Structural Studies of Enzyme Directed Evolution

The previous sections described structural studies of antibody maturation as a method to understand the evolution of binding (and catalysis) in the immune system. The technique of directed evolution parallels the process of affinity maturation. Both methods use random mutagenesis and gene shuffling, followed by screening and/or selection to identify mutants with the desired function. In contrast to affinity maturation, 

Structural Analysis of Adaptive Mechanisms to Evolutionary Pressures 

Bacillus subtilis /zNB esterase is a member of the a./(3 hydrolase fold family (Moore and Arnold, 1996 Ollis et al., 1992). The canonical a/j3 hydrolase fold consists of a mostly parallel eight-stranded [3 sheet surrounded on both sides by a helices (Nardini and Dijkstra, 1999). p B esterase contains 489 amino acids arranged in a central thirteen-stranded f3 sheet that is surrounded by fifteen a helices (Fig. 12, see color insert). Similar to the structure of acetylcholine esterase (Sussman et al., 1991), a large fraction of the pSB esterase structure has no defined secondary structure (52% random coil, 33% a helix, and 14% /3 sheet). This high degree of random coil structure is allowed in the a/(3 hydrolase fold, where large insertions in loops were found to be tolerated while still maintaining catalytic activity (Nardini and Dijkstra, 1999). 

56G8 Mutations Location in structure Generation Proposed role for 5-6C8 mutations 

L144M buried, surface loop 3 Reorients helix 6, which stabilizes the 265-275 loop. Interacts with loops also affected by 

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