Residues required for activity in Escherichia coli o-succinylbenzoate synthase (OSBS) are not conserved in all OSBS enzymes.

Understanding how enzyme specificity evolves will provide guiding principles for protein engineering and function prediction. The o-succinylbenzoate synthase (OSBS) family is an excellent model system for elucidating these principles because it has many highly divergent amino acid sequences that are <20% identical, and some members have evolved a second function. The OSBS family belongs to the enolase superfamily, members of which use a set of conserved residues to catalyze a wide variety of reactions. These residues are the only conserved residues in the OSBS family, so they are not sufficient to determine reaction specificity. Some enzymes in the OSBS family catalyze another reaction, N-succinylamino acid racemization (NSAR). NSARs cannot be segregated into a separate family because their sequences are highly similar to those of known OSBSs, and many of them have both OSBS and NSAR activities. To determine how such divergent enzymes can catalyze the same reaction and how NSAR activity evolved, we divided the OSBS family into subfamilies and compared the divergence of their active site residues. Correlating sequence conservation with the effects of mutations in Escherichia coli OSBS identified two nonconserved residues (R159 and G288) at which mutations decrease efficiency ≥200-fold. These residues are not conserved in the subfamily that includes NSAR enzymes. The OSBS/NSAR subfamily binds the substrate in a different orientation, eliminating selective pressure to retain arginine and glycine at these positions. This supports the hypothesis that specificity-determining residues have diverged in the OSBS family and provides insight into the sequence changes required for the evolution of NSAR activity.

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: improved thermostability by a single-isoleucine mutant.

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability.

Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.