Crystal structure of α-1,4-glucan lyase, a unique glycoside hydrolase family member with a novel catalytic mechanism.

α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (ß/α)8-barrel catalytic domain with B and B' subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp(553) and Asp(665) are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite -1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.

Crystallization and preliminary X-ray analysis of carnein, a serine protease from Ipomoea carnea.

Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging-drop vapour-diffusion method. A data set was collected to 2.0 A resolution in-house from a single crystal at 110 K. The crystals belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 126.9, c = 84.6 A, alpha = beta = 90, gamma = 120 degrees. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46 A(3) Da(-1), corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress.

Crystal structure of the copper-containing quercetin 2,3-dioxygenase from Aspergillus japonicus.

Quercetin 2,3-dioxygenase is a copper-containing enzyme that catalyzes the insertion of molecular oxygen into polyphenolic flavonols. Dioxygenation catalyzed by iron-containing enzymes has been studied extensively, but dioxygenases employing other metal cofactors are poorly understood. We determined the crystal structure of quercetin 2,3-dioxygenase at 1.6 A resolution. The enzyme forms homodimers, which are stabilized by an N-linked heptasaccharide at the dimer interface. The mononuclear type 2 copper center displays two distinct geometries: a distorted tetrahedral coordination, formed by His66, His68, His112, and a water molecule, and a distorted trigonal bipyramidal environment, which additionally comprises Glu73. Manual docking of the substrate quercetin into the active site showed that the different geometries of the copper site might be of catalytic importance.

Structural and mutational characterization of the catalytic A-module of the mannuronan C-5-epimerase AlgE4 from Azotobacter vinelandii.

Alginate is a family of linear copolymers of (1-->4)-linked beta-d-mannuronic acid and its C-5 epimer alpha-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a right-handed parallel beta-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The beta-helix is composed of four parallel beta-sheets, comprising 12 complete turns, and has an amphipathic alpha-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp(152), an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr(149), His(154), and Asp(178) as being essential for activity. Tyr(149) probably acts as the proton acceptor, whereas His(154) is the proton donor in the epimerization reaction.

The X-ray structure of the haloalcohol dehalogenase HheA from Arthrobacter sp. strain AD2: insight into enantioselectivity and halide binding in the haloalcohol dehalogenase family.

Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheA(AD2) from Arthrobacter sp. strain AD2 at 2.0-A resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 shows that HheA(AD2) has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheA(AD2) can bind both enantiomers of the haloalcohol substrate 1-p-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheA(AD2). Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheA(AD2), in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.

Anaerobic enzyme.substrate structures provide insight into the reaction mechanism of the copper-dependent quercetin 2,3-dioxygenase.

Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic process, two CC bonds are broken and concomitantly carbon monoxide is released. The x-ray structures of Aspergillus japonicus 2,3QD anaerobically complexed with the substrate kaempferol and the natural substrate quercetin have been determined at 1.90- and 1.75-A resolution, respectively. Flavonols coordinate to the copper ion as monodentate ligands through their 3OH group. They occupy a shallow and overall hydrophobic cavity proximal to the metal center. As a result of a van der Waals contact between the most outward flavonol A-ring and Pro(164), a flexible loop in front of the active site becomes partly ordered. Interestingly, flavonols bound to 2,3QD are bent at the C2 atom, which is pyramidalized. The increased local sp(3) character at this atom may stabilize a carbon-centered radical activated for dioxygen attack. Glu(73) coordinates the copper through its O epsilon 1 atom. The short distance of about 2.55 A between its O epsilon 2 atom and the flavonol O3 atom suggests that a hydrogen bond exists between the two atoms, indicating that Glu(73) can act as a base in flavonol deprotonation and that it retains the proton. Structure-based geometric considerations indicate O(2) binding to the flavonol C2 atom as the preferred route for flavonol dioxygenation.

Crystal structure and carbohydrate-binding properties of the human cartilage glycoprotein-39.

The human cartilage glycoprotein-39 (HCgp-39 or YKL40) is expressed by synovial cells and macrophages during inflammation. Its precise physiological role is unknown. However, it has been proposed that HCgp-39 acts as an autoantigen in rheumatoid arthritis, and high expression levels have been associated with cancer development. HCgp-39 shares high sequence homology with family 18 chitinases, and although it binds to chitin it lacks enzymatic activity. The crystal structure of HCgp-39 shows that the protein displays a (beta/alpha)8-barrel fold with an insertion of an alpha + beta domain. A 43-A long carbohydrate-binding cleft is present at the C-terminal side of the beta-strands in the (beta/alpha)8 barrel. Binding of chitin fragments of different lengths identified nine sugar-binding subsites in the groove. Protein-carbohydrate interactions are mainly mediated by stacking of side chains of aromatic amino acid residues. Surprisingly, the specificity of chitin binding to HCgp-39 depends on the length of the oligosaccharide. Although chitin disaccharides tend to occupy the distal subsites, longer chains bind preferably to the central subsites in the groove. Despite the absence of enzymatic activity, long chitin fragments are distorted upon binding, with the GlcNAc at subsite -1 in a boat conformation, similar to what has been observed in chitinases. The presence of chitin in the human body has never been documented so far. However, the binding features observed in the complex structures suggest that either chitin or a closely related oligosaccharide could act as the physiological ligand for HCgp-39.

Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer.

Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 A resolution. It comprises two domains. The N-terminal domain has a beta-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an alpha-helical type I cytochrome c with His(608) and Met(647) as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 A, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.

Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1.

Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop vapour-diffusion experiments against 50-70% saturated ammonium sulfate solution at pH 6.5-7.3. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 104.5, c = 121.4 A, and contain two monomers in the asymmetric unit. The crystals diffract to 3.0 A resolution with X-rays from a Cu Kalpha rotating-anode generator.