Cloning, expression and biochemical characterization of mitochondrial and cytosolic malate dehydrogenase from Phytophthora infestans

Mycological research Pub Date : 2009-06-01 DOI:10.1016/j.mycres.2009.02.012

Patricia E. López-Calcagno , Johanna Moreno , Luis Cedeño , Luis Labrador , Juan L. Concepción , Luisana Avilán

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引用次数: 19

Abstract

The genes of the mitochondrial and cytosolic malate dehydrogenase (mMDH and cMDH) of Phytophthora infestans were cloned and overexpressed in Escherichia coli as active enzymes. The catalytic properties of these proteins were determined: both enzymes have a similar specific activity. In addition, the natural mitochondrial isoenzyme was semi-purified from mycelia and its catalytic properties determined: the recombinant mitochondrial isoform behaved as the natural enzyme. A phylogenetic analysis indicated that mMDH, present in all stramenopiles studied, can be useful to study the relationships between these organisms. MDH with the conserved domain MDH_cytoplasmic_cytosolic is absent in some stramenopiles as well as in fungi. This enzyme seems to be less related within the stramenopile group. The Phytophthora cMDHs have an insertion of six amino acids that is also present in the stramenopile cMDHs studied, with the exception of Thalassiosira pseudonana cMDH, and is absent in other known eukaryotic cMDHs.

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疫霉线粒体和细胞质苹果酸脱氢酶的克隆、表达及生化特性研究

克隆了疫霉菌线粒体苹果酸脱氢酶和细胞质苹果酸脱氢酶(mMDH和cMDH)基因，并在大肠杆菌中作为活性酶过表达。测定了这两种蛋白的催化性能:两种酶具有相似的特异活性。此外，从菌丝体中半纯化了天然线粒体同工酶，并确定了其催化性能:重组线粒体同工酶表现为天然酶。系统发育分析表明，mMDH存在于所有研究的叠层生物中，可以用于研究这些生物之间的关系。具有保守结构域MDH_cytoplasmic_cytosolic的MDH在一些层桩和真菌中不存在。这种酶似乎在堆菌群中关系不大。疫霉cMDHs插入了6个氨基酸，除了假海藻cMDH外，这些氨基酸也存在于所研究的层菌cMDHs中，并且在其他已知的真核cMDHs中不存在。

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Mycological research

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