Biochemical and genetic characterization of the membrane-associated malate dehydrogenase (acceptor) from Corynebacterium glutamicum.
In addition to a cytoplasmic, NAD-dependent malate dehydrogenase (EC 1.1.1.37), Corynebacterium glutamicum possesses a highly active membrane-associated malate dehydrogenase (acceptor) (EC 1.1.99.16). This enzyme also takes part in the citric acid cycle. It oxidizes L-malate to oxaloacetate and donates electrons to ubiquinone-1 and other artificial acceptors or, via the electron transfer chain, to oxygen. NAD is not an acceptor and the natural direct acceptor for the enzyme is most likely a quinone. The enzyme is therefore called malate:quinone oxidoreductase, abbreviated to Mqo. Mqo is a peripheral membrane protein and can be released from the membrane by addition of chelators. The solubilized form was partially purified and characterized biochemically. FAD is probably a tightly but non-covalently bound prosthetic group, and the enzyme is activated by lipids. A C. glutamicum mutant completely lacking Mqo activity was isolated. It grows poorly on several substrates tested. The mutant possesses normal levels of cytoplasmic NAD-dependent malate dehydrogenase. A plasmid containing the gene from C. glutamicum coding for Mqo was isolated by complementation of the Mqo-negative phenotype. It leads to overexpression of Mqo activity in the mutant. The nucleotide sequence of the mqo gene was determined and is the first sequence known for this enzyme. The derived protein sequence is similar to hypothetical proteins from Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis.
Molenaar
D
D
Biotechnologisches Zentrallabor, Heinrich-Heine-Universität, Düsseldorf, Germany. molenaar@rz.uni-duesseldorf.de
van der Rest
M E
ME
Petrović
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D005798:Genes, Bacterial; D008055:Lipids / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010071:Oxaloacetates / Q000378:metabolism; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013379:Substrate Specificity; D014451:Ubiquinone / Q000378:metabolism / Q000494:pharmacology
D008055:Lipids; D008293:Malates; D009838:Oligodeoxyribonucleotides; D010071:Oxaloacetates; D014451:Ubiquinone; D005182:Flavin-Adenine Dinucleotide; C030298:malic acid; D008291:Malate Dehydrogenase; C081608:malate dehydrogenase (acceptor); C025203:Ubiquinone Q1