Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.     

Crystal structure of pyruvate kinase from Geobacillus stearothermophilus

The pyruvate kinase (PK) from a moderate thermophile, Geobacillus stearothermophilus, is an allosteric enzyme activated by AMP and ribose 5-phosphate but not fructose 1, 6-bisphosphate (FBP), which is a common activator of PKs. It has an extra C-terminal sequence (ECTS), which contains a highly conserved phosphoenolpyruvate (PEP) binding motif, but its function and structure remain unclear. To elucidate the structural characteristics of the effector-binding site and the ECTS, the crystal structure of the C9S/C268S mutant of the enzyme was determined at 2.4 A resolution. The crystal belonged to space group P6(2)22, with unit cell parameters a, b = 145.97 A, c = 118.03 A. The enzyme was a homotetramer and its overall domain structure was similar to the previously solved structures except that the ECTS formed a new domain (C' domain). The structure of the C' domain closely resembled that of the PEP binding domain of maize pyruvate phosphate dikinase. A sulphate ion was found in a pocket in the effector-binding C domain. This site corresponds to the 6-phosphate group-binding site in yeast PK bound FBP and seems to be the effector-binding site. Through comparison of the structure of the putative effector-binding site to that of the FBP binding site of the yeast enzyme, the structural basis of the effector specificity of the G. stearothermophilus PK is discussed.     

Crystal structures of the conserved tRNA-modifying enzyme GidA: implications for its interaction with MnmE and substrate

GidA is a flavin-adenine-dinucleotide (FAD)-binding protein that is conserved among bacteria and eucarya. Together with MnmE, it is involved in the addition of a carboxymethylaminomethyl group to the uridine base in the wobble position (nucleotide 34) of tRNAs that read split codon boxes. Here, we report the crystal structures of the GidA proteins from both Escherichia coli and Chlorobium tepidum. The structures show that the protein can be divided into three domains: a first FAD-binding domain showing the classical Rossmann fold, a second α/β domain inserted between two strands of the Rossmann fold, and an α-helical C-terminal domain. The domain inserted into the Rossmann fold displays structural similarity to the nicotinamide-adenine-dinucleotide-(phosphate)-binding domains of phenol hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, and, correspondingly, we show that GidA binds NADH with high specificity as an initial donor of electrons. GidA behaves as a homodimer in solution. As revealed by the crystal structures, homodimerization is mediated via both the FAD-binding domain and the NADH-binding domain. Finally, a large patch of highly conserved, positively charged residues on the surface of GidA leading to the FAD-binding site suggests a tRNA-binding surface. We propose a model for the interaction between GidA and MnmE, which is supported by site-directed mutagenesis. Our data suggest that this interaction is modulated and potentially regulated by the switch function of the G domain of MnmE.     

Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11

Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.     

Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism

This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (α/β)₈ TIM-barrel domain; and (iii) the small (α + β) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (−4)(−3)(−2)(−1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)₆-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F_o − F_c omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.     

Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 A resolution: mechanistic implications

The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD phasing, model building, and crystallographic refinement to a resolution of 2.0 A. The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 A, and the model stereochemistry is satisfactory. The basic fold of the enzyme is that of a beta8/alpha8 (TIM) barrel. The barrel is centrally located, with an N-terminal alpha-helical domain flanking one side. An inserted beta-sheet domain folds against the opposite side of the barrel, and an alpha-helical C-terminal domain forms a plug which caps the active site. Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A, requiring Mg(2+). The structure reveals an enzyme-substrate complex with glyoxylate and Mg(2+) which coordinates the aldehyde and carboxylate functions of the substrate. Two strictly conserved residues, Asp631 and Arg338, are proposed to provide concerted acid-base chemistry for the generation of the enol(ate) intermediate of acetyl-coenzyme A, while main-chain hydrogen bonds and bound Mg(2+) polarize glyoxylate in preparation for nucleophilic attack. The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, with the electrophile activated for nucleophilic attack by nearby positive charges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A. An active site aspartate is, however, the only common feature of these two enzymes, and the active sites of these enzymes are produced by quite different protein folds. Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase.     

Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus

Beta-D-Xylosidases are glycoside hydrolases that catalyse the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicelluloses. beta-D-Xylosidases are found in glycoside hydrolase families 3, 39, 43, 52 and 54. The first crystal structure of a GH39 beta-xylosidase revealed a multi-domain organization with the catalytic domain having the canonical (beta/alpha)8 barrel fold. Here, we report the crystal structure of the GH39 Geobacillus stearothermophilus beta-D-xylosidase, inactivated by a point mutation of the general acid-base residue E160A, in complex with the chromogenic substrate molecule 2,5-dinitrophenyl-beta-D-xyloside. Surprisingly, six of the eight active sites present in the crystallographic asymmetric unit contain the trapped covalent glycosyl-enzyme intermediate, while two of them still contain the uncleaved substrate. The structural characterization of these two critical species along the reaction coordinate of this enzyme identifies the residues forming its xyloside-binding pocket as well as those essential for its aglycone recognition.     

Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) catalyzes the interconversion of D-glucose 6-phosphate (G6P) and D-fructose 6-phosphate (F6P) and plays important roles in glycolysis and gluconeogenesis. Biochemical characterization of the enzyme has led to a proposed multistep catalytic mechanism. First, the enzyme catalyzes ring opening to yield the open chain form of the substrate. Then isomerization proceeds via proton transfer between C2 and C1 of a cis-enediol(ate) intermediate to yield the open chain form of the product. Catalysis proceeds in both the G6P to F6P and F6P to G6P directions, so both G6P and F6P are substrates. X-ray crystal structure analysis of rabbit and bacterial PGI has previously identified the location of the enzyme active site, and a recent crystal structure of rabbit PGI identified Glu357 as a candidate functional group for transferring the proton. However, it was not clear which active site amino acid residues catalyze the ring opening step. In this paper, we report the X-ray crystal structure of rabbit PGI complexed with the cyclic form of its substrate, D-fructose 6-phosphate, at 2.1 A resolution. The location of the substrate relative to the side chains of His388 suggest that His388 promotes ring opening by protonating the ring oxygen. Glu216 helps to position His388, and a water molecule that is held in position by Lys518 and Thr214 accepts a proton from the hydroxyl group at C2. Comparison to a structure of rabbit PGI with 5PAA bound indicates that ring opening is followed by loss of the protonated water molecule and conformational changes in the substrate and the protein so that a helix containing amino acids 513-520 moves in toward the substrate to form additional hydrogen bonds with the substrate.     

Crystal structure and ligand binding properties of the truncated hemoglobin from Geobacillus stearothermophilus

A novel truncated hemoglobin has been identified in the thermophilic bacterium Geobacillus stearothermophilus (Gs-trHb). The protein has been expressed in Escherichia coli, the 3D crystal structure (at 1.5 Angstroms resolution) and the ligand binding properties have been determined. The distal heme pocket displays an array of hydrogen bonding donors to the iron-bound ligands, including Tyr-B10 on one side of the heme pocket and Trp-G8 indole nitrogen on the opposite side. At variance with the highly similar Bacillus subtilis hemoglobin, Gs-trHb is dimeric both in the crystal and in solution and displays several unique structural properties. In the crystal cell, the iron-bound ligand is not homogeneously distributed within each distal site such that oxygen and an acetate anion can be resolved with relative occupancies of 50% each. Accordingly, equilibrium titrations of the oxygenated derivative in solution with acetate anion yield a partially saturated ferric acetate adduct. Moreover, the asymmetric unit contains two subunits and sedimentation velocity ultracentrifugation data confirm that the protein is dimeric.     

Crystal structures of the Bacillus stearothermophilus CCA-adding enzyme and its complexes with ATP or CTP

CCA-adding enzymes polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. The 3.0 A resolution crystal structures of the CCA-adding enzyme from Bacillus stearothermophilus and its complexes with ATP or CTP reveal a seahorse-shaped subunit consisting of four domains: head, neck, body, and tail. The head is structurally homologous to the palm domain of DNA polymerase beta but has additional structural features and functions. The neck, body, and tail represent new protein folding motifs. The neck provides a specific template for the incoming ATP or CTP, whereas the body and tail may bind tRNA. Each subunit has one active site capable of switching its base specificity between ATP and CTP, an important component of the CCA-adding mechanism.     

Crystal structure of unsaturated glucuronyl hydrolase complexed with substrate: molecular insights into its catalytic reaction mechanism

Unsaturated glucuronyl hydrolase (UGL), which is a member of glycoside hydrolase family GH-88, is a bacterial enzyme that degrades mammalian glycosaminoglycans and bacterial biofilms. The enzyme, which acts on unsaturated oligosaccharides with an alpha-glycoside bond produced by microbial polysaccharide lyases responsible for bacterial invasion of host cells, was believed to release 4-deoxy-l-threo-5-hexosulose-uronate (unsaturated glucuronic acid, or DeltaGlcA) and saccharide with a new nonreducing terminus by hydrolyzing the glycosidic bond. We detail the crystal structures of wild-type inactive mutant UGL of Bacillus sp. GL1 and its complex with a substrate (unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in DeltaGlcA, based on the structural analysis of the enzyme-substrate complex and biochemical analysis. The proposed catalytic mechanism of UGL is a novel case among known glycosidases. Under the proposed mechanism, Asp-149 acts as a general acid and base catalyst to protonate the DeltaGlcA C4 atom and to deprotonate the water molecule. The deprotonated water molecule attacks the DeltaGlcA C5 atom to yield unstable hemiketal; this is followed by spontaneous conversion to an aldehyde (4-deoxy-l-threo-5-hexosulose-uronate) and saccharide through hemiacetal formation and cleavage of the glycosidic bond. UGL is the first clarified alpha(6)/alpha(6)-barrel enzyme using aspartic acid as the general acid/base catalyst.     

Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases

The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.     

Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 A. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.     

Crystal structure of activated ribulose-1,5-bisphosphate carboxylase complexed with its substrate, ribulose-1,5-bisphosphate

The three-dimensional structure of the complex of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, CO2, Mg2+, and ribulose bisphosphate has been determined with x-ray crystallographic methods to 2.6-A resolution. Ribulose-1,5-bisphosphate binds across the active site with the two phosphate groups in the two phosphate binding sites of the beta/alpha barrel. The oxygen atoms of the carbamate and the side chain of Asp-193 provide the protein ligands to the bound Mg2+ ion. The C2 and the C3 or C4 oxygen atoms of the substrate are also within the first coordination sphere of the metal ion. At the present resolution of the electron density maps, two slightly different conformations of the substrate, with the C3 hydroxyl group "cis" or "trans" to the C2 oxygen, can be built into the observed electron density. The two different conformations suggest two different mechanisms of proton abstraction in the first step of catalysis, the enolization of the ribulose 1,5-bisphosphate. Two loop regions, which are disordered in the crystals of the nonactivated enzyme, could be built into their respective electron density. A comparison with the structure of the quaternary complex of the spinach enzyme shows that despite the different conformations of loop 6, the positions of the Mg2+ ion, and most atoms of the substrate are very similar when superimposed on each other. There are, however, some significant differences at the active site, especially in the metal coordination sphere.     

Crystal structures of T. b. rhodesiense adenosine kinase complexed with inhibitor and activator: implications for catalysis and hyperactivation

Background

The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP) utilizing adenosine triphosphate (ATP) as the preferred phosphoryl donor.

Methods and Findings

Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK): the structure of TbrAK in complex with the bisubstrate inhibitor P¹,P⁵-di(adenosine-5′)-pentaphosphate (AP5A) at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) at 2.8 Å resolution.

Conclusions

The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.     

Crystal structures of Fms1 and its complex with spermine reveal substrate specificity

Fms1 is a rate-limiting enzyme for the biosynthesis of pantothenic acid in yeast. Fms1 has polyamine oxidase (PAO) activity, which converts spermine into spermidine and 3-aminopropanal. The 3-aminopropanal is further oxidized to produce beta-alanine, which is necessary for the biosynthesis of pantothenic acid. The crystal structures of Fms1 and its complex with the substrate spermine have been determined using the single-wavelength anomalous diffraction (SAD) phasing method. Fms1 consists of an FAD-binding domain, with Rossmann fold topology, and a substrate-binding domain. The active site is a tunnel located at the interface of the two domains. The substrate spermine binds to the active site mainly via hydrogen bonds and hydrophobic interactions. In the complex, C11 but not C9 of spermine is close enough to the catalytic site (N5 of FAD) to be oxidized. Therefore, the products are spermidine and 3-aminopropanal, rather than 3-(aminopropyl) 4-aminobutyraldehyde and 1,3-diaminoprone.     

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.     

Crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase complexed with cofactors: implications of a flexible loop movement upon substrate binding

The key enzyme in the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be an effective target of antimalarial drugs. Here we report the crystal structure of DXR complexed with NADPH and a sulfate ion from Escherichia coli at 2.2 A resolution. The structure showed the presence of an extra domain, which is absent from other NADPH-dependent oxidoreductases, in addition to the conformation of catalytic residues and the substrate binding site. A flexible loop covering the substrate binding site plays an important role in the enzymatic reaction and the determination of substrate specificity.