Biomedical subjects

K H Kalk

Publications and source records attributed to K H Kalk.

At least 55 records · Page 3Linked to original sources

PubMed1994

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.

Amino Acid Sequence↗

PubMed1994

Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring.

The crystal structures of wild-type p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with the substrate analogues 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate have been determined at 2.3-, 2.5-, and 2.8-A resolution, respectively. In addition, the crystal structure of a Tyr222Ala mutant, complexed with 2-hydroxy-4-aminobenzoate, has been determined at 2.7-A resolution. The structures have been refined to R factors between 14.5% and 15.8% for data between 8.0 A and the high-resolution limit. The differences between these complexes and the wild-type enzyme-substrate complex are all concentrated in the active site region. Binding of substrate analogues bearing a 4-amino group (4-aminobenzoate and 2-hydroxy-4-aminobenzoate) leads to binding of a water molecule next to the active site Tyr385. As a result, a continuous hydrogen-bonding network is present between the 4-amino group of the substrate analogue and the side chain of His72. It is likely that this hydrogen-bonding network is transiently present during normal catalysis, where it may or may not function as a proton channel assisting the deprotonation of the 4-hydroxyl group of the normal substrate upon binding to the active site. Binding of substrate analogues bearing a hydroxyl group at the 2-position (2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate) leads to displacement of the flavin ring from the active site. The flavin is no longer in the active site (the "in" conformation) but is in the cleft leading to the active site instead (the "out" conformation). It is proposed that movement of the FAD out of the active site may provide an entrance for the substrate to enter the active site and an exit for the product to leave.

4-Aminobenzoic Acid↗

PubMed1994

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.

Cellobiose↗

PubMed1994

Doughnut-shaped structure of a bacterial muramidase revealed by X-ray crystallography.

The integrity of the bacterial cell wall depends on the balanced action of several peptidoglycan (murein) synthesizing and degrading enzymes. Penicillin inhibits the enzymes responsible for peptide crosslinks in the peptidoglycan polymer. Enzymes that act solely on the glycosidic bonds are insensitive to this antibiotic, thus offering a target for the design of antibiotics distinct from the beta-lactams. Here we report the X-ray structure of the periplasmic soluble lytic transglycosylase (SLT; M(r) 70,000) from Escherichia coli. This unique bacterial exomuramidase cleaves the beta-1,4-glycosidic bonds of peptidoglycan to produce small 1,6-anhydromuropeptides. The structure of SLT reveals a 'superhelical' ring of alpha-helices with a separate domain on top which resembles the fold of lysozyme. Site-directed mutagenesis and a crystallographic inhibitor-binding study confirmed that the lysozyme-like domain contains the active site of SLT.

Binding Sites↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1994

Structure of partially-activated E. coli heat-labile enterotoxin (LT) at 2.6 A resolution.

Biological toxicity of E. coli heat-labile enterotoxin and the closely related cholera toxin requires that the assembled toxin be activated by proteolytic cleavage of the A subunit and reduction of a disulfide bond internal to the A subunit. The structural role served by this reduction and cleavage is not known, however. We have crystallographically determined the structure of the E. coli heat-labile enterotoxin AB5 hexamer in which the A subunit has been cleaved by trypsin between residues 192 and 195. The toxin is thus partially activated, in that it has been cleaved but the disulfide bond has not been reduced. The structure of the A subunit in the cleaved toxin is substantially the same as that previously observed for the uncleaved AB5 structure, suggesting that although such cleavage is required for biological activity of the toxin it does not by itself cause a conformational change.

Adenosine Diphosphate Ribose↗

PubMed1994

Crystal structure of recombinant human triosephosphate isomerase at 2.8 A resolution. Triosephosphate isomerase-related human genetic disorders and comparison with the trypanosomal enzyme.

The crystal structure of recombinant human triosephosphate isomerase (hTIM) has been determined complexed with the transition-state analogue 2-phosphoglycolate at a resolution of 2.8 A. After refinement, the R-factor is 16.7% with good geometry. The asymmetric unit contains 1 complete dimer of 53,000 Da, with only 1 of the subunits binding the inhibitor. The so-called flexible loop, comprising residues 168-174, is in its "closed" conformation in the subunit that binds the inhibitor, and in the "open" conformation in the other subunit. The tips of the loop in these 2 conformations differ up to 7 A in position. The RMS difference between hTIM and the enzyme of Trypanosoma brucei, the causative agent of sleeping sickness, is 1.12 A for 487 C alpha positions with 53% sequence identity. Significant sequence differences between the human and parasite enzymes occur at about 13 A from the phosphate binding site. The chicken and human enzymes have an RMS difference of 0.69 A for 484 equivalent residues and about 90% sequence identity. Complementary mutations ensure a great similarity in the packing of side chains in the core of the beta-barrels of these 2 enzymes. Three point mutations in hTIM have been correlated with severe genetic disorders ranging from hemolytic disorder to neuromuscular impairment. Knowledge of the structure of the human enzyme provides insight into the probable effect of 2 of these mutations, Glu 104 to Asp and Phe 240 to Ile, on the enzyme. The third mutation reported to be responsible for a genetic disorder, Gly 122 to Arg, is however difficult to explain. This residue is far away from both catalytic centers in the dimer, as well as from the dimer interface, and seems unlikely to affect stability or activity. Inspection of the 3-dimensional structure of trypanosomal triosephosphate isomerase, which has a methionine at position 122, only increased the mystery of the effects of the Gly to Arg mutation in the human enzyme.

Amino Acid Sequence↗

PubMed1994

Galactose-binding site in Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT).

The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coli is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal structures, including that of an LT:galactose complex which we report here at 2.2 A resolution. The binding surface on the free toxin contains several tightly associated water molecules and a relatively flexible loop consisting of residues 51-60 of the B subunit. During receptor binding this loop becomes tightly ordered by forming hydrogen bonds jointly to the GM1 pentasaccharide and to a set of water molecules which stabilize the toxin:receptor complex.

Bacterial Toxins↗

PubMed1993

Crystallographic and fluorescence studies of the interaction of haloalkane dehalogenase with halide ions. Studies with halide compounds reveal a halide binding site in the active site.

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 catalyzes the conversion of 1,2-dichloroethane to 2-chloroethanol and chloride without use of oxygen or cofactors. The active site is situated in an internal cavity, which is accessible from the solvent, even in the crystal. Crystal structures of the dehalogenase enzyme complexed with iodoacetamide, chloroacetamide, iodide, and chloride at pH 6.2 and 8.2 revealed a halide binding site between the ring NH's of two tryptophan residues, Trp-125 and Trp-175, located in the active site. The halide ion lies on the intersection of the planes of the rings of the tryptophans. The binding of iodide and chloride to haloalkane dehalogenase caused a strong decrease in protein fluorescence. The decrease could be fitted to a modified form of the Stern-Volmer equation, indicating the presence of fluorophors of different accessibilities. Halide binding was much stronger at pH 6.0 than at pH 8.2. Assuming ligand binding to Trp-125 and Trp-175 as the sole cause of fluorescence quenching, dissociation constants at pH 6.0 with chloride and iodide were calculated to be 0.49 +/- 0.04 and 0.074 +/- 0.007 mM, respectively. Detailed structural investigation showed that the halide binding site probably stabilizes the halide product as well as the negatively charged transition state occurring during the formation of the covalent intermediate.

Acetamides↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase.

Crystal structures of haloalkane dehalogenase were determined in the presence of the substrate 1,2-dichloroethane. At pH 5 and 4 degrees C, substrate is bound in the active site without being converted; warming to room temperature causes the substrate's carbon-chlorine bond to be broken, producing a chloride ion with concomitant alkylation of the active-site residue Asp124. At pH 6 and room temperature the alkylated enzyme is hydrolysed by a water molecule activated by the His289-Asp260 pair in the active site. These results show that catalysis by the dehalogenase proceeds by a two-step mechanism involving an ester intermediate covalently bound at Asp124.

Catalysis↗

PubMed1993

Non-covalent binding of the heavy atom compound [Au(CN)2]- at the halide binding site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

The Na[Au(CN)2] heavy atom derivative contributed considerably to the successful elucidation of the crystal structure of haloalkane dehalogenase isolated from Xanthobacter autotrophicus GJ10. The gold cyanide was located in an internal cavity of the enzyme, which also contains the catalytic residues. Refinement of the dehalogenase-gold cyanide complex at 0.25 nm to an R-factor of 16.7% demonstrates that the heavy atom molecule binds non-covalently between two tryptophan residues pointing into the active site cavity. At this same site also chloride ions can be bound. Therefore, inhibition of dehalogenase activity by the Au(CN)-2 presumably occurs by competition for the same binding site as substrates.

Amino Acid Sequence↗

PubMed1993

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)

Acetyltransferases↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

Crystallographic analysis of substrate binding and catalysis in dihydrolipoyl transacetylase (E2p).

The catalytic domain of dihydrolipoyl transacetylase (E2pCD) forms the core of the pyruvate dehydrogenase multienzyme complex and catalyzes the acetyltransferase reaction using acetylCoA as acetyl donor and dihydrolipoamide (Lip(SH)2) as acceptor. The crystal structures of six complexes and derivatives of Azotobacter vinelandii E2pCD were solved. The binary complexes of the enzyme with CoA and Lip(SH)2 were determined at 2.6- and 3.0-A resolutions, respectively. The two substrates are found in an extended conformation at the two opposite entrances of the 30 A long channel which runs at the interface between two 3-fold-related subunits and forms the catalytic center. The reactive thiol groups of both substrates are within hydrogen-bond distance from the side chain of His 610. This fact supports the indication, derived from the similarity with chloramphenicol acetyl transferase, that the histidine side chain acts as general-base catalyst in the deprotonation of the reactive thiol of CoA. The conformation of Asn 614 appears to be dependent on the protonation state of the active site histidine, whose function as base catalyst is modulated in this way. Studies on E2pCD soaked in a high concentration of dithionite lead to the structure of the binary complex between E2pCD and hydrogen sulfite solved at 2.3-A resolution. It appears that the anion is bound in the middle of the catalytic center and is therefore capable of hosting and stabilizing a negative charge, which is of special interest since the reaction catalyzed by E2pCD is thought to proceed via a negatively charged tetrahedral intermediate. The structure of the binary complex between E2pCD and hydrogen sulfite suggests that transition-state stabilization can be provided by a direct hydrogen bond between the side chain of Ser 558 and the oxy anion of the putative intermediate. In the binary complex with CoA, the hydroxyl group of Ser 558 is hydrogen bonded to the nitrogen atom of one of the two peptide-like units of the substrate. Thus, CoA itself is involved in keeping the Ser hydroxyl group in the proper position for transition-state stabilization. Quite unexpectedly, the structure at 2.6-A resolution of a ternary complex in which CoA and Lip(SH)2 are simultaneously bound to E2pCD reveals that CoA has an alternative, nonproductive binding mode. In this abortive ternary complex, CoA adopts a helical conformation with two intramolecular hydrogen bonds and the reactive sulfur of the pantetheine arm positioned 12 A away from the active site residues involved in the transferase reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗

PubMed1993

Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin.

Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 multimer structure, in which the B pentamer has a membrane binding function and the A subunit is needed for enzymatic activity. The LT crystal structure has been solved using a combination of multiple isomorphous replacement, fivefold averaging and molecular dynamics refinement. Phase combination using all these sources of phase information was of crucial importance for the chain tracing. The structure has now been refined to 1.95 A resolution, resulting in a model containing 6035 protein atoms and 293 solvent molecules with a crystallographic R-factor of 18.2% and good stereochemistry. The B subunits are arranged as a highly stable pentamer with a donut shape. Each subunit takes part in approximately 30 inter-subunit hydrogen bonds and six salt bridges with its two neighbors, whilst burying a large surface area. The A subunit has higher temperature factors and less well-defined secondary structure than the B subunits. It interacts with the B pentamer mainly via the C-terminal A2 fragment, which runs through the highly charged central pore of the B subunits. The pore contains at least 66 water molecules, which fill the space left by the A2 fragment. A detailed analysis of the contacts between A and B subunits showed that most specific contacts occur at the entrance of the central pore of the B pentamer, while the contacts within the pore are mainly hydrophobic and water mediated, with the exception of two salt bridges. Only a few contacts exist between the A1 fragment and the B pentamer, showing that the A2 fragment functions as a "linker" of the A and B parts of the protein. Interacting with the A subunit by the B subunits does not cause large deviations from a common B subunit structure, and the 5-fold symmetry is well maintained. A potential NAD(+)-binding site is located in an elongated crevice at the interface of two small sheets in the A1 fragment. At the back of this crevice the functionally important Arg7 makes a hydrogen bond connecting two strands, which seems to be conserved across the ADP-ribosylating toxin family. The putative catalytic residue (A1:Glu112) is located nearby, close to a very hydrophobic region, which packs two loops together. This hydrophobic region may be important for catalysis and membrane translocation.

Amino Acid Sequence↗

PubMed1993

Structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei determined from Laue data.

The three-dimensional structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.12.1.12] from the sleeping-sickness parasite Trypanosoma brucei was solved by molecular replacement at 3.2-A resolution with an x-ray data set collected by the Laue method. For data collection, three crystals were exposed to the polychromatic synchrotron x-ray beam for a total of 20.5 sec. The structure was solved by using the Bacillus stearothermophilus enzyme model [Skarzyński, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187] with a partial data set which was 37% complete. The crystals contain six subunits per asymmetric unit, which allowed us to overcome the absence of > 60% of the reflections by 6-fold density averaging. After molecular dynamics refinement, the current molecular model has an R factor of 17.6%. Comparing the structure of the trypanosome enzyme with that of the homologous human muscle enzyme, which was determined at 2.4-A resolution, reveals important structural differences in the NAD binding region. These are of great interest for the design of specific inhibitors of the parasite enzyme.

Animals↗