Iron is an essential element for various lifeforms but is largely insoluble due to the oxygenation of Earth's atmosphere and oceans during the Proterozoic era. Metazoans evolved iron transport glycoproteins, like transferrin (Tf) and lactoferrin (Lf), to keep iron in a non-toxic, usable form, while maintaining a low free iron concentration in the body that is unable to sustain bacterial growth. To survive on the mucosal surfaces of the human respiratory tract where it exclusively resides, the Gram-negative bacterial pathogen Moraxella catarrhalis utilizes surface receptors for acquiring iron directly from human Tf and Lf. The receptors are comprised of a surface lipoprotein to capture iron-loaded Tf or Lf and deliver it to a TonB-dependent transporter (TBDT) for removal of iron and transport across the outer membrane. The subsequent transport of iron into the cell is normally mediated by a periplasmic iron-binding protein and inner membrane transport complex, which has yet to be determined for Moraxella catarrhalis. We identified two potential periplasm to cytoplasm transport systems and performed structural and functional studies with the periplasmic binding proteins (FbpA and AfeA) to evaluate their role. Growth studies with strains deleted in the fbpA or afeA gene demonstrated that FbpA, but not AfeA, was required for growth on human Tf or Lf. The crystal structure of FbpA with bound iron in the open conformation was obtained, identifying three tyrosine ligands that were required for growth on Tf or Lf. Computational modeling of the YfeA homologue, AfeA, revealed conserved residues involved in metal binding.

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Å resolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Å resolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Å between the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and β-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141β and Glu204β via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

Tartryl-CoA was discovered in the crystal structure of human GTP-specific succinyl-CoA synthetase (SCS).SCS catalyzes the only substrate-level phosphorylation of the citric acid cycle.It catalyzes the reversible reaction: succinyl-CoA + NDP + Pi ⇌ succinate + CoA + NTP in the presence of magnesium ions.Humans have two different SCSs, ATP-specific SCS and GTP-specific SCS.The crystallization experiment included GTP-specific SCS, ADP, CoA, and magnesium ions in the protein solution, while polyethylene glycol 3350 and ammonium tartrate were in the well solution.During crystallization, tartryl-CoA was synthesized from tartrate and CoA, and, instead of being released from the enzyme, tartryl-CoA remained as a bound ligand.The CoA portion binds as expected in the CoA-binding site, but the tartryl portion binds in the phosphate-binding site, close to the catalytic histidine residue.Although succinyl-CoA is structurally similar to tartryl-CoA, succinyl-CoA would not bind to SCS in the same way as tartryl-CoA.The two extra hydroxyl groups of tartrate contribute to the binding of tartryl-CoA.Tartryl-CoA acts as an inhibitor, inhibiting SCS after a single turnover.

Succinyl-CoA synthetase (SCS) catalyzes the only substrate-level phosphorylation step in the tricarboxylic acid cycle. Human GTP-specific SCS (GTPSCS), an αβ-heterodimer, was produced in Escherichia coli. The purified protein crystallized from a solution containing tartrate, CoA and magnesium chloride, and a crystal diffracted to 1.52 Å resolution. Tartryl-CoA was discovered to be bound to GTPSCS. The CoA portion lies in the amino-terminal domain of the α-subunit and the tartryl end extends towards the catalytic histidine residue. The terminal carboxylate binds to the phosphate-binding site of GTPSCS.

ATP-citrate lyase (ACLY) catalyzes production of acetyl-CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)-terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C-terminal portion of ACLY, full-length C. limicola ACLY was cleaved, first non-specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C-terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46βE, K114βD, V113βL and L227βF, and one with two mutations, K46βE/K114βD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20β, Gly111β and Val113β. The O atom of the side chain of Gln20β interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111β. It is the different conformations of the backbone at Gln20β, of the side chain of Gln20β and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227βF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46βE/K114βD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.

Succinyl-CoA synthetase (SCS) catalyzes the only substrate level phosphorylation in the citric acid cycle: Succinyl-CoA + NDP Succinate + CoA + NTP in the presence of magnesium ions.In mammals, SCS is a heterodimer with and subunits and nucleotide-specific isoforms of SCS exist: one is ATP-specific SCS (ATPSCS) and the other one is GTP-specific SCS (GTPSCS).ATPSCS is coded by genes SUCLG1 and SUCLA2 and GTPSCS is coded by genes SUCLG1 and SUCLG2.SUCLG1 codes for the common subunit.The specificity of SCS for either ATP/ADP or GTP/GDP is determined by the subunit (1).Deleterious mutation in the gene SUCLA2 has been reported to cause diseases, such as encephalomyopathy and mitochondrial DNA depletion (2), and mutations in the genes SUCLG1 and SUCLA2 led to elevated levels of methylmalonic acid (3).The structures of GTPSCS and its complexes have been well studied over the decades.But there is no structure of ATPSCS or its complexes in the Protein Data Bank.Knowing the structure of the complex of ATPSCS with ATP-Mg 2+ will be helpful in further understanding the mechanism of the SCS-catalyzed reaction.Previous studies of full length human ATPSCS did not succeed in obtaining high resolution structures.A study of truncated human ATPSCS, with only the ATP-binding domain (Abd-ATPSCS), was proposed in order to reveal the interactions between ATPSCS and ATP-Mg 2+ .Abd-ATPSCS was produced in E. coli BL21(DE3) and purified through a three-step chromatographic purification.Abd-ATPSCS was cocrystallized with ATP and magnesium ions at a high concentration of polyethylene glycol 3350.The crystal diffracted to ~2.8 Å.

ACLY is an enzyme that is localized in the cytosol, where it catalyzes the following reaction:citrate + ATP + CoA acetyl-CoA + oxaloacetate + ADP + Pi Although the tricarboxylic acid (TCA) cycle produces acetyl-CoA, citrate synthase must convert acetyl-CoA into citrate, which can exit the mitochondria through the mitochondrial tricarboxylate transport protein because acetyl-CoA cannot be transported through the membrane.In the cytosol, ACLY converts citrate into acetyl-CoA and oxaloacetate, where the acetyl-CoA can be used for lipid biosynthesis.Since the citrate utilized comes from the catabolism of carbohydrates, ACLY links carbohydrate metabolism in the mitochondria with lipid metabolism that occurs in the cytosol.As well, ACLY may be involved in tumour development due to its role in lipid biogenesis.Thus, understanding the catalytic mechanism will reveal key features of ACLY that researchers can exploit, such as in pharmaceutical industry, or to limit or abolish the link between carbohydrates and lipids.Structural data provides key details about an enzyme's reaction mechanism.Currently, there are structures of the amino terminal portion of ACLY, with substrates citrate, ADP-Mg 2+ bound.However, no structure of the carboxyl-terminal portion is available.This is where the CoA binding site is thought to reside and where the intermediate (citryl-CoA) is cleaved to produce acetyl-CoA and oxaloacetate.The objective is to determine the structure of the carboxyl-terminal portion of ACLY to unravel the catalytic mechanism.X-ray crystallography will be utilized for this project to obtain atomic level resolution, revealing key structural features and catalytic residues.To isolate the carboxyl-terminal portion, the Tobacco Etch Virus (TEV) protease was used.By utilizing site-directed mutagenesis, the TEV protease recognition site (EXXYXQ S/G) was introduced by mutating several residues in the amino acid sequence of ACLY.The recognition site was mutated into various regions of ACLY, to produce four different constructs that produced a carboxyl-terminal portion of different residue length after proteolysis.Additionally, due to the consensus amino acid sequence being EXXYXQ S/G, where proteolysis occurs between the glutamine and serine or glycine and X refers to any hydrophobic residue, the different constructs were made with varying residues in the X positions to determine their affect on protein folding and stability.The carboxyl-terminal portions can then be isolated via specific proteolysis using TEV protease, purified and then crystallized.Data from the crystals were collected from the Canadian Light Source in Saskatoon.By obtaining the structure, the reaction mechanism can be elucidated for ACLY.This could provide researchers information that can prove vital to determining ways to inhibit this enzyme.As well, this provides one step forward to solving the full-length structure of ACLY if both the amino-and carboxyl-terminal portions are available.

Hydroxycitrate from the fruit of Garcinia cambogia [i.e. (2S,3S)-2-hydroxycitrate] is the best-known inhibitor of ATP-citrate lyase. Well diffracting crystals showing how the inhibitor binds to human ATP-citrate lyase were grown by modifying the protein. The protein was modified by introducing cleavage sites for Tobacco etch virus protease on either side of a disordered linker. The protein crystallized consisted of residues 2-425-ENLYFQ and S-488-810 of human ATP-citrate lyase. (2S,3S)-2-Hydroxycitrate binds in the same orientation as citrate, but the citrate-binding domain (residues 248-421) adopts a different orientation with respect to the rest of the protein (residues 4-247, 490-746 and 748-809) from that previously seen. For the first time, electron density was evident for the loop that contains His760, which is phosphorylated as part of the catalytic mechanism. The pro-S carboxylate of (2S,3S)-2-hydroxycitrate is available to accept a phosphoryl group from His760. However, when co-crystals were grown with ATP and magnesium ions as well as either the inhibitor or citrate, Mg2+-ADP was bound and His760 was phosphorylated. The phosphoryl group was not transferred to the organic acid. This led to the interpretation that the active site is trapped in an open conformation. The strategy of designing cleavage sites to remove disordered residues could be useful in determining the crystal structures of other proteins.

In a review paper in "Biochemistry", Blundell et al 1 pointed out that inhibitors binding to aspartic peptidases adopt an extended conformation such that alternate residues are in close proximity (i.e.P2 and P1'; P1 and P3).Covalent linking of the side chains of these alternate residues could have an increase in the potency of the inhibitor by locking it in the bound conformation thereby reducing the loss of conformational entropy on binding.Penicillopepsin is an aspartic peptidase isolated from the fungus Penicillium janthinellum 2 .This paper will discuss the structures of several phosphonate containing inhibitors bound to penicillopepsin.The tetrahedral geometry of the phosphonate moiety of the inhibitors resembles that of the cleavage transition state of the scissile carbonyl-carbon atom that forms during peptide bond hydrolysis.The proximity of the P2 Val and the P1' Phe of the pentapeptide inhibitor phosphonate analog, Isovaleryl-Val-Val-Leu P -O-Phe-CO 2 CH 3 , suggested that a covalent bond between them could be engineered and synthesized if the P2 Val was substituted by an asparagine residue.The structures of the phosphonate-based macrocycle (Ki = 0.10 nM) having an amide link between the P2 Asn and the P1' Phe, and the acyclic analog (Ki = 42 nM) of that inhibitor were determined to 0.95 Å and 1.41 Å resolutions respectively.These structures and the enhanced potency of the macrocyclicinhibitor will be discussed.

Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.