Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor.
Membrane-bound nitrate reductase of Escherichia coli consists of three subunits designated as A, B, and C, with subunit C being the apoprotein of cytochrome b, A hemA mutant that cannot synthesize delta-aminolevulinic acid (ALA) produces a normal, stable, membrane-bound enzyme when grown with ALA. When grown without ALA, this mutant makes a reduced amount of membrane-bound enzyme that is unstable and contains no C subunit. Under the same growth conditions, this mutant accumulates a large amount of a soluble form of the enzyme in the cytoplasm. Accumulation of this cytoplasmic form begins immediately upon induction of the enzyme with nitrate. The cytoplasmic form is very similar to the soluble form of the enzyme obtained by alkaline heat extraction. It is a high-molecular-weight complex with a Strokes radius of 8.0 nm and consists of intact A and B subunits. When ALA is added to a culture growing without ALA, the cytoplasmic form of the enzyme is incorporated into the membrane in a stable form, coincident with the formation of functional cytochrome b. Reconstitution experiments indicate that subunit C is present in cultures grown without ALA but is reduced in amount or unstable. These results indicate that membrane-bound nitrate reductase is synthesized via a soluble precursor containing subunits A and B, which then binds to the membrane upon interaction with the third subunit, cytochrome b.
D000622:Aminolevulinic Acid / Q000096:biosynthesis / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002701:Chloramphenicol / Q000494:pharmacology; D003580:Cytochromes / Q000096:biosynthesis; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D009565:Nitrate Reductases / Q000096:biosynthesis*; D011498:Protein Precursors / Q000096:biosynthesis*; D012995:Solubility
D003580:Cytochromes; D011498:Protein Precursors; D002701:Chloramphenicol; D000622:Aminolevulinic Acid; D009565:Nitrate Reductases