Prokaryotic Expression and Bioinformatics Analysis of Cytosolic Malate Dehydrogenase from Camellia sinensis(Theaceae)

Abstract

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 1 235 bp in length,encoding a protein of 332 amino acids with the putative molecular weight of 35.5 kD. The E.coli Rosetta (DE3) harboring pGEX-MDH was induced by 0.5 mmol·L-1 IPTG at 32℃ for 3 hours,and a 61.5 kD glutathione Stransferase (GST)-fused MDH was obtained in soluble form. The results of NCBI-BLAST revealed that Cs-cMDH shared 88%-93% 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 thirteen β-sheet and thirteen α-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.