Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site

Abstract

An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures.

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1

“…Nevertheless, compared to our previous studies on EstK and EstE1, 16,19,22 we found that EstSP1 has adopted the same strategy as hyperthermophilic EstE1 rather than cold-adapted EstK, showing that cold-adapted enzymes could be specifically categorized into two sets of enzymes, i.e., psychrophilic and psychrotrophic. Furthermore, it gives insight that instead of global flexibility the localized flexibility around active site is sufficient to render psychrophilic characters, which is evident from the mutational and comparative studies of EstSP1.…”

“…22 Additionally, the mutation of Tyr182 to a similar-sized Phe had little effect on EstE1 thermal stability, indicating that the hydrogen bond involving Tyr182 is critical for stabilization of the catalytic His loop but not for thermal stability of the enzyme, as the overall structure of EstE1 was already adapted to high temperatures. 22…”

“…The structural model of EstSP1 based on the crystal structure of a mesophilic esterase Est8 (PDB ID: 4YPV) 26 indicated that Tyr191 stabilizes the catalytic His loop in EstSP1 via a hydrogen bond with Ile287 (Figure 1b), similar to the mechanism of active-site stabilization previously shown for EstK, rPPE, and EstE1. 16,19−22 We utilized site-directed mutagenesis to generate mutations of Tyr191 to form aromatic amino acids with different characteristics (Phe and Trp), as well as a small aliphatic amino acid (Ala) and a charged amino acid (His). Although His is a basic amino acid, the imidazole group of His has an aromatic characteristic at all pH values.…”

Bacterial Hormone-Sensitive Lipases (bHSLs): Emerging Enzymes for Biotechnological Applications

“…In EstE1, two residues of Val and Phe were identified to be important for its dimerization [71]. In addition, Tyr in the wall of the active site was largely responsible for the thermal stability of EstE1 [94]. In Sto-Est, Arg was shown to be critical for maintaining dimer conformation [73].…”

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

“…The low rate of amino-acid conservation at position IV hints that residue X IV could affect the enzymatic activity, given that its side chain has a suitable conformation that does not interfere with the catalytic triad, with amino acids of variable side-chain lengths occurring at position IV, including glutamine [33] and tryptophan [34]. Indeed, residue X IV is suggested to be involved in the optimal configuration of the catalytic triad [32,35] and the enzymatic activity [36][37][38], to participate in substrate binding [15,30,[38][39][40][41] and to affect the thermostability [35] of ABH fold enzymes. Site-directed mutagenesis of residue X IV in different ABH fold enzymes has confirmed its significance in the enzymatic activity, resulting in a wide range of effects upon its replacement: from decreased [32,[35][36][37] and acute loss [38] of enzymatic activity, to improved activity [36,38,41] and increased thermostability [35].…”