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

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.

Identification and Biochemical Characterization of a Thermostable Malate Dehydrogenase from the MesophileStreptomyces coelicolorA3(2)

“…fungi, such as H. lanuginosa (5.8 mM) and M. pusillus (4.44 mM), T. emersonii (1.0 mM), and P. infestans (0.64 mM). 20,25,26,30) With NADH as co-substrate, the K m value for ScMDH (0.083 mM) was very similar to the data for S. aureofaciens MDH (0.085 mM) and P. infestans mMDH (0.086 mM). 20,30) Recombinant ScMDH displayed a similar K m value for NAD þ (0.15 mM), corresponding to enzymes from S. aureofaciens (0.27 mM) and fungi T. emersonii (0.13 mM), C. neoformans (0.11 mM), H. lanuginosa (0.1 mM), and M. pusillus (0.16 mM).…”

“…20,25,26,30) With NADH as co-substrate, the K m value for ScMDH (0.083 mM) was very similar to the data for S. aureofaciens MDH (0.085 mM) and P. infestans mMDH (0.086 mM). 20,30) Recombinant ScMDH displayed a similar K m value for NAD þ (0.15 mM), corresponding to enzymes from S. aureofaciens (0.27 mM) and fungi T. emersonii (0.13 mM), C. neoformans (0.11 mM), H. lanuginosa (0.1 mM), and M. pusillus (0.16 mM). 20,25,26,31) Therefore, ScMDH has higher affinity towards OAA than malate in vitro, as is the case for mMDHs and cMDHs from numerous sources.…”

“…20,25,26,31) Therefore, ScMDH has higher affinity towards OAA than malate in vitro, as is the case for mMDHs and cMDHs from numerous sources. 26,30) Like other MDHs, ScMDH had higher V max and K cat values towards OAA than towards malate. The V max and k cat values for OAA reduction were approximately 400-fold of the values for malate oxidation at 30 C and pH 8.5 respectively.…”

“…29) The K m value of 0.189 mM for OAA by recombinant ScMDH was close to the values determined for MDHs of S. aureofaciens (0.1 mM) and the fungus H. lanuginosa (0.12 mM), 25) but different from the reported K m values for mMDHs from other fungal sources, e.g., T. emersonii and Phytophthora infestans (0.022 mM). 26,30) On the other hand, the K m value for ScMDH with L-malate as substrate (0.494 mM) was similar to the data for the fungus Cryptococcus neoformans (0.25 mM), 31) but distinctly different from the K m values for MDHs from S. aureofaciens (9 mM) and A, Alignment based on the X-ray structures of T. flavus MDH (1BMD) (top) and A. arcticum MDH (1B8P) (bottom). The NAD þ -binding sites (]), substrate binding sites ( ), and active site (H) are shown, and the completely conserved residues are shaded.…”

“…Similar results were obtained for T. emersonii, P. infestans, wheat, and pineapple. 26,30,32,33) Generally, the activity of eukaryotic mMDHs is inhibited by excess OAA, while cMDHs are often inhibited by excess malate. 30,34) Substrate inhibition kinetics showed that ScMDH activity was strongly inhibited by excess OAA (K i ¼ 5:8 mM), similarly to fungus P. infestans mMDH (K i ¼ 2:25 AE 0:3 mM), 30) but was very different from S. aureofaciens MDH, which did not display substrate inhibition at high OAA concentrations.…”