RESUMEN
We solved the 1.8 Å crystal structure of ß-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of ß-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed ß-propeller and a C-terminal ß-sandwich module. The active site is located in the centre of the ß-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 ß-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 Å crystal structure of the ß-fructofuranosidase complex with ß-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.
Asunto(s)
Bifidobacterium/enzimología , beta-Fructofuranosidasa/química , Bifidobacterium/genética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Fructosa/metabolismoRESUMEN
X-ray structures of two compounds isolated from wood knots of coniferous trees, namely dihydrokaempferol (3,5,8,13-tetrahydroxyflavanon) and lariciresinol (3,14-dimetoxy-7,10-epoxylignan-4,15,19-triol), are presented here. Diffraction data for the Dihydrokaempferol crystals were collected on a CAD4 diffractometer and on a synchrotron for the lariciresinol crystal. The investigated compounds inhibit lipid peroxidation and lariciresinol is additionally a good scavenger of superoxide radicals. The structural data presented in this work provide a useful basis for designing more active compounds with potential use as antioxidants.
Asunto(s)
Abies/química , Flavonoides/química , Furanos/química , Lignanos/química , Fenoles/química , Populus/química , Europa (Continente) , Flavonoides/aislamiento & purificación , Furanos/aislamiento & purificación , Lignanos/aislamiento & purificación , Estructura Molecular , Fenoles/aislamiento & purificación , Polifenoles , Difracción de Rayos XRESUMEN
A complex of chagasin, a protein inhibitor from Trypanosoma cruzi, and papain, a classic family C1 cysteine protease, has been crystallized. Kinetic studies revealed that inactivation of papain by chagasin is very fast (k(on) = 1.5 x 10(6) M(-1) x s(-1)), and results in the formation of a very tight, reversible complex (K(i) = 36 pM), with similar or better rate and equilibrium constants than those for cathepsins L and B. The high-resolution crystal structure shows an inhibitory wedge comprising three loops, which forms a number of contacts responsible for the high-affinity binding. Comparison with the structure of papain in complex with human cystatin B reveals that, despite entirely different folding, the two inhibitors utilize very similar atomic interactions, leading to essentially identical affinities for the enzyme. Comparisons of the chagasin-papain complex with high-resolution structures of chagasin in complexes with cathepsin L, cathepsin B and falcipain allowed the creation of a consensus map of the structural features that are important for efficient inhibition of papain-like enzymes. The comparisons also revealed a number of unique interactions that can be used to design enzyme-specific inhibitors. As papain exhibits high structural similarity to the catalytic domain of the T. cruzi enzyme cruzipain, the present chagasin-papain complex provides a reliable model of chagasin-cruzipain interactions. Such information, coupled with our identification of specificity-conferring interactions, should be important for the development of drugs for treatment of the devastating Chagas disease caused by this parasite.
Asunto(s)
Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Papaína/química , Papaína/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/genética , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de los fármacos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/farmacología , Alineación de Secuencia , Especificidad por Sustrato , Trypanosoma cruzi/química , Trypanosoma cruzi/genéticaRESUMEN
Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.
Asunto(s)
Catepsina B/antagonistas & inhibidores , Cistatinas/química , Proteínas Protozoarias/química , Animales , Cistatinas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Proteínas Protozoarias/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Soluciones , Trypanosoma cruziRESUMEN
Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.