Atomic structure of the cubic core of the pyruvate dehydrogenase multienzyme complex.

The highly symmetric pyruvate dehydrogenase multienzyme complexes have molecular masses ranging from 5 to 10 million daltons. They consist of numerous copies of three different enzymes: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and lipoamide dehydrogenase. The three-dimensional crystal structure of the catalytic domain of Azotobacter vinelandii dihydrolipoyl transacetylase has been determined at 2.6 angstrom (A) resolution. Eight trimers assemble as a hollow truncated cube with an edge of 125 A, forming the core of the multienzyme complex. Coenzyme A must enter the 29 A long active site channel from the inside of the cube, and lipoamide must enter from the outside. The trimer of the catalytic domain of dihydrolipoyl transacetylase has a topology identical to chloramphenicol acetyl transferase. The atomic structure of the 24-subunit cube core provides a framework for understanding all pyruvate dehydrogenase and related multienzyme complexes.

Crystal structures of hevamine, a plant defence protein with chitinase and lysozyme activity, and its complex with an inhibitor.

BACKGROUND: Hevamine is a member of one of several families of plant chitinases and lysozymes that are important for plant defence against pathogenic bacteria and fungi. The enzyme can hydrolyze the linear polysaccharide chains of chitin and peptidoglycan. A full understanding of the structure/function relationships of chitinases might facilitate the production of transgenic plants with increased resistance towards a wide range of pathogens. RESULTS: The crystal structure of hevamine has been determined to a resolution of 2.2 A, and refined to an R-factor of 0.169. The enzyme possesses a (beta alpha)8-barrel fold. An inhibitor binding study shows that the substrate-binding cleft is located at the carboxy-terminal end of the beta-barrel, near the conserved Glu127. Glu127 is in a position to act as the catalytic proton donor, but no residue that might stabilize a positively charged oxocarbonium ion intermediate was found. A likely mechanism of substrate hydrolysis is by direct attack of a water molecule on the C1 atom of the scissile bond, resulting in inversion of the configuration at C1. CONCLUSIONS: The structure of hevamine shows a completely new lysozyme/chitinase fold and represents a new class of polysaccharide-hydrolyzing (beta alpha)8-barrel enzymes. Because the residues conserved in the family to which hevamine belongs are important for maintaining the structure of the (beta alpha)8-barrel, all members of the family, including fungal, bacterial and insect chitinases, are likely to share this architecture. The crystal structure obtained provides a basis for protein engineering studies in this family of chitinases.

Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand.

BACKGROUND: Lytic transglycosylases are bacterial muramidases that catalyse the cleavage of the beta- 1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydrobond in the MurNAc residue. These muramidases play an important role in the metabolism of the bacterial cell wall and might therefore be potential targets for the rational design of antibacterial drugs. One of the lytic transglycosylases is Slt35, a naturally occurring soluble fragment of the outer membrane bound lytic transglycosylase B (MltB) from Escherichia coli. RESULTS: The crystal structure of Slt35 has been determined at 1.7 A resolution. The structure reveals an ellipsoid molecule with three domains called the alpha, beta and core domains. The core domain is sandwiched between the alpha and beta domains. Its fold resembles that of lysozyme, but it contains a single metal ion binding site in a helix-loop-helix module that is surprisingly similar to the eukaryotic EF-hand calcium-binding fold. Interestingly, the Slt35 EF-hand loop consists of 15 residues instead of the usual 12 residues. The only other prokaryotic proteins with an EF-hand motif identified so far are the D-galactose-binding proteins. Residues from the alpha and core domains form a deep groove where the substrate fragment GlcNAc can be bound. CONCLUSIONS: The three-domain structure of Slt35 is completely different from the Slt70 structure, the only other lytic transglycosylase of known structure. Nevertheless, the core domain of Slt35 closely resembles the fold of the catalytic domain of Slt70, despite the absence of any obvious sequence similarity. Residue Glu162 of Slt35 is in an equivalent position to Glu478, the catalytic acid/base of Slt70. GlcNAc binds close to Glu162 in the deep groove. Moreover, mutation of Glu162 into a glutamine residue yielded a completely inactive enzyme. These observations indicate the location of the active site and strongly support a catalytic role for Glu162.

The structure of an energy-coupling protein from bacteria, IIBcellobiose, reveals similarity to eukaryotic protein tyrosine phosphatases.

BACKGROUND: . The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation. In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell. RESULTS: . The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 and refined to an R factor of 18.7% (Rfree of 24. 1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys 10) is located at the C-terminal end of the first beta strand. No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases. CONCLUSIONS: . A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved. The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbo-hydrate-specific components of different families cannot complement each other.

The structure of the Escherichia coli phosphotransferase IIAmannitol reveals a novel fold with two conformations of the active site.

BACKGROUND: The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) catalyses the cellular uptake and subsequent phosphorylation of carbohydrates. Moreover, the PTS plays a crucial role in the global regulation of various metabolic pathways. The PTS consists of two general proteins, enzyme I and the histidine-containing protein (HPr), and the carbohydrate-specific enzyme II (EII). EIIs are usually composed of two cytoplasmic domains, IIA and IIB, and a transmembrane domain, IIC. The IIA domains catalyse the transfer of a phosphoryl group from HPr to IIB, which phosphorylates the transported carbohydrate. Knowledge of the structures of the IIA proteins may provide insight into the mechanisms by which the PTS couples phosphorylation reactions with carbohydrate specificity. RESULTS: We have determined the crystal structure of the Escherichia coli mannitol-specific IIA domain, IIAmtl (M(r) 16.3 kDa), by multiple anomalous dispersion analysis of a selenomethionine variant of IIAmtl. The structure was refined at 1.8 A resolution to an R factor of 19.0% (Rfree 24.2%). The enzyme consists of a single five-stranded mixed beta sheet, flanked by helices on both sides. The phosphorylation site (His65) is located at the end of the third beta strand, in a shallow crevice lined with hydrophobic residues. The sidechains of two conserved active-site residues, Arg49 and His111, adopt two different conformations in the four independent IIAmtl molecules. Using a solution structure of phosphorylated HPr, and a combination of molecular modelling and NMR binding experiments, structural models of the HPr-IIAmtl complex were generated. CONCLUSIONS: The fold of IIAmtl is completely different from the structures of other IIA proteins determined so far. The two conformations of Arg49 and His111 might represent different states of the active site, required for the different phosphoryl transfer reactions in which IIAmtl is involved. A comparison of the HPr-IIAmtl model with models of HPr in complex with other IIA enzymes shows that the overall interaction mode between the two proteins is similar. Differences in the stabilisation of the invariant residue Arg17 of HPr by the different IIA proteins might be part of a subtle mechanism to control the hierarchy of carbohydrate utilisation by the bacterium.

Structure of papain refined at 1.65 A resolution.

Papain is a sulfhydryl protease from the latex of the papaya fruit. Its molecules consist of one polypeptide chain with 212 amino acid residues. The chain is folded into two domains with the active site in a groove between the domains. We have refined the crystal structure of papain, in which the sulfhydryl group was oxidized, by a restrained least-squares procedure at 1.65 A to an R-factor of 16.1%. The estimated accuracy in the atomic co-ordinates is 0.1 A, except for disordered atoms. All phi/psi angles for non-glycine residues are found within the outer limit boundary of a Ramachandran plot and this provides another check on the quality of the model. In the alpha-helical parts of the structure, the C = O bonds are directed more away from the helix axis than in a classical alpha-helix, leading to somewhat longer hydrogen bonds, 2.98 A, compared to 2.89 A. The hydrogen-bonding parameters and conformational angles in the anti-parallel beta-sheet structure show a large diversity. Hydrogen bonds in the core of the sheet are generally shorter than those at the more twisted ends. The average value is 2.91 A. The hydrogen bond distance Ni+3-Oi in turns is relatively long and the geometry is far from linear. Hydrogen bond formation, therefore, is perhaps not an essential prerequisite for turn formation. Although the crystallization medium is 62% (w/w) methanol in water, only 29 out of 224 solvent molecules can be regarded with any certainty as methanol molecules. The water molecules play an important role in maintaining structural stability. This is specially true for internal water. Twenty-one water molecules are located in contact areas between adjacent papain molecules. It seems as if the enzyme is trapped in a grid of water molecules with only a limited number of direct interactions between the protein molecules. The residues in the active site cleft belong to the most static parts of the structure. In general, disorder in atomic positions increases when going from the interior of the protein molecule to its surface. This behavior was quantified and it was found that the point of minimum disorder is near the molecular centroid.

Crystallization of thermitase, a thermostable subtilisin, from a sodium formate solution by means of an automated procedure.

A new crystal form of native thermitase has been obtained using sodium formate as the precipitating agent and employing an automated crystallization procedure. The crystals have the form of tetragonal bipyramids, the longest dimension being about 0.4 mm. The space group is P4(1)2(1)2 or P4(3)2(1)2, with a = 182 A and b = c = 53.3 A. The crystals diffract beyond 2.5 A.

Structure of an engineered porcine phospholipase A2 with enhanced activity at 2.1 A resolution. Comparison with the wild-type porcine and Crotalus atrox phospholipase A2.

The crystal structure of an engineered phospholipase A2 with enhanced activity has been refined to an R-factor of 18.6% at 2.1 A resolution using a combination of molecular dynamics refinement by the GROMOS package and least-squares refinement by TNT. This mutant phospholipase was obtained previously by deleting residues 62 to 66 in porcine pancreatic phospholipase A2, and changing Asp59 to Ser, Ser60 to Gly and Asn67 to Tyr. The refined structure allowed a detailed comparison with wild-type porcine and Crotalus atrox phospholipase A2. The conformation of the deletion region appears to be intermediate between that in those two enzymes. The residues in the active center are virtually the same. An internal hydrophobic area occupied by Phe63 in the wild-type porcine phospholipase A2 is kept as conserved as possible by local rearrangement of neighboring atoms. In the mutant structure, this hydrophobic pocket is now occupied by the disulfide bond between residues 61 and 91. A detailed description of the second binding site for a calcium ion in this enzyme is given.

Structure determination of the glycosomal triosephosphate isomerase from Trypanosoma brucei brucei at 2.4 A resolution.

The three-dimensional crystal structure of the enzyme triosephosphate isomerase from the unicellular tropical blood parasite Trypanosoma brucei brucei has been determined at 2.4 A resolution. This triosephosphate isomerase is sequestered in the glycosome, a unique trypanosomal microbody of vital importance for the energy-generating machinery of the trypanosome. The crystals contain one dimer per asymmetric unit. The structure could be solved by the method of molecular replacement, using the refined co-ordinates of chicken triosephosphate isomerase as a search model. The positions and individual isotropic temperature factors of the 3792 atoms of the complete dimer have been refined by the Hendrickson & Konnert restrained refinement procedure. While tight restraints have been maintained on the bonded distances, the R-factor has dropped to 23.2% for 12317 reflections between 6 A and 2.4 A. A total of 0.6 mg of enzyme was used for establishing the correct crystallization conditions and solving the three-dimensional structure. Although the sequences of trypanosomal and chicken triosephosphate isomerase are identical at only 52% of the 247 common positions, the overall folds are very similar. The architecture of the active sites is virtually the same with 85% of the side-chains being identical. On the other hand, the residues involved in the dimer contacts are the same at only 55% of the positions. Nevertheless, the position of the local 2-fold axis in the chicken and glycosomal dimers is similar. A remarkable feature of glycosomal triosephosphate isomerase is its high overall positive charge. This extra charge is concentrated in four clusters of positively charged side-chains on the surface of the dimer, quite far away from the active site. These clusters may be involved in the mechanism of import of this triosephosphate isomerase into the glycosome.

Structure of porcine pancreatic phospholipase A2 at 2.6 A resolution and comparison with bovine phospholipase A2.

The previously published three-dimensional structure of porcine pancreatic prophospholipase A2 at 3 A resolution was found to be incompatible with the structures of bovine phospholipase A2 and bovine prophospholipase A2. This was unexpected because of the very homologous amino acid sequences of these enzymes. Therefore, the crystal structure of the porcine enzyme was redetermined using molecular replacement methods with bovine phospholipase as the parent model. The structure was crystallographically refined at 2.6 A resolution by fast Fourier transform and restrained least-squares procedures to an R-factor of 0.241. The crystals appeared to contain phospholipase A2 and not prophospholipase A2. Apparently the protein is slowly converted under the crystallization conditions employed. Our investigation shows that, in contrast to the previous report, the three-dimensional structure of porcine phospholipase A2 is very similar to that of bovine phospholipase A2, including the active site. Smaller differences were observed in some residues involved in the binding of aggregated substrates. However, an appreciable conformational difference is in the loop 59 to 70, where a single substitution at position 63 (bovine Val leads to porcine Phe) causes a complete rearrangement of the peptide chain. In addition to the calcium ion in the active site, a second calcium ion is present in the crystals; this is located on a crystallographic 2-fold axis and stabilizes the interaction between two neighbouring molecules.

Crystal structure of bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide.

Bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide was crystallized from 50% (v/v) 2-methyl-2,4-pentanediol. The space group was P3(1)21 with cell dimensions a = b = 46.73 A and c = 102.5 A (1 A = 0.1 nm). Diffraction data were collected by oscillation photography from one single crystal of dimensions 0.2 mm x 0.2 mm x 0.2 mm. The crystal structure was determined to a resolution of 2.5 A by crystallographic refinement of a starting model, which consisted of native bovine pancreatic phospholipase A2 positioned and oriented in the P3(1)21 cell as in the bovine pro-phospholipase A2. The crystallographic R-factor decreased from 0.378 to 0.197 after 70 refinement cycles. For the greater part the three-dimensional structure was very similar to that of native phospholipase. The inhibitor group shows up clearly. However, as in solution, there is no calcium ion bound any more in the active site, and this causes a significant conformational change in the loop from residue 59 to 73. This loop is remote from the calcium binding site. Interestingly, this is the same loop that also shows different conformations in other phospholipase A2 molecules. The inhibitor molecule has hydrophobic interactions with Phe5 and Cys45. Rational design of specific and potent inhibitors of phospholipase A2 catalysis is discussed on the basis of the present three-dimensional structure.

Nitrate binding to Limulus polyphemus subunit type II hemocyanin and its functional implications.

The horseshoe crab, Limulus polyphemus, employs hemocyanin as an oxygen carrier in its hemolymph. This hemocyanin displays cooperative oxygen binding and heterotropic allosteric regulation by protons, chloride ions and divalent cations. Here, we report the crystal structure of Limulus polyphemus subunit type II hemocyanin with a nitrate ion bound in the interface of its first and second domains. Interestingly, the nitrate-binding site coincides with the binding site for the allosteric effector chloride. Oxygen-binding data indeed indicate that nitrate, like chloride, reduces the oxygen affinity of this hemocyanin. The observed binding of two distinct anions to a single site suggests that several other anions may also bind at this site. This opens the intriguing possibility that bicarbonate, which is structurally similar to nitrate and closely linked to respiration, can act as an allosteric effector that lowers the oxygen affinity. Such an effect could be another factor in the repertoire of allosteric regulators of this hemocyanin; however, the physiological implications will be a challenge to decipher, since there exists a complex interplay of effects between bicarbonate, chloride, pH and divalent cations.

Structural investigations of calcium binding and its role in activity and activation of outer membrane phospholipase A from Escherichia coli.

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that catalyses the hydrolysis of phospholipids. Enzymatic activity is regulated by reversible dimerisation and calcium-binding. We have investigated the role of calcium by X-ray crystallography. In monomeric OMPLA, one calcium ion binds between two external loops (L3L4 site) at 10 A from the active site. After dimerisation, a new calcium-binding site (catalytic site) is formed at the dimer interface in the active site of each molecule at 6 A from the L3L4 calcium site. The close spacing and the difference in calcium affinity of both sites suggests that the L3L4 site may function as a storage site for a calcium ion, which relocates to the catalytic site upon dimerisation. A sequence alignment demonstrates conservation of the catalytic calcium site but evolutionary variation of the L3L4 site. The residues in the dimer interface are conserved as well, suggesting that all outer membrane phospholipases require dimerisation and calcium in the catalytic site for activity. For this family of phospholipases, we have characterised a consensus sequence motif (YTQ-X(n)-G-X(2)-H-X-SNG) that contains conserved residues involved in dimerisation and catalysis.

Refined X-ray structures of haloalkane dehalogenase at pH 6.2 and pH 8.2 and implications for the reaction mechanism.

The crystal structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 has been refined at 1.9 A resolution at two different pH values, the pH of crystallization (pH 6.2) and the pH of optimal activity (pH 8.2), to final R-factors of 16.8% and 16.4%, respectively. Both models show good stereochemical quality. Two non-glycine residues have main-chain torsion angles that are located outside the "allowed" regions in a Ramachandran plot. One of them is the nucleophilic residue Asp124, which, together with the two other active site residues His289 and Asp260, is situated in an internal, predominantly hydrophobic cavity. The other residue, Asn148, helps stabilize the conformations of two of these active-site residues, Asp124 and Asp260. Comparison of the models at pH 6.2 and pH 8.2 revealed one major structural difference. At pH 6.2, a salt-bridge is present between the N epsilon 2 atom of His289 and the O delta 1 atom of Asp124, while at pH 8.2, this salt-bridge is absent, indicating that the N epsilon 2 atom of the histidine residue is mostly deprotonated at the pH of optimum activity. This is in agreement with the putative reaction mechanism in which the O delta 1 atom of Asp124 performs a nucleophilic attack on the substrate, resulting in an intermediate ester. This ester is subsequently cleaved by a hydrolytic water molecule. The high-resolution data sets clearly show the exact position of this water molecule. It is in an ideal position for donating a proton to the N epsilon 2 atom of His289 and subsequently cleaving the covalently bound intermediate ester, releasing the alcohol product. Detailed investigation of both refined models showed a number of unusual structural features. Four out of 11 helices contain an internal proline residue other than in the first turn. Two other alpha-helices have adopted in their central part a 3(10) conformation. A novel four-residue turn between a helix and a strand, the alpha beta 4 turn, is located at the site of the bend in the central eight-stranded beta-sheet of the dehalogenase structure.

Refined crystal structure of the catalytic domain of dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii at 2.6 A resolution.

Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal structure of the catalytic domain, i.e. residues 382 to 637, of Azotobacter vinelandii E2p (E2pCD) was solved by multiple isomorphous replacement and refined by energy minimization procedures. The final model contains 2182 protein atoms and 37 ordered water molecules. The R-factor is 18.7% for 10,344 reflections between 10.0 and 2.6 A resolution. The root-mean-square shift deviation from the ideal values is 0.017 A for bond lengths and 3.3 degrees for bond angles. The N-terminal residues 382 to 394 are disordered and not visible in the electron density map, otherwise all residues have well-defined density. The catalytic domain forms an oligomer of 24 subunits, having octahedral 432 symmetry. In the E2pCD crystals, the 24 subunits are related by the crystallographic symmetry. The cubic arrangement of subunits gives rise to a large hollow cube with edges of 120 A. The faces of the cube have pores of diameter of 30 A. The true building block of the cube is the E2p trimer, eight of which occupy the corners of the cube. Two levels of intermolecular contacts can be distinguished: (1) the extensive interactions between 3-fold related subunits leading to a tightly associated trimer; and (2) the interactions along the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer. Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT) and comprises a central beta-sheet surrounded by five alpha-helices. The comparison of the two proteins indicates a large rotation of the N-terminal residues 395 to 426 of E2pCD, which reshapes the substrate binding site and extends the interaction between threefold related subunits. The catalytic centre consists of a 30 A long channel extending from the "inner" side of the trimer to the "outer" side, where inner and outer refer to the position in the 24-meric cubic core of the pyruvate dehydrogenase complex and correspond with CoA and lipoamide binding sites, respectively. The active site is formed by the residues with the lowest mobility as indicated by the atomic B-factors. Five proline residues surround the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 A resolution. Analysis of redox and thermostability properties.

The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric flavoenzyme with a molecular mass of 106,000 daltons, was solved by the molecular replacement method and refined to an R-factor of 19.4% at 2.8 A resolution. The root-mean-square difference from ideal values for bonds and angles is 0.019 A and 3.8 degrees, respectively. The structure is closely related to that of the same flavoprotein from Azotobacter vinelandii. The root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence identity. The residues in the active site are identical, while 89% of the interface residues are the same in the two enzymes. A few structural variations provide the basis for the differences in thermostability and redox properties between the two homologous proteins. Particularly, in the A. vinelandii molecule a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring, unpaired to any H-bond donor, probably providing an explanation for the lower stability of the A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface loops, which previously could not be accurately positioned in the A. vinelandii structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the basis of the P. fluorescens structure, the six loops could be correctly defined also in the A. vinelandii enzyme. This is an unusual case where similar refinement methodologies applied to two crystal forms of closely related proteins led to electron density maps of substantially different quality. The correct definition of these surface residues is likely to be an essential step for revealing the structural basis of the interactions between lipoamide dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme complex.

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.

Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form.

The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.

Crystallization of enzyme IIB of the cellobiose-specific phosphotransferase system of Escherichia coli.

Crystals of enzyme IIB of the cellobiose-specific phosphotransferase system have been obtained from 15% polyethylene glycol 4000 using both streak-seeding and macroseeding techniques at 4 degrees. Crystals were grown with the hanging drop method of vapour diffusion. Addition of 2-propanol and benzamidine/HCl proved essential to obtain single crystals suitable for X-ray analysis. The crystals diffract to 1.8 A resolution and have the monoclinic space group P2(1), with cell dimensions a = 53.6 A, b = 31.7 A, c = 60.0 A and beta = 101.7 degrees. From a self-rotation function it seems likely that there are two molecules in the asymmetric unit related by a non-crystallographic 2-fold axis.

Crystal structure of a porcine pancreatic phospholipase A2 mutant. A large conformational change caused by the F63V point mutation.

The highly homologous bovine and porcine pancreatic phospholipase A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an alpha-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 3(10)-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine.HCl. The F63V mutant was crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 79.88 A, b = 65.23 A, c = 52.62 A, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 A resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.