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

Abstract

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.

Lignocellulases: a review of emerging and developing enzymes, systems, and practices

“…Contrary to the remark by Zhang (2011), Cota et al (2013) reported based on comparison between the recombinant protein yield and the hydrolytic activity achieved that the production of chimeric enzymes is more efficient (in terms of cost and catalytic efficiency) than wild-type proteins and could be more profitable in streamlining biomass conversion strategies than separate production of single enzyme. Cota et al (2013) further reported that the mode of operation of their chimera was exactly similar to that of the parental enzymes. Moreover, Moraïs et al (2012) reported that their designer cellulosome system from Thermobifida fusca exhibited "equal or superior" activity to that of the free system.…”

“…However, the successful expression of the essential cellulolytic enzymes (i.e., endoglucanases, exoglucanases, and β-glucosidases) on a single peptide chain, in a processive order, such that their proportionate quantities favor maximum hydrolysis efficiency has been highly challenging (Tozakidis et al 2016). Also, the determination of simple and reliable structural organization of the chimeric domains has been a significant drawback in the construction of a protein chimera, but the advent of small-angle X-ray scattering (SAXS) with flexible analytical models (e.g., molecular dynamics (MD) and Monte Carlo simulations) has provided not only successful computational data validation approaches, but also accurate fitting of the scattering profile due to their potential to explore the protein conformation in space (Cota et al 2013).…”

“…This may be due to factors such as changes in the microenvironments of the active sites, possible unproductive competition between functionally similar enzymes, difficulties in component arrangement as well as the nature of the peptide linker. Cota et al (2013) investigated and assembled a complex xylanase-lichenase (XylLich) chimera-both enzymes from Bacillus subtilis-through all-atom molecular dynamics simulations. Contrary to the remark by Zhang (2011), Cota et al (2013) reported based on comparison between the recombinant protein yield and the hydrolytic activity achieved that the production of chimeric enzymes is more efficient (in terms of cost and catalytic efficiency) than wild-type proteins and could be more profitable in streamlining biomass conversion strategies than separate production of single enzyme.…”

“…Cota et al (2013) investigated and assembled a complex xylanase-lichenase (XylLich) chimera-both enzymes from Bacillus subtilis-through all-atom molecular dynamics simulations. Contrary to the remark by Zhang (2011), Cota et al (2013) reported based on comparison between the recombinant protein yield and the hydrolytic activity achieved that the production of chimeric enzymes is more efficient (in terms of cost and catalytic efficiency) than wild-type proteins and could be more profitable in streamlining biomass conversion strategies than separate production of single enzyme. Cota et al (2013) further reported that the mode of operation of their chimera was exactly similar to that of the parental enzymes.…”

Clostridium thermocellum thermostable lichenase with circular permutations and modifications in the N-terminal region retains its activity and thermostability

“…An end-to-end fusion is frequently used when engineering bifunctional proteins to connect two enzymes [8,[13][14][15][16] using different approaches, for example, by optimization of peptide linkers [14] or through all-atom molecular dynamics simulations [16]. However, the chimeric enzymes constructed with the help of this method are frequently more susceptible to proteolytic degradation and structural instability [17].…”