Shah Tina D, Hickey Meghan C, Capasso Kathryn E, Palenchar Jennifer B
Department of Chemistry, Villanova University, Villanova, PA 19085, United States.
Mol Biochem Parasitol. 2011 Oct;179(2):100-6. doi: 10.1016/j.molbiopara.2011.07.001. Epub 2011 Jul 7.
A putative β-hydroxybutyrate dehydrogenase (βHBDH) ortholog was identified in Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. The trypanosome enzyme has greater sequence similarity to bacterial sources of soluble βHBDH than to membrane-bound Type I βHBDH found in higher eukaryotes. The βHBDH gene was cloned from T. brucei genomic DNA and active, recombinant His-tagged enzyme (His(10)-TbβHBDH) was purified to approximate homogeneity from E. coli. βHBDH catalyzes the reversible NADH-dependent conversion of acetoacetate to D-3-hydroxybutyrate. In the direction of D-3-hydroxybutyrate formation, His(10)-TbβHBDH has a k(cat) value of 0.19 s(-1) and a K(M) value of 0.69 mM for acetoacetate. In the direction of acetoacetate formation, His(10)-TbβHBDH has a k(cat) value of 11.2 s(-1) and a K(M) value of 0.65 mM for D-3-hydroxybutyrate. Cofactor preference was examined and His(10)-TbβHBDH utilizes both NAD(H) and NADP(H) almost equivalently, distinguishing the parasite enzyme from other characterized βHBDHs. Furthermore, His(10)-TbβHBDH binds NAD(P)(+) in a cooperative fashion, another unique characteristic of trypanosome βHBDH. The apparent native molecular weight of recombinant His(10)-TbβHBDH is 112 kDa, corresponding to tetramer, as determined through size exclusion chromatography. RNA interference studies in procyclic trypanosomes were carried out to evaluate the importance of TbβHBDH in vivo. Upon knockdown of TbβHBDH, a small reduction in parasite growth was observed suggesting βHBDH has an important physiological role in T. brucei.
在布氏锥虫（一种导致非洲昏睡病的单细胞真核寄生虫）中鉴定出一种假定的β-羟基丁酸脱氢酶（βHBDH）直系同源物。与在高等真核生物中发现的膜结合I型βHBDH相比，锥虫中的这种酶与可溶性βHBDH的细菌来源具有更高的序列相似性。从布氏锥虫基因组DNA中克隆了βHBDH基因，并从大肠杆菌中纯化出活性重组His标签酶（His(10)-TbβHBDH），纯度接近均一。βHBDH催化乙酰乙酸盐与D-3-羟基丁酸之间依赖于NADH的可逆转化。在生成D-3-羟基丁酸的方向上，His(10)-TbβHBDH对乙酰乙酸盐的k(cat)值为0.19 s(-1)，K(M)值为0.69 mM。在生成乙酰乙酸盐的方向上，His(10)-TbβHBDH对D-3-羟基丁酸的k(cat)值为11.2 s(-1)，K(M)值为0.65 mM。研究了辅因子偏好性，发现His(10)-TbβHBDH几乎同等程度地利用NAD(H)和NADP(H)，这使该寄生虫酶与其他已鉴定的βHBDH有所区别。此外，His(10)-TbβHBDH以协同方式结合NAD(P)(+)，这是锥虫βHBDH的另一个独特特征。通过尺寸排阻色谱法测定，重组His(10)-TbβHBDH的表观天然分子量为112 kDa，对应于四聚体。在原循环锥虫中进行了RNA干扰研究，以评估TbβHBDH在体内的重要性。敲低TbβHBDH后，观察到寄生虫生长略有减少，这表明βHBDH在布氏锥虫中具有重要的生理作用。
