Structure and Mechanism of a Cold-Adapted Bacterial Lipase

Abstract

The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic–psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus , which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability.

Biophysical characterization and analysis of a mesophilic chorismate mutase fromB. pumilus

“…Compared to the related chorismate mutase enzyme from B. subtilis (BsCM), which shares a 69% sequence identity and 93% similarity to BpCM, the thermodynamic activation parameters also indicate that like BsCM, BpCM is likely a mesophilic enzyme, despite other enzymes from B. pumilus exhibiting psychrophilic behavior. 35, 36 The activation free energy for BpCM is less than 1 kcal mol -1 lower than reported values for BsCM, both experimentally and computationally. The activation enthalpies of both enzymes are the same within reported errors, thus the difference in Δ G ‡ is likely a result of BpCM having a lower T Δ S ‡ of about 0.6 kcal mol -1 at 298 K.…”

“…The thermodynamic activation parameters predicted from the Arrhenius plot approach applied to both simulation and experimental results are in excellent agreement with each other. 35,36 The activation free energy for BpCM is less than 1 kcal mol -1 lower than reported values for BsCM, both experimentally and computationally. The activation enthalpies of both enzymes are the same within reported errors, thus the difference in ∆G ‡ is likely a result of BpCM having a lower T∆S ‡ of about 0.6 kcal mol -1 at 298 K.…”

A Multienzyme Cascade Pathway Immobilized in a Hydrogen‐Bonded Organic Framework for the Conversion of CO2

“…In addition, enzymes are susceptible to the effects of temperature thus leading to the loss of enzyme activity. [22] Figure 6b shows that FFFP@HOF-101 maintains a cascade catalytic efficiency of more than 77% despite temperatures up to 50 °C. In contrast, the cascade catalytic efficiency of the free FFFP pathway at 50 °C was below 20%.…”

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues

“…The need to establish the specific features that aid their catalytic functions at low temperatures compared to their mesophilic and thermophilic counterparts makes it necessary to analyse the available data on these lipolytic enzymes. The general catalytic mechanism of lipase and esterase is that of serine hydrolases enzyme that involves a nucleophilic attack on the substrate during the acylation step, which forms a covalent complex of enzyme and substrate, followed by the diacylation step in which the enzyme-substrate complex is hydrolysed by a molecule of water [ 128 , 129 ].…”

“…In a recent study on the structural basis of cold-adaptation of two orthologous mesophilic-psychrophilic bacterial lipases, van der Ent, Lund [ 128 ] observed a limited number of mutations (34 out of 181 residues) that were responsible for their thermal adaptation. Only single amino acid was found close to the substrate binding site, and the remaining mutations were found farther away on the enzyme surface.…”