Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungusTalaromyces emersonii

Abstract

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52°C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The fulllength m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni 2+ -nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.Keywords: malate dehydrogenase; filamentous fungus; bio-specific affinity chromatography; thermophilic eukaryote; Talaromyces emersonii.Malate dehydrogenase (MDH) catalyzes the pyridine nucleotide-dependent interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. It is also thought to have a role in the malate/aspartate shuttle across the inner mitochondrial membrane. In most eukaryotic cells there are two major isoenzymes of MDH, cytosolic MDH (c-MDH) and mitochondrial MDH (m-MDH) [1].The majority of m-MDHs isolated to date from eukaryotic sources are homodimers of identical subunits, with a monomer size of 34 kDa. Mitochondrial MDH displays a complex regulatory pattern that involves allosteric activation [2], membrane interaction [3] and formation of multienzyme complexes [4] with enzymes such as citrate synthase, aspartate aminotransferase and other mitochondrial components [5][6][7][8][9][10]. Much of the previous research has focused on the comparison of primary and three-dimensional structures of mitochondrial and cytoplasmic forms of MDH isolated from a single source, e.g. pig heart tissue [11,12]. In contrast to bacterial, vertebrate and invertebrate m-MDH, a paucity of information exists on fungal m-MDH with significantly more known about yeast m-MDH than homologs from filamentous fungal species. Previous research by Dalhaus et al. [13] and Goward and Nicholls [14] has proposed that investigation...

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).…”

“…2B), similarly to the MDHs from three thermophilic fungi, H. lanuginosa (50 C), M. pusillus (50 C), and T. emersonii (52 C). 25,26) Thermal inactivation studies have indicated that ScMDH is thermostable, with an estimated t 1=2 of 120 min at 50 C (Fig. 3B), much more thermostable than MDHs from thermophilic fungi reported so far, 27) in spite of the fact that it is less thermostable than MDHs from a few extremely thermophile bacteria.…”

“…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. 26,30) Like other MDHs, ScMDH had higher V max and K cat values towards OAA than towards malate.…”

“…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.…”

“…coprophile; and !30 min for those from T. emersonii (t 1=2 ¼ 30 min) and M. pusillus (t 1=2 ¼ 60 min). [25][26][27] Therefore, ScMDH is a significantly thermostable enzyme from the mesophilic soil organism S. coelicolor A3(2).…”