THE ROLE OF THE C-TERMINAL, RESIDUES W201 AND Y400 OF ?-AMYLASE BAQA FROM BACILLUS AQUIMARIS MKSC 6.2 IN STARCH BINDING.

• Main Author: Ulpiyana, Ayra
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/87830
• Institution: Institut Teknologi Bandung

?-Amylase (1,4-?-D-glucan-glucanohydrolase, EC 3.2.1.1) is an enzyme that hydrolyzes ?-1,4 glycosidic bonds in starch from within the molecule, producing linear or branched oligosaccharides. The ?-amylase structure comprises three main domains, namely domain A which functions as the catalytic domain, domain B which is responsible for Ca²? ion binding, and domain C which stabilizes the catalytic domain. Based on amino acid sequence similarity, ?-amylase is classified under Glycoside Hydrolase Family 13 (GH13). This family is further divided into 47 subgroups based on conserved residues, substrate specificity, and hydrolysis products. The GH13_45 subfamily exhibits three distinctive features, including a tryptophan pair between CSR-V and CSR-II, the conserved LPDlx motif (leucine, proline, aspartic acid, leucine, and any amino acid residue) in CSR-V, and five aromatic amino acids located at the end of domain C. Several members of the GH13_45 subfamily have a hydrophobic C-terminal domain, such as the ASKA ?-amylase from Anoxybacillus SK3-4, GTA from Geobacillus thermoleovorans CCB_US3_UF5, BaqA from Bacillus aquimaris MKSC 6.2, and BmaN1 from Bacillus megaterium NL3. BaqA ?-amylase is a member of the GH13_45 family that hydrolyzes starch through aromatic residues predicted as surface binding sites (SBS), resembling the SBSs of barley AMY1. In AMY1, two SBSs have been identified, namely SBS1 which consists of tryptophan-278 and tryptophan-279 located on the surface of domain A, and SBS2 which is represented by tyrosine-380 in domain C. In BaqA, the tryptophan-201 and tryptophan-202 pair in domain A resembles the SBS found in AMY1. Additionally, BaqA contains tyrosine-400 in domain C, oriented towards the protein surface, is a strong candidate for SBS2, similar to tyrosine-380 in AMY1. This study aims to analyze the role of the C-terminal region, tryptophan-201, and tyrosine-400 residues in BaqA for starch binding. The objectives were achieved through experimental and bioinformatics approaches. The experimental approach involved the expression, purification, and physicochemical characterization of BaqA?C and BaqA?C_W201A. Additionally, site-directed mutagenesis was performed on the tyrosine-400 residue, substituting it with alanine, serine, and tryptophan to produce the variants BaqA?C_Y400A, BaqA?C_Y400S, and BaqA?C_Y400W. Protein expression and crude activity assays were conducted for BaqA?C and the C-terminal mutant variants. The bioinformatics approach included molecular docking simulations and molecular dynamics simulations of all protein variants. BaqA?C expressed in E. coli ArcticExpress (DE3) produces a protein with a molecular weight of 58 kDa. BaqA?C exhibits optimal activity at 50 °C, pH 6.5, and a NaCl concentration of 500 mM. BaqA?C shows halotolerance, retaining 60% of its activity at a NaCl concentration of 1.5 M. BaqA?C remains stable for up to 6 hours after preincubation at 50 °C. BaqA?C hydrolyzes soluble starch with a specific activity of 6.4 U/mg and degrades raw corn starch and cassava starch, producing reducing sugars of 2.7 mM and 3.1 mM, corresponding to Degree of Hydrolysis (DH) values of 1.1% and 1.3%, respectively. Enzyme kinetic parameters show that BaqA?C effectively functions at high substrate concentrations, with a Km of 36.24 mg.mL?1, Vmax of 10.5 µg.min?1, kcat of 412 s?1 dan kcat/Km of 11.4 mL.mg?1 s?1. From these findings, it was concluded that the removal of 34 amino acid residues at the C-terminus induced changes in pH and temperature optima, increased the amount of reducing sugars produced from the hydrolysis of various types of starch, and enhanced stability, halotolerant properties, and kinetic parameter values in BaqA?C compared to BaqA observed in previous studies. Expression of BaqA?C_W201A in E. coli ArcticExpress (DE3) produced a protein with a molecular weight of approximately 58 kDa. The specific activity of BaqA?C_W201A for hydrolyzing soluble starch was 4.5 U/mg. The kinetic parameters of BaqA?C_W201A revealed a Km of 55.9 mg.mL?1, Vmax of 157 µg.min?1, kcat of 668 s?1, dan kcat/Km of 11.9 mL.mg?1 s?1 for soluble starch. BaqA?C_W201A was capable of hydrolyzing raw corn starch and cassava starch, producing reducing sugars of 1.61 mM (0.72% DH) for corn starch and 0.9 mM (0.4% DH) for cassava starch. Scanning Electron Microscope (SEM) analysis showed that the surface of corn and cassava starch hydrolyzed by BaqA?C_W201A exhibited fewer pores and/or less peeling compared to starch hydrolyzed by BaqA?C, indicating a reduction in the effectiveness of raw starch hydrolysis by BaqA?C_W201A. This reduction in activity was supported by changes in secondary structure composition, analyzed using Circular Dichroism (CD). BaqA?C showed 16.8% ?-helix, 37.2% ?-sheet, and 32% ?-turn structures. In contrast, BaqA?C_W201A exhibited 5.2% ?-helix, 34.8% ?-sheet, and 14.5% ?-turn, indicating structural changes that likely contributed to the decreased hydrolytic activity. Molecular dynamics simulations revealed that ligand binding at the active site of BaqA?C_W201A caused changes in ligand orientation and influenced the flexibility of tyrosine-179 in the active site. This was confirmed through Root Mean Square Fluctuation (RMSF) analysis. Additionally, the mutation of tryptophan-201 increased the hydrogen bond distance between tyrosine-179 and acarbose, resulting in decreased ligand interaction stability. Molecular mechanics generalized Born surface area (MM-GBSA) analysis indicated that BaqA?C exhibited stronger interactions with acarbose compared to BaqA?C_W201A. The binding affinity of BaqA?C to acarbose was -8.8 kcal/mol, whereas the binding affinity of BaqA?C_W201A to acarbose was -7.9 kcal/mol. This decrease in binding affinity is consistent with experimental results, which showed that the tryptophan-201 mutation reduced the hydrolytic activity of BaqA?C_W201A across various types of starch. The starch-binding residue in the C-terminal domain of BaqA?C was identified through cavity analysis of the BaqA?C structural model and alignment of conserved residues at the C-terminal region among members of the GH13_45 subfamily. Ligand-binding cavity analysis using the POCASA tool revealed the presence of a cavity in the C-terminal domain with the second-largest volume (222 ų), following the cavity in domain A, which has a volume of 439 ų. Amino acid sequence alignment of the GH13_45 subfamily showed that tyrosine-400 in BaqA is a conserved residue within this subfamily. Preliminary analysis of tyrosine-400 as a starch-binding site in the C-domain through molecular dynamics simulations showed that mutating tyrosine-400 to tryptophan in BaqA?C_Y400W in the absence of acarbose increased the flexibility of residues 161–179 and 318–325 in domain A, which form loop and ?-helix structures. Energetically, the mutation of tyrosine-400 to tryptophan enhanced the stability of interaction with acarbose bound to domain C, as indicated by the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) analysis. The ?G value for BaqA?C_Y400W was ?24.45 kcal/mol, demonstrating better acarbose affinity compared to BaqA?C, which had a ?G value of ?23.24 kcal/mol. Site directed mutagenesis was performed on tyrosine-400 by replacing the 5'-TAT-3' nucleotide sequence encoding tyrosine at positions 1198–1200 in the baqA?C gene with 5'-CGC-3', 5'-AGC-3', and 5'-ACC-3', encoding alanine, serine, and tryptophan, respectively, to produce the variants BaqA?C_Y400A, BaqA?C_Y400S, and BaqA?C_Y400W. Expression of these mutants in E. coli ArcticExpress (DE3) produced soluble and active proteins with a molecular weight of 58 kDa. Crude activity assays showed that BaqA?C exhibited a specific activity of 3.6 U/mg toward soluble starch, which was not significantly different from BaqA?C_Y400A (3.9 U/mg). However, the specific activity significantly increased in BaqA?C_Y400W to 6.8 U/mg, while mutation to serine in BaqA?C_Y400S caused a decrease in activity to 2.3 U/mg.The increase in specific activity observed in BaqA?C_Y400W was consistent with molecular dynamics simulations, which indicated that the interaction of BaqA?C_Y400W with acarbose was more stable compared to the other mutants. This enhancement is thought to be influenced by changes in the flexibility of residues in the catalytic domain resulting from the tyrosine-400 to tryptophan mutation. The C-terminal region of BaqA influences its stability and halotolerance properties. BaqA?C exhibits better salt tolerance and stability compared to the full-length BaqA. The tryptophan-201 residue in the A domain of BaqA?C plays a role in starch binding. Additionally, the mutation of this residue to alanine affects the residues surrounding the active site, particularly tyrosine-179, and increases the bonding distance with the substrate, which is presumed to contribute to the reduction in hydrolytic activity. Meanwhile, the mutation of tyrosine-400 in BaqA?C affects the flexibility of residues in domain A. Substituting tyrosine-400 with tryptophan increases the flexibility of residues in the catalytic domain, which is likely responsible for the enhanced hydrolytic activity observed in BaqA?C_Y400W. Further studies through crystallization are expected to provide deeper insights into the interactions between starch and starch-binding residues in BaqA?C. Additionally, the role of tyrosine-179 in the active site can be further investigated through site-directed mutagenesis, expression, purification, and physicochemical analysis.