Haemophilus influenzae 의 acetohydroxyacid synthase 재조합 단백질 발현 , 정제 및 특성

Abstract

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6)는 필수 아미노산 Valine, Leucine 그리고 Isoleucine의 생합성에 있어 첫 단계를 촉매하는 효소이다. Valine, Leucine 그리고 Isoleucine의 생합성 과정의 첫 단계에서 thiamin diphosphate- (ThDP-)과 FAD를 보조 효소로 가진다. 이 단백질은 자체만으로 활성을 나타내는 catalytic subunit과 효소활성을 활성화 시키는 regulatory subunit으로 구성되어있다. Heamophilus influenzae의 AHAS holoenzyme의 유전자를 pETDuet 발현벡터에 삽입시켜 대장균 BL21(DE3)에서 발현시켰다. Hi Trap chelating HP column을 사용하여 발현 시킨 단백질을 정제 하였다. 정제하여 얻은 단백질의 분자량은 SDS-PAGE 전기영동법을 이용하여 catalytic subunit 과 regulatory subunit의 64 and 18 kDa를 확인하였다. AHAS의 효소 활성은 96-well microplate을 사용하여 측정하였다 Holoenzyme의 specific activity는 3.2 U/mg이었다 또한 pyruvate의 Km은 4.8 mM이었고, ThDP and Mg^(2+) 의 K_(0.5) 는 각 각 4.26 mM, 127 mM이었다. 또한 tryptophan fluorescence의 Quenching을 이용한 ThDP의 Kd는 3.0mM이었다. AHAS에 대한 새로운 저해제를 사용하였을때 Heamophilus influenzae의 AHAS Holoenzyme에 대한 저해정도는 IC_(50) (50% inhibitory concentration)은 0.036 - 2.7 mM를 나타내었다. 이 결과는 H. iInfluenzae의 새로운 저해제로 개발될 수 있는 가능성을 보여주었다.; Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyzes the first step in the biosynthesis of the branched-chain amino acids. AHAS is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The enzyme has a large subunit containing the catalytic machinery and a small subunit with a regulatory role. The small subunit may affect feedback regulation, enzymatic activity, and stability. The holoenzyme of AHAS gene from Heamophilus influenzae was cloned into the bacterial expression vector pETDuet and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by Ni^(2+) -charged Hi Trap chelating HP column. The molecular mass of catalytic and regulatory subunits are around 64 and 18 kDa, rspectively. The specific activity of holoenzyme was 3.2 U/mg. The Km value for pyruvate was 4.8 mM, whereas the K_(0.5) values for cofactor (ThDP and Mg^(2+)) were 4.26 mM and 127 mM, respectively. The dissociation constant of ThDP were 3.0mM by tryptophan fluorescence quenching. The Kcat and Kcat/Km were 4.417 s^(-1) and 904.93 M^(-1) sec^(-1). The kinetic parameters of H. influenzae AHAS were determined and an enzyme activity assay system was designed using 96-well microplate. A new class of AHAS inhibitor was identified with the IC_(50) (50% inhibitory concentration) in the range of 0.036 - 2.7 μM. The results of this inhibitor screening can be used for designing of new anti H. iInfluenzae drug. We will continue to discover a potential hit compound against AHAS using rapid initial screening of chemical libraries and further we will test in vivo activity of hit compound as anti H. influenzae drug.