Two glucoamylases Glu and Gla produced by variant strains of yeast Saccharomycopsis fibuligera, HUT 7212 and KZ, respectively. The mature enzymes consist of 492 amino acid residues. An alignment of their amino acid sequences revealed a high homology, with seven amino acid residue alterations causing differences in the specific activity and thermal stability between both enzymes. Previous biochemical tests indicated that a significant part of both, Glu and Gla, renewed their catalytic activity after thermal denaturation. Glucoamylase Gla, in contrast with glucoamylase Glu, has lower catalytic activity but higher stability and better ability to re-nature [1].
Previous stability studies are complemented now by the study of thermal denaturation and determination of melting point using a method of dynamic light scattering. The method is based on the measurement of light scattered on the protein particles in the solution and determination of particle size. Melting point was defined as the temperature at which the size of the molecules rapidly increased due to thermal unfolding. DLS measurements revealed different behaviour of Gla and Glu enzymes during thermal denaturation.
Up to now, only tertiary structure of glucoamylase Glu in the complex with its inhibitors acarbose (PDB code 2F6D) or TRIS was determined (PDB code 1AYX, 2FBA) [2, 3]. In spite of the fact, that Gla and Glu are very similar, structure determination of glucoamylase Gla was not successful for a long time due to crystal twinning and/or low crystal quality. Finally, after further optimization of crystallization conditions, a diffraction quality crystal of Gla was obtained. A complete data set to 1.77 Å resolution was collected using synchrotron radiation at the X-13 beamline at EMBL, DORIS storage ring, DESY, Hamburg. The crystal was monoclinic with P21 space group and unit cell dimensions a = 66.5, b = 81.5, c = 83.4 Å ß = 109.94 °. There are two protein molecules in the asymmetric unit. For comparison, crystals of Glu prepared at very similar conditions, were orthorhombic with P212121 space group, and only one protein molecule in the asymmetric unit. The two molecules in Gla structure are arranged in head to tail manner with active sites on the opposite sides. Five of seven altered amino acid residues are located on the surface of the molecule and participate also in intermolecular contact formation. Intermolecular contacts between A and B molecule in the asymmetric unit as well as crystal contacts are analysed and compared with those in Glu structure. As expected, the presented tertiary structure of glucoamylase Gla is very close to that of Glu, however, the small changes on the molecular surface can be used for explanation of different physico-chemical properties of the two enzymes.
This work was supported by the Slovak Academy Research Grant Agency VEGA No. 2/0190/14.