Homology modeling and docking studies of δ19-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8

Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerant Pseudomonas sp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed in Escherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant Pseudomonas sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, −6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with the in vivo activity of the Δ9-fatty acid desaturase on the membrane phospholipids

Ion interaction and hydrogen bonds as main features of protein thermostability in mutated T1 recombinant lipase originated from Geobacillus zalihae

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 °C and 76.0 °C in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in β-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance

Redesigning of Geobacillus zalihae T1 lipase based on spacegrown crystal structure

A microgravity environment is a favorable condition meant for growing a protein crystal, due to less sedimentation and convection. These factors would have benefited the protein crystal in terms of morphologies, crystal quality and appearance, which are important in producing a high quality electron density map. Nevertheless, the differences of structural architecture and features in protein related to the formation of hydrogen bonds and ion interactions remains unclear. In order to understand the relative contributions of a space atomic model in protein structure stability, it was necessary to compare the structure with the one grown on earth condition. There are existing limitations about manipulation of structural information for production of new enzyme due to insufficient analysis of both structures. Therefore, an earth and space condition crystal structures from a thermostable T1 lipase of Geobacillus zalihae were analyzed and compared. It was anticipated that the differences in hydrogen bonds and ion interactions are the main contributing factors towards protein stabilization. A molecular dynamics simulations approach was used to study differences of atomic fluctuations and conformational changes of both T1 lipase structures. From here, the structures stability was determined by a set of parameters comprising root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) in which the results showed a more stable space-grown structure compared to the earth-grown structure due to the presence of more hydrogen bonds. According to the in silico data, hydrogen bond interactions at position Asp43, Thr118, Glu250 and Asn304 and ion interaction at position Glu226 were chosen to imitate the space-grown crystal structure. Following that, the impact of combined interactions in mutated structure of T1 lipase was studied. The molecular interactions of five single mutants and the one that combined all five mutations, 5M were predicted based on structural changes and energy landscape by GROMACS simulation package. Site directed mutagenesis was applied on wild-type HT1 (wt-HT1) lipase to generate five single mutants (D43E, T118N, E226D, E250L and N304E), in which these sites were further combined by a gene synthesis to generate a new mutant showing five mutation points (D43E/T118N/E226D/E250L/N304E). The native lipase wt- HT1, single mutants and 5M mutant lipases were purified by affinity chromatography showing a recovery between 49.6 to 59.9% and a purification fold of 2.5 to 3.3. All lipases exhibited high activity at 60 to 80 °C. Mutants E250L and N304E shifted in optimum temperature to 80 °C as compared to wt-HT1 lipase. All lipases showed high activity at alkaline conditions of pH 6.0 to 9.0. The thermostability study indicates the mutant E226D as the most stable lipase having prolonged half-life (T1/2) values and melting temperature. A T1/2 value of E226D was found at 28 hours, 165 minutes and 47 minutes at 60 °C, 70 °C and 80 °C, respectively where the mutant reportedly showing a melting temperature (Tm) of 77.4 ± 2.6 °C. In contrast, mutation of all five positions in the 5M mutant failed to increase the stability of lipase as the half-life at 60 °C exhibited a decline from 9 hours to 6 hours. At 70 °C and 80 °C, the half-life was found to be 23 minutes and 8 minutes, respectively. The melting temperature decreased 3.3 °C to 67.6 ± 0.8 °C. The presence of metal ions, especially calcium ion, had a positive effect on the stability of D43E, T118N, E250L and 5M lipases, which increased as more calcium was added. Meanwhile, Zn2+, Cu2+, Mg2+ and Fe3+ ions inhibited the activity of lipases. In addition, the activities of D43E, T118N and 5M lipases increased in the presence of DMSO. All lipases showed a good hydrolysis rate in natural oil, except for coconut oil. All lipases shown to have loss in activities in the presence of surfactants and sodium dodecyl sulfate (SDS). In the presence of calcium ion, the stability of 5M mutant and wt-HT1 lipases were increased towards high temperatures and organic solvents. The presence of calcium prolonged the half-life of 5M and wt-HT1, and increased the Tm at 8.4 and 12.1 °C, respectively. The combination of substituted amino acid had produced a highly stable mutant hydrolyzing oil in selected organic solvents such as DMSO, n-hexane and n-heptane. To correlate mutations in 5M mutant with its structural transition, 5M mutant lipase was subjected to crystallization in 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.5 supplemented with 0.2 M sodium chloride (NaCl). The protein structure was elucidated at resolution 2.64 Å with 90.9% completeness. The crystal structure of 5M mutant consists of two asymmetric units that are similar to each other, with RMSD value of 0.7789 Å after superimpositions of chains A and B. The structure analysis revealed that 5M failed to introduce hydrogen bonds and ionic interaction at the intended positions. The cumulative mutations also resulted in decreasing in molecular interactions such as hydrogen bonds and interactions. The impacts of the mutations resulted in decreasing in stability and half-life of lipase against high temperature. As a conclusion, it is difficult to emulate the cumulative interactions happened in the space-grown T1 lipase as shown by mutant 5M. Nonetheless, lipases containing a single mutant of D43E and E226D were found to be successful in introducing and increasing the mutant stability, where the stability of protein structure was highly dependent on the role of hydrogen bonds and ion interactions

Changes of thermostability, organic solvent, and pH stability in Geobacillus zalihae HT1 and its mutant by calcium ion

Thermostable T1 lipase from Geobacillus zalihae has been crystallized using counter-diffusion method under space and Earth conditions. The comparison of the three-dimensional structures from both crystallized proteins show differences in the formation of hydrogen bond and ion interactions. Hydrogen bond and ion interaction are important in the stabilization of protein structure towards extreme temperature and organic solvents. In this study, the differences of hydrogen bond interactions at position Asp43, Thr118, Glu250, and Asn304 and ion interaction at position Glu226 was chosen to imitate space-grown crystal structure, and the impact of these combined interactions in T1 lipase-mutated structure was studied. Using space-grown T1 lipase structure as a reference, subsequent simultaneous mutation D43E, T118N, E226D, E250L, and N304E was performed on recombinant wild-type T1 lipase (wt-HT1) to generate a quintuple mutant term as 5M mutant lipase. This mutant lipase shared similar characteristics to its wild-type in terms of optimal pH and temperature. The stability of mutant 5M lipase improved significantly in acidic and alkaline pH as compared to wt-HT1. 5M lipase was highly stable in organic solvents such as dimethyl sulfoxide (DMSO), methanol, and n-hexane compared to wt-HT1. Both wild-type and mutant lipases were found highly activated in calcium as compared to other metal ions due to the presence of calcium-binding site for thermostability. The presence of calcium prolonged the half-life of mutant 5M and wt-HT1, and at the same time increased their melting temperature (Tm). The melting temperature of 5M and wt-HT1 lipases increased at 8.4 and 12.1 °C, respectively, in the presence of calcium as compared to those without. Calcium enhanced the stability of mutant 5M in 25% (v/v) DMSO, n-hexane, and n-heptane. The lipase activity of wt-HT1 also increased in 25% (v/v) ethanol, methanol, acetonitrile, n-hexane, and n-heptane in the presence of calcium. The current study showed that the accumulation of amino acid substitutions D43E, T118N, E226D, E250L, and N304E produced highly stable T1 mutant when hydrolyzing oil in selected organic solvents such as DMSO, n-hexane, and n-heptane. It is also believed that calcium ion plays important role in regulating lipase thermostability

Structure elucidation and docking analysis of 5M mutant of T1 lipase Geobacillus zalihae

5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 Å with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl–esters

Molecular Dynamic Simulation of Space and Earth-Grown Crystal Structures of Thermostable T1 Lipase Geobacillus zalihae Revealed a Better Structure

Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacillus zalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition

Main structural targets for engineering lipase substrate specificity

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases

Altering the Regioselectivity of T1 Lipase from <i>Geobacillus zalihae</i> toward <i>sn</i>-3 Acylglycerol Using a Rational Design Approach

The regioselectivity characteristic of lipases facilitate a wide range of novel molecule unit constructions and fat modifications. Lipases can be categorized as sn-1,3, sn-2, and random regiospecific. Geobacillus zalihae T1 lipase catalyzes the hydrolysis of the sn-1,3 acylglycerol chain. The T1 lipase structural analysis shows that the oxyanion hole F16 and its lid domain undergo structural rearrangement upon activation. Site-directed mutagenesis was performed by substituting the lid domain residues (F180G and F181S) and the oxyanion hole residue (F16W) in order to study their effects on the structural changes and regioselectivity. The novel lipase mutant 3M switches the regioselectivity from sn-1,3 to only sn-3. The mutant 3M shifts the optimum pH to 10, alters selectivity toward p-nitrophenyl ester selectivity to C14-C18, and maintains a similar catalytic efficiency of 518.4 × 10−6 (s−1/mM). The secondary structure of 3M lipase comprises 15.8% and 26.3% of the α-helix and β-sheet, respectively, with a predicted melting temperature (Tm) value of 67.8 °C. The in silico analysis was conducted to reveal the structural changes caused by the F180G/F181S/F16W mutations in blocking the binding of the sn-1 acylglycerol chain and orientating the substrate to bond to the sn-3 acylglycerol, which resulted in switching the T1 lipase regioselectivity