Investigating the Role of Asp203 and His294 in the Catalytic Mechanism of BmaN1 Alpha Amylase Fina Khaerunnisa Frima (a,b*), Reza Aditama (a), Ihsanawati (a), Dessy Natalia (a)
a) Chemistry Department, Institut Teknologi Bandung, Bandung, Indonesia.
b) Chemistry Department, Institut Teknologi Sumatera, Lampung Selatan, Indonesia.
Abstract
BmaN1 is an alpha amylase produced by a sea anemone associated Bacillus megaterium NL3, which was isolated from from Kakaban Lake, East Kalimantan, Indonesia. Based on the similarity of amino acid sequences, BmaN1possesses aberrant catalytic residues within GH13 family and has recently been classified into the subfamily GH13_45. The amino acid sequence alignment and structure modelling of BmaN1, using alpha amylase from Geobacillus thermoleovorans (GTA, PDB code: 4E2O) as a template, revealed that only Glu231 was in the conserved position and functions as a proton donor. However, the other two of triad catalytic residues are different. Firstly, His294 functions as a transition state stabilizer in the conserved aspartate position. Additionally, Asp203 of BmaN1 is shifted to the i+1 position from the conserved nucleophile position, while Lys202 occupies the conserved nucleophile aspartate of GH13 alpha amylase. To investigate the role of catalytic residue Asp203 and His294, a truncated form of BmaN1 designated as BmaN1dC lacking 45 residues at the C-terminus was used. The aim of this research was to study the function of Asp203 and His294 in the catalytic mechanism. Site directed mutagenesis was performed to generate amino acid substitution Asp203Asn and His294Asp in BmaN1dC. BmaN1dC mutants was then produced in Escherichia coli ArcticExpress (DE3) and purified using Ni-NTA affinity column chromatography. Subsequently, the activity of BmaN1dC variants in starch hydrolysis was determined and it was found that BmaN1dC Asp203Asn and His294Asp lost up to 71% and 69% of their activity, respectively. These findings highlight the critical role of Asp203 and His294 in the catalytic mechanism of BmaN1.
