isocitrate dehydrogenase (IDH) in complex with Mn²⁺ and isocitrate (PDB ID: 1ITW) from Azotobacter vinelandii is extremely similar in primary, secondary, and tertiary structure to IDH from Pseudomonas aeruginosa, but has a different quaternary structure. This comparison protein was found using the PSI-BLAST tool from the National Library of Medicine and the Dali Server from the University of Finland. Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST) is used to search databases for sequence similarities to a given query protein. The results are given in the form of E values, where a lower E value indicates a higher degree of similarity between proteins. An E value less than 0.05 is considered significant (6). The E value of the comparison of IDH in Azotobacter vinelandii and IDH in Pseudomonas aeruginosa is 0.0, indicating that they have extremely similar primary structures. The Dali Server also compares proteins but looks for similarities in tertiary structure rather than sequence. The degree of structural similarity is signified as a Z score, in which a score over 2 is considered significant (7). The Z score for the comparison of IDH in Azotobacter vinelandii and IDH in Pseudomonas aeruginosa is 50.1, indicating very significant similarities in tertiary structure.

The crystal structure of the monomeric IDH from Azotobacter vinelandii complexed with isocitrate and Mn²⁺ was determined to a resolution of 1.95 Å. The structure was found to be two distinct domains with different sizes. The small domain, denoted domain I, contains both the N-terminal and C-terminal segments. It is composed of five β-strands and 14 α-helices. Near the C-terminal segment, there is a helix-rich domain consisting of eight α-helices. The large domain, denoted domain II, is composed of 16 β-strands and 13 α-helices. Three of the 16 β-strands form a parallel β-sheet, and eight of the 16 β-strands form a unique arm-like structure that protrudes from domain II. There is also a central sheet region which spans both domains and forms an open twisted β-sheet composed of eight parallel and two antiparallel β-strands. The substrate and Mn²⁺ binding site is located at the bottom of the hydrophobic cleft between the two domains (8). The substrate binding site is where isocitrate attaches to the enzyme through hydrogen bonds, and it is located at Ser-132, Asn-135, Arg-139, Arg-145, Arg-547, Tyr-420, and Lys-255. Mn²⁺ is coordinated by O₂ and O₇ of isocitrate, Asp-350 and Asp-548 of IDH and two water molecules in an octahedral manner (8).

Azotobacter vinelandii possesses monomeric IDH abundantly in the cytoplasm. Since Azotobacter vinelandii is used for nitrogen fixing, the high concentration of IDH in the enzyme indicates its role in energy production and nitrogen fixation. Unlike in Pseudomonas aeruginosa, in Azotobacter vinelandii, IDH is the only enzyme that regulates the TCA cycle while ICL regulates the glyoxylate shunt. However, the IDH in both organisms have extremely similar primary, secondary, and tertiary structures, only differing in quaternary structure. The main difference is that IDH from Pseudomonas aeruginosa has two asymmetrical subunits, while IDH from Azotobacter vinelandii has four asymmetrical subunits (5). There are two copies of the two different subunits of IDH from Azotobacter vinelandii in one monomer of IDH. Since the IDH molecules from both organisms have substrate binding sites on subunit A, and there are two of those subunits in the Azotobacter vinelandii IDH, it may be slower for Azotobacter vinelandii IDH to function as an enzyme since both substrate binding sites must be occupied (8). Therefore, Azotobacter vinelandii will produce energy at a lower rate than Pseudomonas aeruginosa, which is consistent with the life cycles of both organisms since Pseudomonas aeruginosa requires a large amount of energy to rapidly replicate. Both IDH proteins are considered monomers due to lack of interface and have one domain that is larger than the other. The active site is between the domains for both proteins as well, and their functions are nearly identical.

Because IDH is used to regulate the TCA cycle and the glyoxylate shunt, it is useful to study to develop drugs that can prevent IDH from ever being inhibited. This would force the organism to only use the TCA cycle, which would prevent growth and therefore stop the disease from spreading. In a mutant of Pseudomonas aeruginosa in which ICL was absent, a mouse pulmonary infection was cleared within 48 hours, indicating that the glyoxylate shunt is an excellent target for adjuvant interventions (1). This is especially useful in the case of Pseudomonas aeruginosa, which is resistant to many drugs and antibiotics.