Assembling a xylanase–lichenase chimera through all-atom molecular dynamics simulations

• Published in: Biochimica et biophysica acta Vol. 1834; no. 8; pp. 1492 - 1500
• Main Authors: Cota, Junio, Oliveira, Leandro C., Damásio, André R.L., Citadini, Ana P., Hoffmam, Zaira B., Alvarez, Thabata M., Codima, Carla A., Leite, Vitor B.P., Pastore, Glaucia, de Oliveira-Neto, Mario, Murakami, Mario T., Ruller, Roberto, Squina, Fabio M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2013
• Subjects: Bacillus subtilis; Bacillus subtilis - enzymology; biofuels; catalytic activity; chimerism; Computational characterization; Computer Simulation; Endo-1,4-beta Xylanases - chemistry; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - metabolism; engineering; enzymes; Experimental validation; genes; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Models, Molecular; Molecular dynamics; Molecular Dynamics Simulation; Multifunctional enzyme; polypeptides; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; recombinant proteins; Scattering, Small Angle; Small-angle X-ray scattering; solvents; surface area; temperature; X-radiation; Molecular dynamics; Experimental validation; Multifunctional enzyme; Small-angle X-ray scattering; Computational characterization
• ISSN: 1570-9639; 0006-3002; 1878-1454; 1878-2434
• DOI: 10.1016/j.bbapap.2013.02.030

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase–lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes. •A computational prediction of a multifunctional chimera disposal is proposed.•The methodology provides an important time saving tool for rational design.•The constructed chimera kept the catalytic properties of the wild-type proteins.•The model experimentally validated can reduce costs on enzyme production.