An improved strategy for the crystallization of Leishmania mexicana pyruvate kinase.

The inclusion of novel small molecules in crystallization experiments has provided very encouraging results and this method is now emerging as a promising alternative strategy for crystallizing 'problematic' biological macromolecules. These small molecules have the ability to promote lattice formation through stabilizing intermolecular interactions in protein crystals. Here, the use of 1,3,6,8-pyrenetetrasulfonic acid (PTS), which provides a helpful intermolecular bridge between Leishmania mexicana PYK (LmPYK) macromolecules in the crystal, is reported, resulting in the rapid formation of a more stable crystal lattice at neutral pH and greatly improved X-ray diffraction results. The refined structure of the LmPYK-PTS complex revealed the negatively charged PTS molecule to be stacked between positively charged (surface-exposed) arginine side chains from neighbouring LmPYK molecules in the crystal lattice.

Allosteric mechanism of pyruvate kinase from Leishmania mexicana uses a rock and lock model.

Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6 degrees rigid body rocking motion of the A- and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.

Crystal structures of Leishmania mexicana phosphoglycerate mutase suggest a one-metal mechanism and a new enzyme subclass.

The structures of Leishmania mexicana cofactor-independent phosphoglycerate mutase (Lm iPGAM) crystallised with the substrate 3-phosphoglycerate at high and low cobalt concentrations have been solved at 2.00- and 1.90-A resolutions. Both structures are very similar and the active site contains both 3-phosphoglycerate and 2-phosphoglycerate at equal occupancies (50%). Lm iPGAM co-crystallised with the product 2-phosphoglycerate yields the same structure. Two Co(2+) are coordinated within the active site with different geometries and affinities. The cobalt at the M1 site has a distorted octahedral geometry and is present at 100% occupancy. The M2-site Co(2+) binds with distorted tetrahedral geometry, with only partial occupancy, and coordinates with Ser75, the residue involved in phosphotransfer. When the M2 site is occupied, the side chain of Ser75 adopts a position that is unfavourable for catalysis, indicating that this site may not be occupied under physiological conditions and that catalysis may occur via a one-metal mechanism. The geometry of the M2 site suggests that it is possible for Ser75 to be activated for phosphotransfer by H-bonding to nearby residues rather than by metal coordination. The 16 active-site residues of Lm iPGAM are conserved in the Mn-dependent iPGAM from Bacillus stearothermophilus (33% overall sequence identity). However, Lm iPGAM has an inserted tyrosine (Tyr210) that causes the M2 site to diminish in size, consistent with its reduced metal affinity. Tyr210 is present in trypanosomatid and plant iPGAMs, but not in the enzymes from other organisms, indicating that there are two subclasses of iPGAMs.

Sulphate removal induces a major conformational change in Leishmania mexicana pyruvate kinase in the crystalline state.

We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening (4 A) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.

Characterization of the cofactor-independent phosphoglycerate mutase from Leishmania mexicana mexicana. Histidines that coordinate the two metal ions in the active site show different susceptibilities to irreversible chemical modification.

Phosphoglycerate mutase (PGAM) activity in promastigotes of the protozoan parasite Leishmania mexicana is found only in the cytosol. It corresponds to a cofactor-independent PGAM as it is not stimulated by 2,3-bisphosphoglycerate and is susceptible to EDTA and resistant to vanadate. We have cloned and sequenced the gene and developed a convenient bacterial expression system and a high-yield purification protocol. Kinetic properties of the bacterially produced protein have been determined (3-phosphoglycerate: K(m) = 0.27 +/- 0.02 mm, k(cat) = 434 +/- 54 s(-1); 2-phosphoglycerate: K(m) = 0.11 +/- 0.03 mm, k(cat) = 199 +/- 24 s(-1)). The activity is inhibited by phosphate but is resistant to Cl(-) and SO(4) (2-). Inactivation by EDTA is almost fully reversed by incubation with CoCl(2) but not with MnCl(2), FeSO(4), CuSO(4), NiCl(2) or ZnCl(2). Alkylation by diethyl pyrocarbonate resulted in irreversible inhibition, but saturating concentrations of substrate provided full protection. Kinetics of the inhibitory reaction showed the modification of a new group of essential residues only after removal of metal ions by EDTA. The modified residues were identified by MS analysis of peptides generated by trypsin digestion. Two substrate-protected histidines in the proximity of the active site were identified (His136, His467) and, unexpectedly, also a distant one (His160), suggesting a conformational change in its environment. Partial protection of His467 was observed by the addition of 25 micro m CoCl(2) to the EDTA treated enzyme but not of 125 micro m MnCl(2), suggesting that the latter metal ion cannot be accommodated in the active site of Leishmania PGAM.

Expression, purification, crystallization and preliminary crystallographic analysis of Leishmania mexicana phosphoglycerate mutase.

Bacterially expressed 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) from Leishmania mexicana with a six-His tag fused at its C-terminus was expressed from plasmid pET28a after IPTG induction in Escherichia coli cells and gave a yield of 20 mg of highly purified iPGAM per litre of cell culture. Crystals of the protein complexed with 3-phosphoglycerate were obtained by the hanging-drop method of vapour diffusion with PEG 4000 as the precipitating agent in the presence of cobalt chloride and diffracted synchrotron radiation to beyond 1.90 A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 72.27, c = 129.68 A. A model of Bacillus stearothermophilus iPGAM (33% identity) was used to provide an initial molecular-replacement solution. X-ray data to 2.05 A for the structure of L. mexicana iPGAM complexed with 2-phosphoglycerate have also been collected.

The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis.

The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions.