The bacterial hydrolase FTT258 from the highly pathogenic bacterium Francisella tularensis exists in two distinct open and closed conformations. Based on comparison to the human homologue (human acyl protein thioesterase), this large structural rearrangement between the open and closed forms was proposed to control the catalytic activity and membrane binding activity of FTT258.  The objective of this study was to determine the specific amino acid residues in the flexible �3 loop that control this conformational change. To evaluate this objective, we conducted an alanine mutagenesis scan of each of the seven residues in the �3 loop and compared the enzyme activity and membrane-binding activity for each of the FTT258 variants. Based on kinetic measurements, the central tryptophan residue (Trp66) is essential to the enzyme activity of FTT258, because the resulting protein variant causes a near complete loss in enzyme activity. Other amino acids localized near the essential Trp (Met63, Arg64, Tyr67, and Asp68) also significantly decreased the hydrolase activity of FTT258, indicating a critical target area for controlling the enzyme activity and likely structural rearrangement. Combinatorial variants containing the Trp66 and Tyr67 substitutions with the other important residues showed enzyme activity similar to the Trp66 variant alone, suggesting a controlling role for this residue. Membrane-binding experiments with each of these variants failed to show significant changes from the wild-type protein. The compilation of our results indicate that the main role of this conformational change of FTT258 is related to controlling enzyme activity and not membrane binding.

Membrane binding of FTT258, a bacterial carboxylesterase from <i>F. tularensis</i>

248th National Meeting (2014)

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Membrane binding of FTT258, a bacterial carboxylesterase from F. tularensis