Enhancing H2O2 resistance of an esterase from Pyrobaculum calidifontis by structure-guided engineering of the substrate binding site.

Green technologies are attracting increasing attention in industrial chemistry where enzymatic reactions can replace dangerous and environmentally unfriendly chemical processes. In situ enzymatic synthesis of peroxycarboxylic acid is an attractive alternative for several industrial applications although concentrated H2O2 can denature the biocatalyst, limiting its usefulness. Herein, we report the structure-guided engineering of the Pyrobaculum calidifontis esterase (PestE) substrate binding site to increase its stability and perhydrolysis activity. The L89R/L40A PestE mutant showed better tolerance toward concentrated H2O2 compared with wild-type PestE, and retained over 72% of its initial activity after 24-h incubation with 2 M H2O2. Surprisingly, the half-life (t 1/2, 80 °C) of PestE increased from 28 to 54 h. The k cat/K m values of the mutant increased 21- and 3.4-fold toward pentanoic acid and H2O2, respectively. This work shows how protein engineering can be used to enhance the H2O2 resistance and catalytic efficiency of an enzyme.

Adaptation to a high-tungsten environment: Pyrobaculum aerophilum contains an active tungsten nitrate reductase.

Nitrate reductases (Nars) belong to the DMSO reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits nitrate reductase (Nar) activity even at WO(4)(2-) concentrations that are inhibitory to bacterial Nars. In this report, we establish that the enzyme purified from cells grown with 4.5 µM WO(4)(2-) contains W as the metal cofactor but is otherwise identical to the Mo-Nar previously purified from P. aerophilum grown at low WO(4)(2-) concentrations. W is coordinated by a bis-molybdopterin guanine dinucleotide cofactor. The W-Nar has a 2-fold lower turnover number (633 s(-1)) but the same K(m) value for nitrate (56 µM) as the Mo-Nar. Quinol reduction and nitrate oxidation experiments monitored by EPR with both pure W-Nar and mixed W- and Mo-Nar preparations suggest a monodentate ligation by the conserved Asp241 for W(V), while Asp241 acts as a bidentate ligand for Mo(V). Redox titrations of the Mo-Nar revealed a midpoint potential of 88 mV for Mo(V/IV). The E(m) for W(V/IV) of the purified W-Nar was estimated to be -8 mV. This relatively small difference in midpoint potential is consistent with comparable enzyme activities of W- and Mo-Nars. Unlike bacterial Nars, the P. aerophilum Nar contains a unique membrane anchor, NarM, with a single heme of the o(P) type (E(m) = 126 mV). In contrast to bacterial Nars, the P. aerophilum Nar faces the cell's exterior and, hence, does not contribute to the proton motive force. Formate is used as a physiological electron donor. This is the first description of an active W-containing Nar demonstrating the unique ability of hyperthermophiles to adapt to their high-WO(4)(2-) environment.