Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis.

BACKGROUND: Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are well-recognized antiparasitic drug targets. HGPRT is also a paradigmatic representative of the phosphoribosyltransferase family of enzymes, which includes other important biosynthetic and salvage enzymes and drug targets. To better understand the reaction mechanism of this enzyme, we have crystallized HGPRT from the apicomplexan protozoan Toxoplasma gondii as a ternary complex with a substrate and a substrate analog. RESULTS: The crystal structure of T. gondii HGPRT with the substrate Mg2+-PRPP and a nonreactive substrate analog, 9-deazaguanine, bound in the active site has been determined at 1.05 A resolution and refined to a free R factor of 15.4%. This structure constitutes the first atomic-resolution structure of both a phosphoribosyltransferase and the central metabolic substrate PRPP. This pre-transition state complex provides a clearer understanding of the structural basis for catalysis by HGPRT. CONCLUSIONS: Three types of substrate deformation, chief among them an unexpected C2'-endo pucker adopted by the PRPP ribose ring, raise the energy of the ground state. A cation-pi interaction between Tyr-118 and the developing oxocarbenium ion in the ribose ring helps to stabilize the transition state. Enforced substrate propinquity coupled with optimal reactive geometry for both the substrates and the active site residues with which they interact contributes to catalysis as well.

Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides.

BACKGROUND: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine. RESULTS: The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2. CONCLUSIONS: Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.

The refined crystallographic structure of a DD-peptidase penicillin-target enzyme at 1.6 A resolution.

The D-alanyl-D-alanine peptidase from Streptomyces sp. R61 is a 37,500 dalton exocellular enzyme that has served as a model for membrane-bound peptidases that are involved in bacterial cell wall biosynthesis. Inhibition of these enzymes by beta-lactam antibiotics ultimately leads to bacterial cell death. The X-ray crystal structure of the R61 D-alanyl-D-alanine peptidase has been solved using multiple isomorphous replacement, simulated annealing and least squares refinement. The space group and unit cell parameters are P2(1)2(1)2(1) with a = 51.1 A, b = 67.3 A and c = 102.4 A. The structure has been refined using 2 sigma data to 1.6 A resolution with a crystallographic R-factor of 0.148. The model contains 347 residues (2938 atoms) and 254 solvent molecules. The overall temperature factor is 9.6 A2, and the estimated coordinate error is 0.14 A. The protein consists of a single polypeptide chain organized into two regions. One region contains a nine-stranded antiparallel beta-sheet with helices on both faces; this region includes both the amino and carboxyl termini. The second region is all helical. Sixty percent of the residues occur in helices or beta-sheet. The reactive Ser62 is found between the two regions of the enzyme at the amino end of the protein's longest-helix which begins with one turn of 3(10) helix and continues with four turns of alpha-helix. The active site is an elongated pocket that contains four basic and four aromatic residues. An oxyanion hole is formed by Ser62 NH and Thr301 NH. The pocket also contains the few key residues that are conserved in all penicillin-binding proteins and beta-lactamases. Two of these residues, Lys65 and Tyr159, are among the 16 side-chains that take on multiple conformations in the R61 crystal structure. Three of the 12 proline rings adopt two conformations which we believe has not been previously reported. There is no anionic acid equivalent to the catalytic Glu166 found in Class A beta-lactamases. Two ordered water molecules (O507 and O644) are found buried in the active site and hydrogen-bonded to each other (2.6 A). O507 could potentially act as the hydrolytic water molecule for deacylation.

Three-dimensional structure of (Na+ + K+)-ATPase revealed by electron microscopy of two-dimensional crystals.

Prolonged incubation of membrane fragments containing homogeneous (Na+ + K+)-ATPase with Mg2+, K+ and VO-3 at 4 degrees C resulted in formation of two-dimensional crystals of this enzyme with unit cell parameters: a = 66 A, b = 118 A, gamma = 108 degrees. The crystals correspond to the two-sided plane group p21. By combining tilted electron microscopic views of the crystals, a three-dimensional structure of (Na+ + K+)- ATPase was calculated at approximately 20 A resolution. The unit cell is formed by two (alpha beta)-promoters which are in contact in their central parts. The structure was compared with chemical modification and immunochemical data; the arrangement of intra- and extramembrane domains was proposed.

Two-dimensional crystallization of reaction centers from Chloroflexus aurantiacus.

Two-dimensional crystals of photosynthetic reaction centers from Chloroflexus aurantiacus were obtained from protein-lipid-detergent micelles by detergent dialysis. The size of crystals was up to 2 microns. Some of them were multilayered crystals. However, other crystal forms were also observed. Preliminary image processing analysis showed that crystals of one crystal form referred to two-sided plane group p2 and had the following unit cell parameters: a = 17.6 nm, b = 18.0 nm, gamma = 84 degrees. The contour map of the crystal stain-excluding region was calculated by the Fourier-filtering procedure at about 2 nm resolution.

[Crystals of free aspartate aminotransferase]. / Issledovanie kristallov svobodnoi aspartat-aminotransferazy.

The crystals of free cytosolic chicken aspartate aminotransferase were subjected to X-ray investigation at 2.7 A. One subunit of the dimeric molecule crystalline enzyme is in the open conformation and the other is in the closed conformation.

[X-ray structural study of leucine aminopeptidase with a resolution of 4 Angstroms]. / Rentgenostrukturnoe issledovanie leitsinaminopeptidazy s razresheniem 4 A.

An X-ray crystallographic structure determination has been carried out on bovine lens leucine aminopeptidase at 4.0 A resolution by using a combination of isomorphous replacement and solvent flattening. The two heavy atom derivatives used were obtained by soaking crystals in ethyl mercury chloride, which bound at four sites, and phenyl mercury acetate, which bound at one site in the monomer. The electron density map reveals that the enzyme hexameric oligomer, arranged in 32 symmetry, has a triangular barrel appearance and dimensions, of height 88 A and maximal width 118 A in barrel equatorial plane. Each subunit in an elongated ellipsoid of approximate length 92 A. Subunits contacts have been described. From an analysis of the map each subunit appears to contain some 36% alpha-helix and is organized into two distinct globular domains. Direct location of zinc cluster and competitive inhibitor binding site are presented.

[Study of crystals of aspartate aminotransferase complexed with D-aspartate]. / Issledovanie kristallov kompleksa aspartat-aminotransferazy s D-aspartatom.

We report here the first X-ray studies of the complex of cytosolic aspartate aminotransferase from chicken heart with D-aspartate at 2.5 A resolution. Crystals of the complex were grown by cocrystallization (space group is P2(1)2(1)2(1), parameters: a = 62.48 A, b = 117.71 A, c = 124.38 A). They contain one dimeric molecule in the asymmetric unit. The X-ray analysis proves that attachment of D-aspartate induces considerable conformational changes in the active sites of two subunits of the enzyme: both subunits of the complex are in the closed conformation, the interaction of the enzyme with D-aspartate induces a substantial turn (about 90 degrees) of the coenzyme in one subunit, the coenzyme ring is deformed, considerable conformational changes are determined for Phe-18 and Glu-141. Apparently, the amino group of the substrate is a trigger of the conformational changes in the active site of the enzyme.

[X-ray structural study of the crystalline structure of human progastricsin by a molecular replacement method]. / Rentgenostrukturnoe issledovanie kristallicheskoi struktury progastriksina cheloveka metodom molekuliarnogo zameshcheniia.

The human progastricsin crystal structure has been solved by the molecular replacement method. The intensities of reflections from native progastricsin crystals were measured at the 4.0 A resolution by the omega-scan method with a Nicolet P3 diffractometer operated in automatic regime. To determine the orientation and position of progastricsin molecules in the unit cell, programme packages MERLOT and BRUTE were applied running on a MicroVAX-II computer. Prior to the translation search, several rotation function peaks were subjected to a rigid body refinement against the correlation coefficient between the observed and calculated structure factors. This approach clearly identified the correct orientation of the molecule. The solution obtained from the BRUTE translation function map was refined by the 6-dimensional correlation search and then by programme CORELS. The human progastricsin molecules packing in the crystal unit ell is described.

[The complex of aspartate aminotransferase with D-aspartate]. / Issledovanie kompleksa aspartat-aminotranferazy s D-aspartatom.

We report here the x-ray studies of the complex cytosolic aspartate aminotransferase from chicken heart with D-aspartate at 2,7 A resolution. Crystals of the complex was prepared by diffusing D-aspartate into free enzyme crystals; their space group is P 2(1)2(1)2(1) with cell dimensions (A): a = 62.59; b = 117.83; c = 124.38. They contain one dimeric molecule in the asymmetric unit. The x-ray crystallographic analysis proves that the connection of the D-aspartate induces small conformational changes in the active site of two subunits of the enzyme: considerable conformational changes are determined for His 189, Phe 360, Tyr 70, Arg 292, Phe 18 and Glu 141.

[A new form of aspartate aminotransferase crystals]. / Novaia forma kristallov aspartat-aminotransferazy.

A new crystal form of chicken cytosolic aspartate aminotransferase (EC 2.6.1.1) has been grown using a mixture of ammonium sulfate with ethanol as a precipitant. Crystals of the enzyme belong to the space group P 2(1)2(1)2(1) having the following unit cell dimensions: a = 62.38 A, b = 117.41 A, c = 124.34 A. There is one molecule of the enzyme in the asymmetric unit. The crystals diffract at 1.8 A resolution.

Binding of cephalothin and cefotaxime to D-ala-D-ala-peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum beta-lactamases.

Two clinically-important beta-lactam antibiotics, cephalothin and cefotaxime, have been observed by X-ray crystallography bound to the reactive Ser62 of the D-alanyl-D-alanine carboxypeptidase/transpeptidase of Streptomyces sp. R61. Refinement of the two crystal structures produced R factors for 3 sigma (F) data of 0.166 (to 1.8 A) and 0.170 (to 2.0 A) for the cephalothin and cefotaxime complexes, respectively. In each complex, a water molecule is within 3.1 and 3.6 A of the acylated beta-lactam carbonyl carbon atom, but is poorly activated by active site residues for nucleophilic attack and deacylation. This apparent lack of good stereochemistry for facile hydrolysis is in accord with the long half-lives of cephalosporin intermediates in solution (20-40 h) and the efficacy of these beta-lactams as inhibitors of bacterial cell wall synthesis. Different hydrogen binding patterns of the two cephalosporins to Thr301 are consistent with the low cefotaxime affinity of an altered penicillin-binding protein, PBP-2x, reported in cefotaxime-resistant strains of Streptococcus pneumoniae, and with the ability of mutant class A beta-lactamases to hydrolyze third-generation cephalosporins.

X-ray studies of enzymes that interact with penicillins.

The technique of X-ray diffraction has been successfully applied to enzymes associated with peptidoglycan biosynthesis. The technique has taught us a great deal about the structures and catalytic mechanisms of penicillin-binding proteins and beta-lactamases. An insight into the structural basis for antibiotic resistance is given.

Structure of the SHV-1 beta-lactamase.

The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established. The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A. The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution. Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively. Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A. Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet. All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1. However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1. A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104.

Crystallization and preliminary X-ray study of OXA-1, a class D beta-lactamase.

The Escherichia coli OXA-1 oxacillinase, a beta-lactamase which provides resistance to beta-lactam antibiotics (penicillins and cephalosporins), has been crystallized. A member of the class D family of serine beta-lactamases, OXA-1 is especially active against the penicillins oxacillin and cloxacillin and is now found in 10% of E. coli clinical isolates. Crystals grown from PEG 8000 at pH 7.5 diffract to 1.5 A resolution at 100 K and have space group P1 (Z = 2), with unit-cell parameters a = 36.0, b = 51.6, c = 72.9 A, alpha = 70.2, beta = 84.1, gamma = 81.5 degrees.

Inhibition of the SHV-1 beta-lactamase by sulfones: crystallographic observation of two reaction intermediates with tazobactam.

Two species resulting from the reaction of the SHV-1 class A beta-lactamase with the sulfone inhibitor tazobactam have been trapped at 100 K and mapped by X-ray crystallography at 2.0 A resolution. An acyclic form of tazobactam is covalently bonded to the catalytic Ser70 side chain, and a second species, a five-atom vinyl carboxylic acid fragment of tazobactam, is bonded to Ser130. It is proposed that the electron density map of the crystal is a composite picture of two complexes, each with only a single bound species. It is estimated that the two complexes exist in the crystal in approximately equal populations. Results are discussed in relation to the mechanism-based inhibition of class A beta-lactamases by the similar inhibitors sulbactam and clavulanic acid.

Structure of the extended-spectrum class C beta-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion.

A class C beta-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improved hydrolytic activity for oxyimino beta-lactam antibiotics has been analyzed by X-ray crystallography to 1.8 A resolution. Relative to the wild-type P99 beta-lactamase, this natural mutant contains a highly unique tandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4 kDa chromosomal beta-lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate in space group P2(1)2(1)2 with cell dimensions a = 78.0 A, b = 69.5 A, and c = 63.1 A. The crystal structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20 for all nonzero data from 8 to 1.8 A. Deviations of model bonds and angles from ideal values are 0.008 A and 1.4 degrees, respectively. Overlay of alpha-carbon atoms in the GC1 and P99 beta-lactamases results in an rms deviation of 0.6 A. Largest deviations occur in a loop containing Gln120 and in the Omega loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width of the opening to the substrate binding cavity, as measured from the 318-324 beta-strand to two loops containing Gln120 and Tyr150 on the other side, is 0.6-1.4 A wider than in P99. It is suggested that conformational flexibility in the expanded Omega loop, and its influence on adjacent protein structure, may facilitate hydrolysis of oxyimino beta-lactams by making the acyl intermediate more open to attack by water. Nevertheless, backbone atoms in core catalytic site residues Ser64, Lys67, Tyr150, Asn152, Lys318, and Ser321 deviate only 0.4 A (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 at pH 8, is consistent with the requirement for a lower than normal pK(a) for this residue.