Divergent Evolution of Ligand Binding in the o-Succinylbenzoate Synthase Family

摘要

Thermobifida fusca o-succinylbenzoate synthase (OSBS), a member of the enolase superfamily that catalyzes a step in menaquinone biosynthesis, shares 22% and 28% amino acid sequence identity with two previously characterized OSBS enzymes from Escherichia coli and Amycolatopsis sp. T-1-60, respectively. These values are considerably lower than typical sequence identities among homologous proteins that have the same function. To determine how such divergent enzymes catalyze the same reaction, we solved the structure of T. fusca OSBS and identified amino acids that are important for ligand binding. We discovered significant differences in structure and conformational flexibility between T. fusca OSBS and other members of the enolase superfamily. In particular, the 20s loop, a flexible loop in the active site that permits ligand binding and release in most enolase superfamily proteins, has a four-amino acid deletion and is well ordered in T. fusca OSBS. Instead, flexibility of a different region allows the substrate to enter from the other side of the active site. T. fusca OSBS was more tolerant of mutations at residues that were critical for activity in E. coli OSBS. Also, replacing active site amino acids found in one protein with the amino acids that occur at the same place in the other protein reduces catalytic efficiency. Thus, the extraordinary divergence between these proteins does not appear to reflect a higher tolerance of mutations. Instead, large deletions outside the active site were accompanied by alteration of active site size and electrostatic interactions, resulting in small but significant differences in ligand binding.