Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis

Neisseria meningitidis causes high transmissible disease known as invasive meningococcal disease (IMD) that is fatal. The best way to treat IMD is by antibiotics treatment. However, N. meningitidis has developed resistance to currently available antibiotics. Therefore, it is essential to discover ne...

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Main Author:	Xiang, Cheong Jing
Format:	Thesis
Language:	English
Published:	2022
Subjects:	QR75-99.5 Bacteria
Online Access:	http://eprints.usm.my/58371/1/Cheong-24%20pages.pdf
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spelling	my-usm-ep.583712023-05-15T02:55:49Z Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis 2022-09 Xiang, Cheong Jing QR75-99.5 Bacteria Neisseria meningitidis causes high transmissible disease known as invasive meningococcal disease (IMD) that is fatal. The best way to treat IMD is by antibiotics treatment. However, N. meningitidis has developed resistance to currently available antibiotics. Therefore, it is essential to discover new antimicrobial agents to combat the antibiotic resistant N. meningitidis strains. Choline kinase in N. meningitidis (NmCK) encoded by licA gene is one of the enzymes involved in cell membrane synthesis and inhibition of this enzyme in other bacteria has shown promising antimicrobial effect. However, basic biochemical properties and inhibitors of NmCK are still unknown. The present study aims to identify and modify important amino acid residues for NmCK catalysis that could be targeted for inhibition by performing two-step PCR site directed mutagenesis. Two aspartates (D153 and D170) in the CK motif of NmCK have been identified as highly conserved and chosen for mutagenesis into alanine. Two step PCR site-directed mutagenesis has successfully produced the full length D153A NmCK gene sequence without any unwanted mutation. Two fragments of D170A mutation have also been produced for subsequent combination into full length NmCK. Molecular modeling of wild type and D153A mutant NmCK was also performed and the model structures showed that the mutation of single amino acid did not change the overall structure of this enzyme. However, it is hypothesized that the mutation could alter the choline binding pocket. In conclusion, the D153A NmCK gene can be used for expression of mutant protein to study the importance of this residue in catalysis and inhibitor binding. The model structures generated can be used for molecular docking studies with potential inhibitors. 2022-09 Thesis http://eprints.usm.my/58371/ http://eprints.usm.my/58371/1/Cheong-24%20pages.pdf application/pdf en public masters Universiti Sains Malaysia Pusat Pengajian Sains Kesihatan
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	QR75-99.5 Bacteria
spellingShingle	QR75-99.5 Bacteria Xiang, Cheong Jing Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis
description	Neisseria meningitidis causes high transmissible disease known as invasive meningococcal disease (IMD) that is fatal. The best way to treat IMD is by antibiotics treatment. However, N. meningitidis has developed resistance to currently available antibiotics. Therefore, it is essential to discover new antimicrobial agents to combat the antibiotic resistant N. meningitidis strains. Choline kinase in N. meningitidis (NmCK) encoded by licA gene is one of the enzymes involved in cell membrane synthesis and inhibition of this enzyme in other bacteria has shown promising antimicrobial effect. However, basic biochemical properties and inhibitors of NmCK are still unknown. The present study aims to identify and modify important amino acid residues for NmCK catalysis that could be targeted for inhibition by performing two-step PCR site directed mutagenesis. Two aspartates (D153 and D170) in the CK motif of NmCK have been identified as highly conserved and chosen for mutagenesis into alanine. Two step PCR site-directed mutagenesis has successfully produced the full length D153A NmCK gene sequence without any unwanted mutation. Two fragments of D170A mutation have also been produced for subsequent combination into full length NmCK. Molecular modeling of wild type and D153A mutant NmCK was also performed and the model structures showed that the mutation of single amino acid did not change the overall structure of this enzyme. However, it is hypothesized that the mutation could alter the choline binding pocket. In conclusion, the D153A NmCK gene can be used for expression of mutant protein to study the importance of this residue in catalysis and inhibitor binding. The model structures generated can be used for molecular docking studies with potential inhibitors.
format	Thesis
qualification_level	Master's degree
author	Xiang, Cheong Jing
author_facet	Xiang, Cheong Jing
author_sort	Xiang, Cheong Jing
title	Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis
title_short	Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis
title_full	Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis
title_fullStr	Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis
title_full_unstemmed	Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis
title_sort	site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis
granting_institution	Universiti Sains Malaysia
granting_department	Pusat Pengajian Sains Kesihatan
publishDate	2022
url	http://eprints.usm.my/58371/1/Cheong-24%20pages.pdf
_version_	1776101218434678784

Site directed mutagenesis on highly conserved amino acid residues of choline kinase gene from neisseria meningitidis

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