Malate dehydrogenase from the green gliding bacterium Chloroflexus aurantiacus is phylogenetically related to lactic dehydrogenases.
The gene encoding malate dehydrogenase (MDH) from Chloroflexus aurantiacus was cloned, sequenced, and analyzed. The mdh gene corresponded to a polypeptide of 309 amino acids with a molecular mass of 32,717 Da. The primary structure and the coenzyme-binding domain showed a high degree of similarity to lactate dehydrogenase (LDH), whereas the conserved amino acids that participate in substrate binding were those typical of MDHs. Using PCR techniques, the mdh gene was cloned in the expression vector pET 11a, and large amounts of active C. aurantiacus MDH were produced in Escherichia coli after induction with isopropyl beta-D-thiogalactoside. The expressed enzyme thus obtained was purified and retained full activity at 55 degrees C. High levels of expression of mdh were also observed when the gene and its flanking sequences were cloned into pUC18/19, indicating that the putative sigma 70 promoter sequences found upstream of the C. aurantiacus mdh functioned in E. coli. When these sequences were deleted, the expression in E. coli was reduced dramatically.
Synstad
B
B
Department of Biology, University of Oslo, Norway.
Emmerhoff
O
O
Sirevåg
R
R
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment
D015342:DNA Probes; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase