Computational Design of Oligosaccharide-Producing Levansucrase from Bacillus licheniformis RN-01 to Increase Its Stability at High Temperature

Abstract

Levan-type fructooligosaccharides (LFOs) and levan can potentially be used as ingredients in prebiotics, skincare products, and antitumor agents. The Y246S mutant of Bacillus licheniformis RN-01 levansucrase (oligosaccharide-producing levansucrase, OPL) was reported to productively synthesize LFOs; however, OPL’s thermostability is low at high temperatures. To enhance OPL structural stability, this study employed molecular dynamics (AMBER) to identify a highly flexible region, as measured by its average root-mean-square fluctuation (RMSF) value, on the OPL surface and computational protein design (Rosetta) to rigidify and increase favorable interactions to increase its structural stability. AMBER identified region nine (residues 277–317) as a highly flexible region that was selected for design because it has the highest number of residues and the second-highest average RMSF, and it is farthest from the active site. Rosetta designed 14 mutants with the best ΔΔG value in each position, where three mutants have better ΔG than OPL. To determine whether their flexibilities and stabilities are lower than those of OPL, all 14 designed mutants were simulated at high temperature (500 K), and we found that K296E, G309S, and A310W mutants were predicted to be more stable and could retain their native structures better than OPL. Our results suggest that enhanced structural stabilities of these mutants are caused by increased hydrogen bond strengths of the designed residues and their neighboring residues. This study designed K296E, G309S, and A310W mutants of OPL with high potential for stability improvement, and they could potentially be used for the effective production of LFOs.

Mechanistic Insights into Roseoflavin Synthesis by N,N-8-Demethyl-8-aminoriboflavin Dimethyltransferase (RosA): Molecular Dynamics Simulations and Residue Conservation Analysis

“…Sodium ions (Na + ) were then added to neutralize the systems. Minimization was performed to remove unfavorable interactions. − PMEMD module of AMBER20 with the SHAKE algorithm was used to simulate the system under the periodic boundary conditions , with a simulation time step of 0.002 ps and the coordinates of trajectory were saved every 5000 steps (10 ps). The temperatures were controlled using the Langevin dynamic technique with a collision frequency of 1 ps –1 .…”

Computational design of Lactobacillus Acidophilus α-L-rhamnosidase to increase its structural stability

“…Using protein ff14SB force field parameters [ 42 ] in AMBER18 [ 43 ], each model was immersed in an isometric truncated octahedral box of TIP3P water molecules with the buffer distance of 13 Å. The five-step minimization procedure [ 29 , 30 , 44 – 56 ] was performed to reduce unfavorable interactions of each model. All steps used 2,500 steps of steepest descent and 2,500 steps of conjugate gradient with different restraints on the proteins.…”

“…Hydrogen bond occupations were then calculated to analyze hydrogen bond interactions, a hydrogen bond was considered to occur if the following criteria were met: (i) a proton donor-acceptor distance ≤ 3.5 Å and (ii) a donorH-acceptor bond angle ≥ 120° [ 44 , 45 , 53 , 55 ]. The strengths of hydrogen bond interactions were classified into four levels depending on hydrogen bond occupations: (i) strong hydrogen bond interactions (hydrogen bond occupations > 75%) (ii) medium hydrogen bond interactions (75% ≥ hydrogen bond occupations > 50%) (iii) weak hydrogen bond interactions (50% ≥ hydrogen bond occupations > 25%) and (iv) very weak hydrogen bond interactions (25% ≥ hydrogen bond occupations > 10%) [ 29 , 30 , 48 , 50 ].…”

“…Moreover, the substitution of amino acid residues on the surface of an enzyme is more preferable to those in its core structure because these mutations have less detrimental effects on enzymatic activity [ 27 , 28 ]. This approach has been employed to successfully improve the thermostabilities of many enzymes such as Bacillus circulans xylanase [ 28 ], Bacillus licheniformis RN-01 levansucrase [ 29 , 30 ], Escherichia coli AppA phytase [ 27 ], Pseudomonas putida NRRL-18668 nitrile hydratase [ 31 ], bacterial α-Carbonic anhydrase [ 32 ] and aldo-keto reductases [ 33 ]. Furthermore, computational techniques were also employed to improve the thermostability of fungal α‐L‐rhamnosidase [ 34 ].…”

Biochemical and ligand binding properties of recombinant Xenopus laevis cortical granule lectin-1

“…LEaP module in AMBER18 with GLYCAM06j-1 force field and ff14SB parameters [ 58 ] was used to immerse the representative binding orientations of all systems in isomeric truncated octahedral boxes of TIP3P water molecules with the 13 Å buffer distance. The five-step minimization procedure [ 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ] was employed to reduce the unfavorable interaction of the system. In this study, all steps used 2,500 steps of steepest descent and 2,500 steps of conjugate gradient.…”