Cloning, Expression And Enzymatic Characterization Of Two Malate Dehydrogenases

Malate dehydrogenase (MDH) is involved in various cellular processes, which catalyzes the reversible interconversion of oxaloacetate and malate linked to the oxidation/reduction of dinucleotide coenzymes. The enzymes play a key role in many metabolic pathways such as TCA cycle, photosynthesis, C4 cycle and so on. According to the coenzyme specificity, subcellular localization and biological function, plant MDHs can be divided into five classes: chloroplast NADP-dependent MDH (chMDH), mitochondrial NAD-dependent MDH (mMDH), microbody NAD-dependent MDH, chloroplast NAD-dependent MDH (cnMDH) and cytosolic NAD-dependent MDH (cMDH). Cytosolic NAD-dependent MDH (cMDH) has not been extensively characterized in plants.The complete gene of cytosolic malate dehydrogenase (cMDH) from Camellia sinensis, called Cs-cMDH, was obtained by RT-PCR and rapid amplification of cDNA ends (GenBank accession number GQ845406). This gene was 1235 bp in length, encoding a protein of 332 amino acids with the putative molecular weight of 35.5 kDa. The E. coli Rosetta (DE3) harboring pGEX-MDH was induced by 0.5 mM IPTG at 32℃for 3 hours, and a 61.5 kDa glutathione S-transferase (GST)-fused MDH was obtained in soluble form. The results of NCBI-BLAST revealed that Cs-cMDH shared 88⁹3% of amino acid sequence identity with other cMDH from different higher plants. According to the multiple sequence alignment based on the three-dimensional structure of protein, Cs-cMDH was predicted to be a dimer with thirteenβ-sheet and thirteenα-helix of each subunit. Cs-cMDH contains typical fingerprint sequence (G12AAGQIG18) as all MDHs. The amino acid D43 in Cs-cMDH is conserved in all NAD-MDHs. Cs-cMDH also has some conserved sequence units homologous to other NAD-MDHs, such as NAD+ binding sites, catalytic motif and substrate binding sites. Moreover, Cs-cMDH contains six Cys which are highly conserved in all plant NAD-cMDHs. Therefore, Cs-cMDH was inferred to be NAD-dependent cMDH. The present study may provide the fundament for the further functional characterization of Cs-cMDH.The entire coding sequence of mdh from Streptomyces lividans TK54, called SlMDH, was amplified using PCR (GenBank accession number EU715400). It contained a 990 bp open reading frame and showed a high similarity to other cytoplasmic MDHs from other species. The mdh gene of S. lividans TK54 was then subcloned into the expression vector pET-28(b) and efficiently expressed in E. coli Rosetta (DE3). The molecular mass of the recombinant SlMDH was about 35.5 kDa by SDS-PAGE analysis. The optimal temperature and pH of catalytic reaction by SlMDH were 45℃and pH 8.0. The Km and kcat values of the purified enzyme were 26.0μM and 1850 s^-1 for NADH, and 79.3μM and 3280 s^-1 for oxaloacetate. Furthermore, the recombinant SlMDH can not be inhibited at high oxaloacetate concentrations and metal ions have no obvious effect on SlMDH.