Site-directed mutagenesis of a novel serine arylesterase from Vibrio mimicus identifies residues essential for catalysis.

Site-directed mutagenesis (SDM) of an arylesterase (the arylesterase) from Vibrio mimicus revealed that residues S29, H153, and D96 constituted a catalytic triad. The use of a serine residue for ester hydrolysis by the arylesterase proves that the enzyme is a novel serine arylesterase. SDM also showed that D28 was necessary for the esterase activity; to our knowledge it is the first time that a residue immediately preceding the active-site serine in esterases was shown biochemically to possess such a property. The results further suggest that D28 plays a role in substrate-binding. Residue 31 was firmly shown to participate in the binding of N-acetyl-D, L-phenylalanine beta-naphthyl ester (NAPNE), an artificial substrate for chymotrypsin. The S31G enzyme showed a 4 fold decrease in the Km for NAPNE over that of wild type enzyme, proving residue 31 is important for substrate-specificity. A mechanism for binding and catalysis of esters by the arylesterase is proposed, which includes the unique role of S31 for aromatic (hydrophobic) acyl-binding. The biochemical properties of the arylesterase suggest that the enzyme stands out as a member of a distinct subfamily within a recently proposed, lipolytic enzyme family.