Adak Subrata, Datta Alok K
Division of Infectious Diseases, Leishmania Group, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India.
Biochem J. 2005 Sep 1;390(Pt 2):465-74. doi: 10.1042/BJ20050311.
Haem-containing enzymes (peroxidase and catalase) are widely distributed among prokaryotes and eukaryotes and play a vital role in H2O2 detoxification. But, to date, no haem-containing enzymatic defence against toxic H2O2 has been discovered in Leishmania species. We cloned, expressed and purified an unusual plant-like APX (ascorbate peroxidase) from Leishmania major (LmAPX) and characterized its catalytic parameters under steady-state conditions. Examination of its protein sequence indicated approx. 30-60% identity with other APXs. The N-terminal extension of LmAPX is characterized by a charged region followed by a stretch of 22 amino acids containing a transmembrane domain. To understand how the transmembrane domain influences the structure-function of LmAPX, we generated, purified and extensively characterized a variant that lacked the transmembrane domain. Eliminating the transmembrane domain had no impact on substrate-binding affinity but slowed down ascorbate oxidation and increased resistance to H2O2-dependent inactivation in the absence of electron donor by 480-fold. Spectral studies show that H2O2 can quickly oxidize the native enzyme to compound (II), which subsequently is reduced back to the native enzyme by an electron donor. In contrast, ascorbate-free transmembrane domain-containing enzyme did not react with H2O2, as revealed by the absence of compound (II) formation. Our findings suggest that the single copy LmAPX gene may play an important role in detoxification of H2O2 that is generated by endogenous processes and as a result of external influences such as the oxidative burst of infected host macrophages or during drug metabolism by Leishmania.
含血红素的酶（过氧化物酶和过氧化氢酶）广泛分布于原核生物和真核生物中，在过氧化氢解毒过程中发挥着至关重要的作用。但是，迄今为止，尚未在利什曼原虫属中发现针对有毒过氧化氢的含血红素酶促防御机制。我们从硕大利什曼原虫中克隆、表达并纯化了一种不同寻常的类植物抗坏血酸过氧化物酶（LmAPX），并在稳态条件下对其催化参数进行了表征。对其蛋白质序列的研究表明，它与其他抗坏血酸过氧化物酶的同源性约为30%-60%。LmAPX的N端延伸区域的特征是有一个带电荷的区域，其后是一段包含跨膜结构域的22个氨基酸序列。为了了解跨膜结构域如何影响LmAPX的结构功能，我们构建、纯化并全面表征了一个缺失跨膜结构域的变体。去除跨膜结构域对底物结合亲和力没有影响，但减缓了抗坏血酸的氧化，并在没有电子供体的情况下使对过氧化氢依赖性失活的抗性提高了480倍。光谱研究表明，过氧化氢能迅速将天然酶氧化为化合物（II），随后该化合物被电子供体还原为天然酶。相比之下，不含抗坏血酸的含跨膜结构域的酶不与过氧化氢反应，这通过未形成化合物（II）得以证明。我们的研究结果表明，单拷贝的LmAPX基因可能在由内源性过程产生的以及受外部影响（如被感染宿主巨噬细胞的氧化爆发或利什曼原虫药物代谢过程中）产生的过氧化氢解毒中发挥重要作用。
