The biotin protein MadF of the malonate decarboxylase from Malonomonas rubra.
The gene for the biotin protein MadF of the Na+-pumping malonate decarboxylase from Malonomonas rubra was expressed in Escherichia coli together with the gene for the biotin ligase birA. MadF was partially purified from cell lysates by ammonium sulfate precipitation. Almost pure biotin protein was obtained by subsequent gel chromatography. With recombinant MadF, malonate decarboxylase activity of M. rubra cell extracts previously inactivated by avidin was recovered. Thus, the biological activity of recombinant MadF was proven. Despite the coexpression of birA, MadF was poorly biotinylated. This effect was not caused by an insufficient cofactor supply due to elevated protein levels at constant biotin uptake rates. Attempts to improve the cofactor incorporation were made by site-directed mutagenesis, by coexpression of madK, and by N-terminal elongation of MadF. These measures improved the fraction of MadF containing biotin to maximally 5%. These results might indicate the existence of a biotin ligase in M. rubra with an altered substrate specificity different from that of BirA.
Berg
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M
Mikrobiologisches Institut der ETH Zürich, ETH-Zentrum, Schmelzbergstr. 7, CH-8092 Zürich.
Dimroth
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P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D002262:Carboxy-Lyases / Q000737:chemistry*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D016415:Sequence Alignment
D001426:Bacterial Proteins; C116069:MadF protein, Malonomonase rubra; D001710:Biotin; D002262:Carboxy-Lyases; C071852:malonate decarboxylase