Structure-function studies of two polysaccharide-degrading enzymes: Bacillus stearothermophilus α-amylase and Trichoderma reesei cellobiohydrolase II

Amylases and cellulases are important enzymes both for the global carbon cycle on earth and for biotechnical applications. They are capable of degrading polysaccharides, which are chemically simple polymers of repeating glucose units that form very complex and water-insoluble macroscopic structures. The enzymatic degradation of starch and cellulose is poorly understood at the molecular level. The cloning and DNA sequence determination of amylaseand cellulase-encoding genes from various organisms allows the use of modern techniques of molecular biology to study and alter their function. In the present investigation two different approaches were used to study the relationship between enzyme structure and function. In the first of these a random mutagenesis method developed in this study was applied. This method is applicable even without structural knowledge of a protein. The complete Bacillus stearothermophilus α-amylase gene was subjected to random mutagenesis, which was optimised to produce a single amino acid change at a time in the corresponding enzyme. Nearly 100 different mutant α-amylases were produced in an Escherichia coli host. The mutated enzymes had different activities on substrate plates used in screening. The location of the mutation(s) was analysed by sequencing the mutated gene. A molecular model of Bacillus stearothermophilus α-amylase was constructed on the basis of sequence alignment and the known structure of a fungal α-amylase from Aspergillus oryzae. The protein is expected to fold into three domains (A, B and C), like many other α-amylases. The data obtained from the analysis of the random mutants was compared to the constructed three-dimensional model. A reasonably good overall correlation was obtained between the mutant data and the model. Two areas on the α-amylase structural model were identified as being important for the activity on polymeric substrate: the open active site cleft situated between domains A and B and containing conserved amino acids known to be important for catalysis and multiple binding of glucose units in other α-amylases; and an interface between the catalytic domain A and domain C about 30 Å away from the active site groove. The three-dimensional structure of Trichoderma reesei cellobiohydrolase II (CBHII) catalytic domain has been determined. The catalytic domain has an α/β barrel fold similar to that of the α-amylase catalytic domain A. Two stable surface loops generate a 20 Å long tunnel for substrate binding and catalysis. The active site tunnel contains four defined binding sites (A-D) for glucosyl units. In the