ALLOPHANE INCREASES THE PROTEIN-LEVELS OF SEVERAL CEPHAMYCIN BIOSYNTHETIC-ENZYMES IN NOCARDIA LACTAMDURANS

Addition of allophane, a phosphate-trapping agent, to two different st rains of Nocardia lactamdurans exerted a large stimulatory effect on c ephamycin biosynthesis. The biosynthesis of cephamycin is inhibited by inorganic phosphate at concentrations above 5 mM and allophane revers ed this inhibitory effect. Allophane-supplemented cultures showed incr eased activities and/or an extended life in the cell of four cephamyci n biosynthetic enzymes: isopenicillin N synthase, the two-protein-comp onent 7 alpha-cephem methoxylase (7 alpha-cephem hydroxylase and 7-hyd roxycephem methyltransferase) and 3'-hydroxymethylcephem 0-carbamoyltr ansferase. However, the first enzyme of the pathway, lysine 6-aminotra nsferase, was not stimulated by allophane. Allophane-supplemented cult ures showed increased protein levels of (i) alpha-aminoadipyl-cysteiny l-valine synthetase (the condensing multienzyme that forms the tripept ide intermediate), and (ii) the two proteins involved in the 7 alpha-c ephem methoxylase, as shown by immunoblotting with antibodies against each of these proteins. Phosphate repressed the de novo synthesis of t hese proteins but did not increase their degradation. These results in dicated that allophane stimulates expression of the cluster of genes e xtending from the pcbAB gene (encoding alpha-aminoadipyl-cysteinyl-val ine synthetase) to cmcI-cmcJ (encoding the two-protein methoxylase) an d cmcH (encoding the 0-carbamoyltransferase).