Characteristics and regulation of a phospholipid-activated malate oxidase from Escherichia coli.

A malate oxidase which uses L-malate and FAD as substrates is produced in mutant strains of Escherichia coli lacking the activity of NAD-dependent malate dehydrogenase. Expression of the oxidase is independent of the presence or absence in the mutants of a material cross-reacting with antibodies against malate dehydrogenase. It is surmised that products of malate dehydrogenase are responsible for the effective repression of malate oxidase. Malate oxidase has been partially purified from a strain of E. coli which is highly derepressed for this enzyme. The enzyme is shown to transfer electrons to various artificial substrates such as ferricyanide, thiazolyl blue tetrazolium, and dichlorophenolindophenol. Among naturally occurring compounds, only vitamin K1, but not cytochrome c, is reduced by the enzyme. The enzyme is localized on the inner face of the cytoplasmic membrane to which it is loosely bound and can be easily solubilized by sonication or membrane disruption. The enzyme is powerfully activated by nonionic detergents, some lipids, phosphatidylglycerol, cardiolipin, and asolectin (a mixture of phosphatidylethanolamine, phosphatidylcholine, and several minor lipids). The phospholipids do not affect the K,,, of malate or ferricyanide, but drastically alter the K, for FAD. In the absence of the phospholipids, FAD yields negatively cooperative plots with a Hill coefficient of 0.5, but in the presence of the phospholipids, the plots are converted to hyperbolas with a Hill coefficient of 1.0. The enzyme is inhibited by adenosine mono-, di-, and triphosphates and NAD, but not NMN. The inhibition is competitive against FAD.