1 research outputs found
Hotspots and Mechanisms of Action of the Thermostable Framework of a Microbial Thermolipase
The lipase TrLipB from Thermomicrobium
roseum is highly thermostable. However, its thermostable
skeleton and mechanism
of action should be investigated for industrial applications. Toward
this, TrLipB was crystallized using the hanging-drop vapor diffusion
method and subjected to X-ray diffraction at 2.0 Ã… resolution
in this study. The rigid sites, such as the prolines on the relatively
flexible loops on the enzyme surface, were scanned. Soft substitutions
of these sites were designed using both molecular dynamics (MD) simulation
and site-directed mutagenesis. The thermostability of several substitutions
decreased markedly, while the catalytic efficiencies of the P9G, P127G,
P194G, and P300G mutants reduced substantially; additionally, the
thermostable framework of the double mutant, P194G/P300G, was considerably
perturbed. However, the substitutions on the lid of the enzyme, including
P49G and P48G, promoted the catalytic efficiency to approximately
150% and slightly enhanced the thermostability below 80 °C. In
MD simulations, the P100G, P194G, P100G/P194G, P194G/P300G, and P100G/P194G/P300G
mutants showed high B-factors and RMSD values, whereas the secondary
structures, radius of gyration, H-bonds, and solvent accessible surface
areas of these mutants were markedly affected. Our observations will
assist in understanding the natural framework of a stable lipase,
which might contribute to its industrial applications