A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase

Abstract

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k 1 , k ؊1 , k 2 , and k 3 ) along with activation energies (E 1 , E ؊1 , E 2 , and E 3 ) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from k cat ‫؍‬ k 3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to k cat ‫؍‬ k 2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyland alcohol-binding sites. Esterase 2 (EST2)1 from the thermophilic eubacterium Alicyclobacillus acidocaldarius is a thermostable serine hydrolase belonging to the hormone-sensitive lipase family of the esterase/lipase superfamily (1-3). Members of this family are ubiquitous (see the ESTHER database), comprise true lipases and amidases other than carboxylesterases, and are still poorly characterized. For some of them a role in lipid catabolism or detoxification has been proposed (4, 5). EST2 hydrolyzes monoacyl esters of different acyl chain length, triglycerides of short acyl chain, and different compounds of pharmacological and industrial interest (3, 6). The est2 gene has been overexpressed in Escherichia coli, and the protein, a monomeric B-type carboxylesterase of about 34 kDa, was purified and characterized. The enzyme displays an optimal temperature at 70°C and maximal activity with p-nitrophenyl (pNP) esters having an acyl chain of six to eight carbon atoms (3). Biochemical and mutagenic studies have allowed the identification of residues of the catalytic triad and oxyanion hole (7).The EST2 crystal structure was recently solved by our group as a covalently bound Hepes adduct (8), thus making it possible to obtain an unambiguous view of the active site and to detect the binding pockets for the acyl and alcohol chains of the ester substrate. On the basis of these structural data, EST2 variants with preferential specificity toward monoacyl esters with acyl chain length greater than eight carbon atoms were designed and generated by site-directed and saturation mutagenesis (9). M211S and R215L mutants and M211S/R215L double mutant were obtained, all having the desired properties in a mixed propan-2-ol/water medium (9). In particular, a 6-fold increase of the specificity constant of the double mutant for pNP-dodecanoate was o...

Crystal structure of an S‐formylglutathione hydrolase from Pseudoalteromonas haloplanktis TAC125

“…By contrast, a very small cavity, bordered by residues Leu54, Ile173, Trp181, Phe227, and Leu232 is located close to this catalytic serine. The structural superposition of PhEst with other esterases in complex with substrate analogues 17,18 allowed the cavity to be identified as the acyl binding pocket and the cleft as the alcohol/thiol binding pocket of the ester substrate. Interestingly, as already observed for other FGHs whose three dimensional structure has been solved, the acyl binding pocket is very small and allows for the accommodation of substrates with very short acyl chains, like formyl-or acetylesters.…”

Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius

“…Its catalytic triad has been identified (i.e., Ser155, Asp252 and His282) [4]; the enzymatic activity [2,5], and the conformational stability against temperature and chemical denaturants [6][7][8] have been investigated in detail. The 3D structure of EST2 has been solved by X-ray diffraction to 2.6 Å resolution, PDB code 1EVQ [9,10], and is shown in Figure 1a. The protein possesses the classical α/β hydrolase fold with a central β-sheet, consisting of eight strands highly twisted, surrounded by nine α-helices; a topology model of the structure is shown in Figure 1b.…”

A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but No Transferase Activity

“…In both esterases and HTAs, the catalytic triad performs catalysis, and residues in the oxyanion hole are involved in substrate binding (7,8,13,18). In the structures of both wild-type Est22 and the L374D mutant, the three catalytic residues, i.e., Ser 175 , Asp 340 , and His 373 , form a hydrogen bonding network.…”

“…During catalysis, the catalytic Ser residue acts as a nucleophile to attack the carbonyl carbon of the susceptible ester and the Asp/Glu residue interacts with the His residue to facilitate proton transfer from the catalytic Ser to His, thereby enhancing the nucleophilicity of Ser (5,6). At least two residues spatially close to the catalytic Ser residue are involved in the formation of the oxyanion hole, and their main-chain nitrogen atoms stabilize the acyl-enzyme intermediate in the reaction (7,8). The catalytic cavity of esterases also contains other residues that form interactions with substrates to orientate them.…”