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Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function–in particular towards large substrates. [ABSTRACT FROM AUTHOR]

The attractiveness of lipases as industrial biocatalysts underlines the importance of understanding their molecular action in detail, helping future efforts to improve performance and applicability. Lipases represent a special class of carboxyl ester hydrolases, which act on long-chained water insoluble acyl-glycerols at the water-lipid interface. This heterogeneous catalysis makes it very difficult to monitor the structural changes taking place in the enzyme during activation in its preferred milieu. In this review, we cover recent developments toward a deeper understanding of the interfacial activation phenomenon of lipases and discuss a selection of biophysical methods suitable for studying lipase activation. For many lipases, such as that from Thermomyces lanuginosus, a structural domain called the 'lid' is directly involved in the activation process. This has been shown through alteration of the lid-residue composition using rational design. The resulting lid-mutants have highlighted the lid's role in governing enzymatic activity and interfacial activation. Moreover, a recently developed fluorescence-based technique called the Tryptophan Induced Quenching method has proved to enable the direct measurement of lipase activation in water-ethanol mixtures, and thus represents an intriguing method to gain further insight into the structural movements taking place at the water-lipid interface. Practical applications: Lipases are important industrial biocatalysts widely used in industrial applications including detergent, leather, paper, fuel, fine chemicals, baking, cosmetics, and food. Knowledge of structural changes of lipase upon activation and identification of the amino acids involved in lipase activation is crucial for design of new variants with improved or new properties. Rational design of the lid-region in lipases has an interesting potential for optimization of lipase function and adapting them to different environments. In order to leverage on the design of new lipase variants and their altered function, methods that enable direct measurement of lipase action directly in solution, in real time, are desired. In this context, fluorescence based methods have shown great potential and provides interesting perspectives for probing the delicate movements occurring in lipases upon activation. Interfacial activation of lipases and the lid movements studied by rational design, fluorescence quenching methods, and solvent effect - polarity driven activation. [ABSTRACT FROM AUTHOR]

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) as only two 113Cd signals were found in 113Cd2113Cd2SOD. The difference between the chemical shifts of 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase. [ABSTRACT FROM AUTHOR]