X-ray crystallographic study of xylopentaose binding to Pseudomonas fluorescens xylanase A

The structure of the complex between a catalytically compromised family 10 xylanase and a xylopentaose substrate has been determined by X-ray crystallography and refined to 3.2 A resolution. The substrate binds at the C-terminal end of the eightfold betaalpha-barrel of Pseudomonas fluorescens subsp. cellulosa xylanase A and occupies substrate binding subsites -1 to +4. Crystal contacts are shown to prevent the expected mode of binding from subsite -2 to +3, because of steric hindrance to subsite -2. The loss of accessible surface at individual subsites on binding of xylopentaose parallels well previously reported experimental measurements of individual subsites binding energies, decreasing going from subsite +2 to +4. Nine conserved residues contribute to subsite -1, including three tryptophan residues forming an aromatic cage around the xylosyl residue at this subsite. One of these, Trp 313, is the single residue contributing most lost accessible surface to subsite -1, and goes from a highly mobile to a well-defined conformation on binding of the substrate. A comparison of xylanase A with C. fimi CEX around the +1 subsite suggests that a flatter and less polar surface is responsible for the better catalytic properties of CEX on aryl substrates. The view of catalysis that emerges from combining this with previously published work is the following: (1) xylan is recognized and bound by the xylanase as a left-handed threefold helix; (2) the xylosyl residue at subsite -1 is distorted and pulled down toward the catalytic residues, and the glycosidic bond is strained and broken to form the enzyme-substrate covalent intermediate; (3) the intermediate is attacked by an activated water molecule, following the classic retaining glycosyl hydrolase mechanism.     

Phosphorylase recognition and phosphorolysis of its oligosaccharide substrate: answers to a long outstanding question.

Phosphorylases are key enzymes of carbohydrate metabolism. Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered. How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non-regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase. The crystal structures of three MalP-oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio-oligosaccharide, 4-S-alpha-D-glucopyranosyl-4-thiomaltotetraose (GSG4), both at 2.9 A resolution; and the 2.1 A resolution ternary complex of MalP with thio-oligosaccharide and phosphate (GSG4-P). The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub-sites -1 to +4. Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate. The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites -1 and +1, the site of catalysis, from the preferred conformation for alpha(1-4)-linked glucosyl polymers.     

Insights into ligand-induced conformational change in Cel5A from Bacillus agaradhaerens revealed by a catalytically active crystal form

Glycoside hydrolases are ubiquitous enzymes involved in a diverse array of biological processes, from the breakdown of biomass, through to viral invasion and cellular signalling. Endoglucanase Cel5A from Bacillus agaradhaerens, classified into glycoside hydrolase family 5, has been studied in a catalytically inactive crystal form at low pH conditions, in which native and complex structures revealed the importance of ring distortion during catalysis. Here, we present the structure of Cel5A in a new crystal form obtained at higher pH values in which the enzyme is active "in-crystal". Native, cellotriosyl-enzyme intermediate and beta-d-cellobiose structures were solved at 1.95, 1.75 and 2.1 A resolution, respectively. These structures reveal two classes of conformational change: those caused by crystal-packing and pH, with others induced upon substrate binding. At pH 7 a histidine residue, His206, implicated in substrate-binding and catalysis, but previously far removed from the substrate-binding cleft, moves over 10 A into the active site cleft in order to interact with the substrate in the +2 subsite. Occupation of the -1 subsite by substrate induces a loop closure to optimise protein-ligand interactions. Cel5A, along with the unrelated family 45 and family 6 cellulases, provides further evidence of substantial conformational change in response to ligand binding for this class of hydrolytic enzyme.     

Crystal structures of amylosucrase from Neisseria polysaccharea in complex with D-glucose and the active site mutant Glu328Gln in complex with the natural substrate sucrose

The structure of amylosucrase from Neisseria polysaccharea in complex with beta-D-glucose has been determined by X-ray crystallography at a resolution of 1.66 A. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 A. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly in the bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained (4)C(1) conformation, and one that binds in the packing interface to a symmetry-related molecule. A third weaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientation of the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrose complex shows one molecule bound in the active site, where the glucosyl moiety is located at the alpha-amylase -1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible access channel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtained through enzyme interactions with the glucosyl ring and that an important part of the enzyme function is a precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328. The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting beta-strand 7 and alpha-helix 7 that is much longer in amylosucrase compared to other enzymes from the alpha-amylase family (family 13 of the glycoside hydrolases).     

Active site specific cadmium(II)-substituted horse liver alcohol dehydrogenase: crystal structures of the free enzyme, its binary complex with NADH, and the ternary complex with NADH and bound p-bromobenzyl alcohol

Three crystal structures have been determined of active site specific substituted Cd(II) horse liver alcohol dehydrogenase and its complexes. Intensities were collected for the free, orthorhombic enzyme to 2.4-A resolution and for a triclinic binary complex with NADH to 2.7-A resolution. A ternary complex was crystallized from an equilibrium mixture of NAD+ and p-bromobenzyl alcohol. The microspectrophotometric analysis of these single crystals showed the protein-bound coenzyme to be largely NADH, which proves the complex to consist of CdII-LADH, NADH, and p-bromobenzyl alcohol. Intensity data for this abortive ternary complex were collected to 2.9-A resolution. The coordination geometry in the free Cd(II)-substituted enzyme is highly similar to that of the native enzyme. Cd(II) is bound to Cys-46, Cys-174, His-67, and a water molecule in a distorted tetrahedral geometry. Binding of coenzymes induces a conformational change similar to that in the native enzyme. The interactions between the coenzyme and the protein in the binary and ternary complexes are highly similar to those in the native ternary complexes. The substrate binds directly to the cadmium ion in a distorted tetrahedral geometry. No large, significant structural changes compared to the native ternary complex with coenzyme and p-bromobenzyl alcohol were found. The implications of these results for the use of active site specific Cd(II)-substituted horse liver alcohol dehydrogenase as a model system for the native enzyme are discussed.     

Substrate recognition by unsaturated glucuronyl hydrolase from Bacillus sp. GL1

Bacterial unsaturated glucuronyl hydrolases (UGLs) together with polysaccharide lyases are responsible for the complete depolymerization of mammalian extracellular matrix glycosaminoglycans. UGL acts on various oligosaccharides containing unsaturated glucuronic acid (DeltaGlcA) at the nonreducing terminus and releases DeltaGlcA through hydrolysis. In this study, we demonstrate the substrate recognition mechanism of the UGL of Bacillus sp. GL1 by determining the X-ray crystallographic structure of its substrate-enzyme complexes. The tetrasaccharide-enzyme complex demonstrated that at least four subsites are present in the active pocket. Although several amino acid residues are crucial for substrate binding, the enzyme strongly recognizes DeltaGlcA at subsite -1 through the formation of hydrogen bonds and stacking interactions, and prefers N-acetyl-d-galactosamine and glucose rather than N-acetyl-d-glucosamine as a residue accommodated in subsite +1, due to the steric hindrance.     

Crystal structure of Thermobifida fusca endoglucanase Cel6A in complex with substrate and inhibitor: the role of tyrosine Y73 in substrate ring distortion

Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(2)-S-Glc(2)), has been determined to 1.5 A resolution. The glycosyl unit in subsite -1 was sterically hindered by Tyr73 and forced into a distorted (2)S(o) conformation. In the enzyme where Tyr73 was mutated to a serine residue, the hindrance was removed and the glycosyl unit in subsite -1 had a relaxed (4)C(1) chair conformation. The relaxed conformation was seen in two complex structures of the mutated enzyme, with cellotetrose (Glc(4)) at 1.64 A and Glc(2)-S-Glc(2) at 1.04 A resolution.     

Role of the N-terminal region of staphylokinase (SAK): evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complex formation

Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), which requires both the conversion of PG to plasmin (Pm) to expose an active site in PG-SAK activator complex and the amino-terminal processing of SAK to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of SAK affects its PG activation capability was investigated by generating SAK mutants, blocked in the native amino-terminal processing site of SAK, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilized PG-SAK binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with PG, Pm or miniplasmin substrate during ternary complex formation as compared to native SAK. Additionally, activator complex formed with PRM3 failed to activate miniplasminogen and exhibited highly diminished activation of substrate PG. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of SAK in accession and processing of substrate by the SAK-Pm activator complex to potentiate the PG activation by enhancing and/or stabilizing the interaction of free PG.     

Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F1 from a Streptomyces sp

Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of beta-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of beta-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and Km values on pH, when the substrate concentration was sufficiently lower than Km, gave the values of 4.1 and 7.2 for the ionization constants, pKe1 and pKe2 of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pKe1 and pKe2, values shifted to higher, to +0.60 and +0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities Ai's in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive Ai values (A1 was 1.10 kcal/mol and A2 was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, the subsites 3 and 4 showed negative Ai values (A3 was -0.21 kcal/mol and A4 was -2.8 kcal/mol).     

Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single-module, cellulose-degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello-oligomers: cellobiose (Glc₂), cellotriose (Glc₃), and cellotetraose (Glc₄), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β-sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello-oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello-oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (⁴C₁) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.     

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.     

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid–base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR–Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.     

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.     

The structure of an inverting GH43 beta-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues

beta-D-Xylosidases are glycoside hydrolases that catalyze the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicellulose. Here we describe the enzyme-substrate crystal structure of an inverting family 43 beta-xylosidase, from Geobacillus stearothermophilus T-6 (XynB3). Each XynB3 monomeric subunit is organized in two domains: an N-terminal five-bladed beta-propeller catalytic domain, and a beta-sandwich domain. The active site possesses a pocket topology, which is mainly constructed from the beta-propeller domain residues, and is closed on one side by a loop that originates from the beta-sandwich domain. This loop restricts the length of xylose units that can enter the active site, consistent with the exo mode of action of the enzyme. Structures of the enzyme-substrate (xylobiose) complex provide insights into the role of the three catalytic residues. The xylose moiety at the -1 subsite is held by a large number of hydrogen bonds, whereas only one hydroxyl of the xylose unit at the +1 subsite can create hydrogen bonds with the enzyme. The general base, Asp15, is located on the alpha-side of the -1 xylose sugar ring, 5.2 Angstroms from the anomeric carbon. This location enables it to activate a water molecule for a single-displacement attack on the anomeric carbon, resulting in inversion of the anomeric configuration. Glu187, the general acid, is 2.4 Angstroms from the glycosidic oxygen atom and can protonate the leaving aglycon. The third catalytic carboxylic acid, Asp128, is 4 Angstroms from the general acid; modulating its pK(a) and keeping it in the correct orientation relative to the substrate. In addition, Asp128 plays an important role in substrate binding via the 2-O of the glycon, which is important for the transition-state stabilization. Taken together, these key roles explain why Asp128 is an invariant among all five-bladed beta-propeller glycoside hydrolases.     

Substrate size dependence of lysozyme-catalyzed reaction

In the study of the mechanism of lysozyme-catalyzed reactions, it has been assumed that the rate constants in the catalytic process, the catalytic activity of catalytic group Glu 35, are independent of the degree of polymerization (size) of the substrate. The characteristics of substrate binding subsite F have recently been reexamined and the substrate binding mode at this subsite has been demonstrated to be more complex than expected from a model based on an X-ray analysis of the lysozyme-substrate complex. In the present study, the time courses of the lysozyme-catalyzed reactions with the substrates chitotetraose [(GlcNAc)4], chitopentaose [(GlcNAc)5], and chitohexaose [(GlcNAc)6], of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc), were obtained experimentally with high-performance liquid chromatography. From the experimental time courses, the values of the rate constants, k+1 (the cleavage of glycosidic linkage) and k-1/k+2 (relative efficiency of transglycosylation), were obtained by a data-fitting method with computer simulation of the lysozyme-catalyzed reaction (A. Masaki et al. (1981) J. Biochem. 90, 1167-1175). As a result, it was found that the k+1 value is dependent on the substrate size and the value of the binding free energy of subsite F is considerably smaller than previously estimated. The substrate size dependence of the k+1 value is considered to relate closely to the fine structure of the binding and catalytic sites.     

Isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix: X-ray structure analysis of a ternary enzyme-substrate complex and thermal stability

Isocitrate dehydrogenase from Aeropyrum pernix (ApIDH) is a homodimeric enzyme that belongs to the beta-decarboxylating dehydrogenase family and is the most thermostable IDH identified. It catalyzes the NADP+ and metal-dependent oxidative decarboxylation of isocitrate to alpha-ketoglutarate. We have solved the crystal structures of a native ApIDH at 2.2 A, a pseudo-native ApIDH at 2.1 A, and of ApIDH in complex with NADP+, Ca2+ and d-isocitrate at 2.3 A. The pseudo-native ApIDH is in complex with etheno-NADP+ which was located at the surface instead of in the active site revealing a novel adenine-nucleotide binding site in ApIDH. The native and the pseudo-native ApIDHs were found in an open conformation, whereas one of the subunits of the ternary complex was closed upon substrate binding. The closed subunit showed a domain rotation of 19 degrees compared to the open subunit. The binding of isocitrate in the closed subunit was identical with that of the binary complex of porcine mitochondrial IDH, whereas the binding of NADP+ was similar to that of the ternary complex of IDH from Escherichiacoli. The reaction mechanism is likely to be conserved in the different IDHs. A proton relay chain involving at least five solvent molecules, the 5'-phosphate group of the nicotinamide-ribose and a coupled lysine-tyrosine pair in the active site, is postulated as essential in both the initial and the final steps of the catalytic reaction of IDH. ApIDH was found to be highly homologous to the mesophilic IDHs and was subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability. Mutational studies revealed that a disulfide bond at the N terminus and a seven-membered inter-domain ionic network at the surface are major determinants for the higher thermostability of ApIDH compared to EcIDH. Furthermore, the total number of ion pairs was dramatically higher in ApIDH compared to the mesophilic IDHs if a cutoff of 4.2 A was used. A calculated net charge of only +1 compared to -19 and -25 in EcIDH and BsIDH, respectively, suggested a high degree of electrostatic optimization, which is known to be an important determinant for increased thermostability.     

Substrate distortion in the Michaelis complex of Bacillus 1,3-1,4-beta-glucanase. Insight from first principles molecular dynamics simulations

The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3-1,4-beta-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the -1 subsite adopts a distorted 1S3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4C1 chair conformation, the 1S3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the beta region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the -1 sugar ring. It thus provides a powerful tool to model E.S complexes for which experimental information is not yet available.     

Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Crystal structures of hen egg-white lysozyme complexes with gadolinium(III) and gadolinium(III)-N-acetyl-D-glucosamine.

Analysis at 0.25 nm resolution of the crystal structures of lysozyme-Gd(III) and lysozyme-Gd(III)-N-acetyl-D-glucosamine (GlcNac), prepared by diffusion methods, show that there are two main binding positions for Gd(III), one of which is close to glutamic acid-35 and the other close to aspartic acid-52. The two sites are 0.36 nm part. There is no evidence for the weak binding of Gd(III) to any of the eight other carboxy groups of lysozyme. In the presence of Gd(III), the binding of GlcNac is similar to that observed for the binding of the beta-anomer in subsite C. There are numerous small conformational changes in the protein on binding (Gd(III) and the sugar, and these have been quantified to a first approximation by real-space refinement. These changes are similar in both structures, and involve, among other small movements, shifts of one of the disulphide bridges by up to 0.05 nm. The movement of residues 70--74 observed in the binary complex of lysozyme-GlcNac [Perkins, Johnson, Machin & Phillips (1978) Biochem. J. 173-617] is not observed in the ternary complex of lysozyme-Gd(III)-GlcNac. The nature of the lysozyme-Gd(III) complex is discussed in the light of evidence from other crystallographic studies and n.m.r. solution studies. Preliminary findings for a lysozyme-Gd(III) complex prepared by co-crystallization methods are reported.