J. Sanz-Aparicio

Structural basis of specificity in tetrameric Kluyveromyces lactis β-galactosidase.

β-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli β-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a β-galactosidase used in food processing.

Three-dimensional Structure of Saccharomyces Invertase ROLE OF A NON-CATALYTIC DOMAIN IN OLIGOMERIZATION AND SUBSTRATE SPECIFICITY

Background: Invertase is a fundamental enzyme for sugar metabolism in yeast and a classical model in early biochemical studies. Results: Invertase shows an unusual octameric quaternary structure composed of two types of dimers. Conclusion: A peculiar pattern of monomer assembly through non-catalytic domain interactions determines invertase specificity. Significance: Unraveling the structural features that rule enzyme modularity casts new light on protein-carbohydrate recognition. Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition.

Addition compounds of dichlorodioxomolybdenum(VI) from hydrochloric acid solutions of molybdenum trioxide. Crystal structure of dichlorodioxodiaquamolybdenum(VI) bis(2,5,8-trioxanonane)

Abstract The behaviour of hydrochloric acid solutions of MoO3 with some common solvents has been explored. MoO2Cl2(H2O)2 separates from solution stabilized as polyether solvates. The crystal structure of [MoO2Cl2(H2O)2(2,5,8-trioxanonane)2] presents octahedral MoO2Cl2(H2O)2 units linked to the polyether chains through hydrogen bonds involving the water molecules. The polyether molecules show a cis-cis conformation.

Structural analysis of Saccharomyces cerevisiae alpha-galactosidase and its complexes with natural substrates reveals new insights into substrate specificity of GH27 glycosidases.

α-Galactosidases catalyze the hydrolysis of terminal α-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae α-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 Å resolution. The monomer folds into a catalytic (α/β)8 barrel and a C-terminal β-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the β-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.

Structural Basis of Increased Resistance to Thermal Denaturation Induced by Single Amino Acid Substitution in the Sequence of Beta-Glucosidase a from Bacillus Polymyxa.

The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. β‐glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well‐characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (α/β)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random‐mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X‐ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N‐terminus of helix α2, to Asp28, located in one of the loops surrounding the active‐site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far‐reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions. Proteins 33:567–576, 1998.

Active-site-mutagenesis study of rat liver betaine-homocysteine S-methyltransferase.

A site-directed-mutagenesis study of putative active-site residues in rat liver betaine-homocysteine S-methyltransferase has been carried out. Identification of these amino acids was based on data derived from a structural model of the enzyme. No alterations in the CD spectra or the gel-filtration chromatography elution pattern were observed with the mutants, thus suggesting no modification in the secondary structure content or in the association state of the proteins. All the mutants obtained showed a reduction of the enzyme activity, the most dramatic effect being that of Glu(159), followed by Tyr(77) and Asp(26). Changes in affinity for either of the substrates, homocysteine or betaine, were detected when substitutions were performed of Glu(21), Asp(26), Phe(74) and Cys(186). Interestingly, Asp(26), postulated to be involved in homocysteine binding, has a strong effect on affinity for betaine. The relevance of these results is discussed in the light of very recent structural data obtained for the human enzyme.

Structural Analysis of Glucuronoxylan-specific Xyn30D and Its Attached CBM35 Domain Gives Insights into the Role of Modularity in Specificity.

Background: Xylanases are crucial in plant cell wall recycling. Results: A glucuronoxylan-specific xylanase is attached to its binding module with moderate flexibility. This CBM35 displays novel structural features regulating specificity. Conclusion: Depolymerization of highly substituted xylans and an oriented interaction with its target substrate are proposed. Significance: Unraveling the mechanisms ruling modularity is essential to understanding the biomass deconstruction and to producing efficient biocatalysts. Glucuronoxylanase Xyn30D is a modular enzyme containing a family 30 glycoside hydrolase catalytic domain and an attached carbohydrate binding module of the CBM35 family. We present here the three-dimensional structure of the full-length Xyn30D at 2.4 Å resolution. The catalytic domain folds into an (α/β)8 barrel with an associated β-structure, whereas the attached CBM35 displays a jellyroll β-sandwich including two calcium ions. Although both domains fold in an independent manner, the linker region makes polar interactions with the catalytic domain, allowing a moderate flexibility. The ancillary Xyn30D-CBM35 domain has been expressed and crystallized, and its binding abilities have been investigated by soaking experiments. Only glucuronic acid-containing ligands produced complexes, and their structures have been solved. A calcium-dependent glucuronic acid binding site shows distinctive structural features as compared with other uronic acid-specific CBM35s, because the presence of two aromatic residues delineates a wider pocket. The nonconserved Glu129 makes a bidentate link to calcium and defines region E, previously identified as specificity hot spot. The molecular surface of Xyn30D-CBM35 shows a unique stretch of negative charge distribution extending from its binding pocket that might indicate some oriented interaction with its target substrate. The binding ability of Xyn30D-CBM35 to different xylans was analyzed by affinity gel electrophoresis. Some binding was observed with rye glucuronoarabinoxylan in presence of calcium chelating EDTA, which would indicate that Xyn30D-CBM35 might establish interaction to other components of xylan, such as arabinose decorations of glucuronoarabinoxylan. A role in depolymerization of highly substituted chemically complex xylans is proposed.

Structural and spectroscopic study of condensed piperidine bicyclanols. 3-Phenethyl-3-azabicyclo[3.2.1]octan-8-α-ol

Abstract The IR and 1 H and 13 C NMR spectra of 3-phenethyl-3-azabicyclo[3.2.1]octan-8-α-ol have been studied in several media, and its crystal structure has been determined by X-ray diffraction. The bicyclic system adopts a chair—envelope conformation with both OH and phenethyl groups in equatorial positions with respect to the piperidine ring. The existence of OH⋯N intermolecular hydrogen bonding in the solid state has been revealed by X-ray and IR data. The results obtained are compared with those previously found for the corresponding β-epimer.

Synthesis and structural study of 9-(2′-hydroxyethyl)-9-azabicyclo[3.3.1]nonan-3α-OL

Abstract The infrared, 1 H and 13 C NMR spectra of 9-(2′-hydroxyethyl)-9-azabicyclo[3.3.1] nonan-3α-ol (I) have been examined in several media. The crystal structure has been determined by X-ray diffraction. The bicycle system adopts in solid state a distorted double chair conformation, being the OH and 2′-hydroxyethyl group attached in axial position with respect to the piperidinol ring. In solution, by the contrary, the title compound adopts a preferred flattened chair-boat conformation with the piperidinol ring in a slight distorted boat form. The unambiguous assignment of all protons of the granatanine system, not described up to date, has been carried out.

Structural and conformational study of 9-(2′-hydroxyethyl)-9-azabicyclo[3.3.1]nonan-3β-ol

Abstract The infrared, 1H and 13C-NMR spectra of 9-(2′-hydroxyethyl)-9-azabicyclo[3.3.1]nonan-3β-ol (I) have been examined in several media. To assist in interpretation of the spectroscopic data, the crystal structure has been determined by X-ray diffraction. The bicyclic system adopts a flattened chair-chair conformation with OH and 2′-hydroxyethyl groups in equatorial and axial position with respect to the piperidinol ring. The crystal structure is stabilized by means of and intermolecular hydrogen bondings. In CDCl3 solution the title compound can be described as an equilibrium between two flattened chair-chair conformations through nitrogen inversion. A slight predominance of the form with the N-substituent axial with respect to the piperidine ring is suggested.