A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework.

Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.

The engineering of a more thermally stable lactate dehydrogenase by reduction of the area of a water-accessible hydrophobic surface.

A site-directed mutant of Bacillus stearothermophilus lactate dehydrogenase (lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been engineered in which the conserved hydrophobic residue isoleucine-250 has been replaced by the more hydrophilic residue asparagine. This isoleucine forms a large part of a water-accessible, hydrophobic surface in the active site of the apo-enzyme which is covered by the B-face of the nicotinamide ring when coenzymes are bound. Reduction in the area of this hydrophobic surface results in the mutant tetramer being more thermally stable than the wild-type enzyme.

Key residues in the allosteric transition of Bacillus stearothermophilus pyruvate kinase identified by site-directed mutagenesis.

The structural gene for pyruvate kinase from Bacillus stearothermophilus has been cloned in Escherichia coli and sequenced. The open reading frame from the ATG start codon to the TAG stop codon is 1482 base-pairs and encodes a peptide of relative molecular mass 52,967. In the expression vector pKK223-3, containing the synthetic tac promoter, the gene is overexpressed in E. coli cells to an estimated level of 30% total soluble cell protein. A purification procedure for the overexpressed protein has been established. The construction and characterization of a pair of mutant proteins has given insight into the structural basis of allosteric regulation in the tetrameric enzyme. Substituting tryptophan for tyrosine at position 466 (mutant Trp466-->Tyr) resulted in an activated form of the enzyme, having a reduced K1/2 for the substrate phosphoenolpyruvate. We propose that the characteristics of this mutant might be the result of bulk removal releasing steric inhibition to the formation of an interdomain salt bridge between Asp356 and Arg444. The regulatory behaviour of the double mutant produced by making the additional substitution aspartate for glutamate at position 356 (Trp466-->Tyr/Asp356-->Glu) corroborates this. The position of the salt bridge is such that it might be pivotal to the conformation of a pocket that is proposed to open up when the active R-conformation is adopted. We suggest that the mechanism of activation of B. stearothermophilus pyruvate kinase by ribose-5-phosphate might hinge on an interaction with, or indirectly through, residue Trp466, removing it from the vicinity of the potential salt bridge between Asp356 and Arg444 and thus effecting a closing together of the protein structure concomitant with an opening up of the pocket region.

Preliminary crystallographic studies on duck ovotransferrin.

Crystals of duck ovotransferrin and duck apo-ovotransferrin have been grown from polyethylene glycol solutions. For both crystals, the space group is P2(1)2(1)2(1), the unit cell dimensions for the ovotransferrin are a = 49.6 A, b = 85.6 A, c = 178.7 A and for the apo-ovotransferrin a = 77.6 A, b = 98.8 A, c = 127.0 A, giving four molecules in the unit cell.

Cooperativity in Bacillus stearothermophilus pyruvate kinase.

The enzyme pyruvate kinase (PK) from the moderate thermophile Bacillus stearothermophilus has been used as a model system with which to investigate the homotropic and heterotropic cooperative interactions of the enzyme. Cooperative ligand binding by the wild-type enzyme was measured using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative structural changes induced by the substrate phosphoenolpyruvate (PEP) are distinct from those induced by the allosteric activator ribose- 5-phosphate (R5P). Furthermore the structural transition induced by the binding of saturating amounts of both PEP and R5P is itself distinct. This conclusion was further substantiated by the production of five mutant proteins in which the R5P- and PEP-induced homotropic cooperative transitions were separated. These results suggest that the cooperativity exhibited by pyruvate kinase from B. stearothermophilus does not conform to a simple two-state model. A putative four-state model is proposed.

Crystallization of a ternary complex of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase was purified from an overexpressing Escherichia coli cell line. The enzyme has been crystallized in several different forms. All of these crystal forms were grown in the presence of NADH, sodium oxamate and fructose 1,6-bisphosphate. Three crystal forms have been characterized, an orthorhombic P2(1)2(1)2 (type III, a = 86 A, b = 105 A, c = 136 A) and two monoclinic P21 forms (type IV, a = 85 A, b = 118 A, c = 136 A, beta = 96 degrees; type V, a = 112 A, b = 85 A, c = 136 A, beta = 91 degrees). Precession photographs from these crystal forms are very alike, suggesting the molecular packing to be similar in all three forms. The P21 type IV crystals diffract to beyond 2 A spacing and are stable to irradiation with X-rays. A complete medium-resolution (4.7 A) dataset has been collected from a single crystal using synchrotron radiation. Rotation function studies with these data show the two tetramers of the asymmetric unit to be in very similar orientations. Higher-resolution data are being collected.

Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme.

The crystal structure of a mutant Bacillus stearothermophilus lactate dehydrogenase, into which an additional loop has been engineered in order to prevent tetramerization of the enzyme, has been solved and refined at 2.4 A. The minimal repeat unit in the crystal is a dimer and the tetramer cannot be generated by any of the crystallographic symmetry operations in P2(1). The loop protrudes out into the solvent, stabilized by a good hydrogen bonding arrangement, and clearly sterically hinders tetramer formation. This is the first structure of B. stearothermophilus lactate dehydrogenase (bsLDH) in which the allosteric activator fructose, 1,6-bisphosphate (FBP) is not present. To investigate the mechanism of allosteric activation in this enzyme we have compared the structure with a ternary complex of B. stearothermophilus lactate dehydrogenase. Many of our observations confirm those reported from a comparison of FBP-bound ternary bsLDH complex with an FBP free LDH from another bacterial source, Bifidobacterium longum. Our results suggest that quaternary structural alterations may have less influence on the mechanism than previously reported. The differences in the quaternary structural behaviour of these two enzymes is discussed.

The crystal structure of human phosphoglucose isomerase at 1.6 A resolution: implications for catalytic mechanism, cytokine activity and haemolytic anaemia.

Phosphoglucose isomerase (PGI) is a multifunctional protein, which, inside the cell, functions as a housekeeping enzyme of glycolysis and gluconeogenesis and, outside the cell, exerts wholly unrelated cytokine properties. We have determined the structure of human PGI to a resolution of 1.6 A using X-ray crystallography. The structure is highly similar to other PGIs, especially the architecture of the active site. Fortuitous binding of a sulphate molecule from the crystallisation solution has facilitated an accurate description of the substrate phosphate-binding site. Comparison with both native and inhibitor-bound rabbit PGI structures shows that two loops move closer to the active site upon binding inhibitor. Interestingly, the human structure most closely resembles the inhibitor-bound structure, suggesting that binding of the phosphate moiety of the substrate may trigger this conformational change. We suggest a new mechanism for catalysis that uses Glu357 as the base catalyst for the isomerase reaction rather than His388 as proposed previously. The human PGI structure has also provided a detailed framework with which to map mutations associated with non-spherocytic haemolytic anaemia.

Structure of a ternary complex of an allosteric lactate dehydrogenase from Bacillus stearothermophilus at 2.5 A resolution.

We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate).

Structure of diferric duck ovotransferrin at 2.35 A resolution.

The structure of diferric duck ovotransferrin (DOT) has been determined and refined at a resolution of 2.35 A. The DOT structure, which contains two iron binding sites, is similar to the known transferrin and lactoferrin structures. The two iron-binding sites, one in the N-terminal lobe and one in the C-terminal lobe of the molecule, are similar but not identical. The main differences between the three known structures lie in the relative orientations of the N- and C-lobes with respect to each other. In the DOT structure the large aromatic side chain of Phe322 in the N-lobe packs against the conserved residue Gly387 in the C-lobe. This interaction is at the centre of the interface between the two lobes and could play a crucial role in determining their relative orientation. Other differences between the structures occur in the surface loops and in the peptide connecting the two lobes. The final crystallographic model consists of 5309 protein atoms (686 residues), two Fe(3+) ions, two (bi)carbonate ions and three carbohydrate moities. 318 water molecules have been added to the model. The final R factor is 0.22 for 25 400 observed reflections between 10 and 2.35 A resolution.

Refined three-dimensional structure of cat-muscle (M1) pyruvate kinase at a resolution of 2.6 A.

The three-dimensional structure of cat-muscle pyoruvate kinase has been refined at a resolution of 2.6 A. The details of the structure permit interpretation of the original heavy-atom studies and give insight into the importance of conserved residues in pyruvate kinases and the allosteric behaviour of the enzyme. There are a small number of essential residues which determine the relative orientations of domains and the precise nature of intersubunit contacts. Arginine residues are particularly important.

Structure of apo duck ovotransferrin: the structures of the N and C lobes are in the open form.

The structure of apo duck ovotransferrin (APODOT) has been determined at a resolution of 4.0 A by the molecular replacement method using the structure of duck ovotransferrin (DOT) as the search model. The DOT structure contains two iron binding sites; one in the N-terminal lobe lying between domains N1 and N2 and one in the C-terminal lobe between domains C1 and C2. Both lobes have a closed structure. Models of various forms of both the N and C lobes were used in the search. The final model was refined to give an R factor of 0.22. The comparison of the structure of APODOT with that of DOT shows that both the N and the C lobes are in an open form, where the N2 and C2 domains undergo large rigid-body rotations of 51.6 and 49.9 degrees relative to the N1 and C1 domains, respectively. The interface between the N and C lobes, which is formed by the N1-C1 contact in the core of the molecule does not change significantly. The DOT molecule may be described in terms of three rigid bodies; the N1 and C1 domains as one rigid body forming the static core of the molecule and the N2 and C2 domains as two other rigid bodies which, on the release of iron, move away from the static core of the molecule to form the open structure of APODOT.