The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima.
Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.
Department of Chemistry, Butler University, Indianapolis, IN 46208, USA.
Matthew K
MK
Hedge
Alexandra M
AM
Gehring
Chinessa T
CT
Adkins
Leigh A
LA
Weston
Luke D
LD
Lavis
R Jeremy
RJ
Johnson
D000115:Acetylesterase / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D004952:Esters; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000115:Acetylesterase