Regulation of the Enzymes of the β‐Ketoadipate Pathway in <i>Moraxella calcoacetica</i> (1968)

Mutants of Moraxella calcoacetica in which the activity of protocatechuate oxygenase is lost or severely impaired synthesize the remaining four enzymes of the protocatechuate coordinate block—carboxymuconate lactonizing enzyme, carboxymuconolactone decarboxylase, β‐ketoadipate enol‐lactone hydrolase I and β‐ketoadipate succinyl‐CoA transferaseI—at high differential rates in the absence of an exogenous inducer. Such mutants can be isolated directly from the wild type, by selection for the loss of protocatechuate oxygenase function. Similar mutants can also be isolated indirectly from the wild type by two‐step selection. The first step involves selection for ability to grow with cis, cis ‐muconate, as a result of a mutation that confers increased permeability both to this compound and to β‐ketoadipate; the second step involves selection for ability to grow with β‐ketoadipate as sole carbon source, as a result of the constitutive synthesis of a β‐ketoadipate succinyl‐CoA transferase. Most mutants selected in this way for ability to grow with β‐ketoadipate synthesize constitutively transferase I and other enzymes of the protocatechuate coordinate block, but have invariably lost protocatechuate oxygenase function. The properties of these constitutive mutants indicate that the greatly elevated rate of synthesis of enzymes of the protocatechuate coordinate block is not caused by mutation of a regulatory gene. Revertants selected for restored protocatechuate oxygenase function revert simultaneously to the inducible state; the magnitude of constitutive enzyme synthesis is directly related to the degree of impairment of protocatechuate oxygenase function; and most constitutive mutants become inducible when grown at a low temperature (20°), even though protocatechuate oxygenase function remains impaired. These traits suggest that the high rate of synthesis of the enzymes of the protocatechuate block is caused by elevated levels within the cell of the inducer, protocatechuate, synthesized from endogenous sources and accumulated as a result of the absence or impairment of protocatechuate oxygenase. The endogenous source of protocatechuate appears to be the hydroaromatic intermediates in the pathway of aromatic biosynthesis. The wild type of Moraxella calcoacetica uses shikimate as a source of carbon and energy. Shikimate is dissimilated via protocatechuate by means of inducible enzymes. The initial step is mediated by shikimate dehydrogenase, an inducible enzyme which can be readily distinguished by its catalytic properties from the biosynthetic enzyme responsible for shikimate synthesis, 5‐dehydroshikimate reductase. Shikimate dehydrogenase is product‐induced by protocatechuate. In mutants lacking protocatechuate oxygenase, it is synthesized, like the enzymes of the protocatechuate block, at high rates in the absence of exogenous inducer, and it is restored to the inducible state when these mutants are cultivated at low temperature, or when they regain protocatechuate oxygenase function by reversion. Accordingly, protocatechuate plays a very important role as an inducer in M. calcoacetica , since it controls the synthesis of all the enzymes responsible for the conversion of shikimate to β‐ketoadipyl‐CoA.