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ABSTRAKTuberkulosis (TB) masih menjadi masalah kesehatan utama di Indonesia. Salah satu
penyebab tingginya kasus TB disebabkan adanya resistensi Mycobacterium
tuberculosis (Mtb) terhadap obat anti tuberkulosis. Isoniazid (INH) yang merupakan
salah satu obat lini pertama dalam pengobatan TB adalah pro-drug yang akan diubah
menjadi bentuk aktifnya melalui aktivitas protein KatG Mtb. Mutasi pada gen katG
yang mengkode protein KatG kemungkinan mempengaruhi aktivitas katalase dan
peroksidase protein sehingga menyebabkan resistensi Mtb terhadap INH. Dalam
studi ini, dilakukan kontruksi plasmid rekombinan protein KatG tipe liar dan protein
KatG dengan mutasi baru pada residu N330D dan H400Y, serta mutasi yang umum
dijumpai pada residu S315T dan S315N. Ekspresi protein KatG rekombinan
dilakukan menggunakan host E. coli. Over ekspresi kelima protein rekombinan KatG
terjadi setelah induksi IPTG. Purifikasi protein KatG rekombinan dilakukan
berdasarkan prinsip kromatografi afinitas menggunakan Nikel sepharose. Setelah
purifikasi diperoleh protein KatG yang murni. Aktivitas katalase dan peroksidase
protein rekombinan KatG diukur pada berbagai konsentrasi substrat yang diperlukan
dalam pengukuran efisiensi katalitik kedua aktivitas protein KatG. Hasilnya
menunjukkan bahwa mutan protein KatG memiliki efisiensi katalitik yang lebih
rendah dari protein KatG tipe liar. Penurunan efisiensi katalitik aktivitas katalase
mutan N330D dan H400Y sebesar 31% dan 37% dan untuk aktifitas peroksidase
sebesar 39% dan 3% dibandingkan KatG tipe liar. Struktur 3 dimensi protein KatG
dari mutan tersebut dibuat menggunakan perangkat Modeller dan divisualisasikan
menggunakan perangkat Pymol. Tidak terdapat adanya perubahan konformasi 3
dimensi protein KatG mutan dibandingkan dengan struktur 3 dimensi protein KatG
tipe liar. Namun, letak residu N330D dan H400Y yang berada dekat daerah aktif
ikatan INH pada protein KatG kemungkinan berpengaruh terhadap penurunan
aktivitas enzimatik protein KatG.
ABSTRACTTuberculosis (TB) is currently a major health problem in Indonesia. One of the many
causes of the high incident of TB is due to the resistance of the Mycobacterium
tuberculosis (Mtb) to anti-TB drugs. Isoniazid (INH), one of the first line anti-TB
drugs for TB treatment, is a pro-drug that is converted to its active form through the
activity of Mtb KatG protein. Mutations in the katG gene encoding KatG may affect
the catalytic efficiency of the catalase and peroxidase activities of the protein that
eventually confers resistance to INH. In this study, recombinant plasmids containing
katG gene that have new mutations on residue N330D dan H400Y, wild type, as well
as mutant proteins with common mutations at residue S315T and S315N were
constructed. Expression of recombinant KatG was performed using E. coli as an
expression host. Over expression of recombinant KatG was facilitated by IPTG
induction. Purification of recombinant KatG was performed using affinity
chromatography employing Nickel sepharose. Pure recombinant proteins were
obtained, and the catalase and peroxidase activities of the recombinant KatG protein
were measured at various concentration of substrates. Result showed that mutant
KatGs have a lower catalytic efficiency for both catalase and peroxidase activities
than the wild type protein. Decreasing catalytic efficiency for catalase of mutants
N330D and H400Y were 31% and 37% than that of wild type KatG, while catalytic
efficiency for peroxidase of mutants N330D and H400Y were 39% and 3% lower
than that of wild type. Three dimensional structures of mutant KatGs were generated
using Modeller and visualized using PyMol softwares. The three dimensional
structural of mutant KatG showed no conformational change compared with that of
wild type KatG. However, the location of residues N330D and H400Y which are in
the close proximity to the active site of KatG for INH binding is likely to have an
effect on the decreased enzymatic activities of mutant KatG proteins."
