Sanna Auer

Structure Function Studies of a Melanocarpus albomyces Laccase Suggest a Pathway for Oxidation of Phenolic Compounds.

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure.

The C‐terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three‐dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C‐terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C‐terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C‐terminus, site‐directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C‐terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL559 was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C‐terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C‐terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C‐terminus of MaL.

The Tryptophan Residue at the Active Site Tunnel Entrance of Trichoderma reesei Cellobiohydrolase Cel7A Is Important for Initiation of Degradation of Crystalline Cellulose

Background: Mutation of Trp-40 in the Cel7A cellobiohydrolase from Trichoderma reesei (TrCel7A) causes a loss of crystalline cellulose-degrading ability. Results: Mutant W40A showed reduced specific activity for crystalline cellulose and diffused the cellulose chain from the entrance of the active site tunnel. Conclusion: Trp-40 is essential for chain end loading to initiate processive hydrolysis of TrCel7A. Significance: The mechanisms of crystalline polysaccharide degradation are clarified. The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain (CBD) are both necessary for full activity on crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on crystalline cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite −7. However, when similar simulations were carried out starting from subsite −5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between crystalline and amorphous celluloses. The CBD is important for enhancing the enzyme population on crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the cellulose surface and/or hindering processive hydrolysis.

CMOS-Integrated Film Bulk Acoustic Resonators for Label-Free Biosensing

The throughput is an important parameter for label-free biosensors. Acoustic resonators like the quartz crystal microbalance have a low throughput because the number of sensors which can be used at the same time is limited. Here we present an array of 64 CMOS-integrated film bulk acoustic resonators. We compare the performance with surface plasmon resonance and the quartz crystal microbalance and demonstrate the performance of the sensor for multiplexed detection of DNA.

An antibody surface for selective neuronal cell attachment

An optimal surface for culturing human embryonic stem cell (hESC)-derived neuronal cells is of high interest. In this study, a specific antibody to a neural cell adhesion molecule (NCAM) was immobilised on a solid surface of polystyrene and used as a selective matrix for culturing of hESC-derived neuronal cells. Thereafter, hESC-derived neurospheres were seeded on the matrix. The neurospheres did not attach to the NCAM antibody containing matrix whereas individual neuronal cells did. The neuronal cell attachment was depended on the NCAM antibody concentration. The neuronal cells were viable on the NCAM antibody containing matrix during an 8 day follow-up and exhibited typical bipolar morphology of immature neurons. Specific binding of the NCAM antigen to an immunoglobulin-polymer coated surface was verified by surface plasmon resonance (SPR) measurements. This study is to our knowledge the first demonstrating the use of an antibody layer as a selective surface for hESC-derived neuronal cells.