Experimental and Modeling Studies of an Unusual Water-Filled Pore Structure with Possible Mechanistic Implications in Family 48 Cellulases

摘要: Molecular dynamics simulations were used to study the possible catalytic role of an unusual conserved water-filled pore structure in family 48 cellulase enzyme Cel48A from Thermobifida fusca. It was hypothesized that this serves as pathway for water molecules consumed hydrolysis catalyzed by reach active site a continuous stream participate processive reactions. Theoretical mutants created which all residues lining made hydrophobic, had effect molecular emptying and preventing any passing through on simulation time scale. Mutants with smaller numbers substitutions nature, could be experimentally site-directed mutagenesis, also identified simulations, these proteins subsequently produced Escherichia coli, expressed purified, but found not fold manner similar wild type protein, determination importance activity. is presence small vacuum responsible instability mutants. In addition, alternate pathways observed would allow enzyme, suggesting hypothesis has functional significance might incorrect.