INCOMPLETE CITRIC-ACID CYCLE OBLIGES AMINOLEVULINIC ACID SYNTHESIS VIA THE C-5 PATHWAY IN A METHYLOTROPH

The enzymic activities of the citric acid cycle and the connected path way of 5-aminolevulinic acid (ALA) formation in the methylotroph Methy lophilus methylotrophus (strain AS1) have been studied. The organism h as the enzymes required for conversion of pyruvate to 2-oxoglutarate. Of these, isocitrate dehydrogenase is unusual because of its preferenc e of NAD as coenzyme over NADP. In addition, the segment of the cycle that oxidizes 2-oxoglutarate to oxaloacetate is incomplete, lacking 2- oxoglutarate and succinate and malate dehydrogenase activities. Furthe rmore, alternative routes of 2-oxoglutarate oxidation to succinate are undetectable. The enzymes of the glyoxylate cycle are also absent. Th is suggests that the cycle in M. methylotrophus has no catabolic role and is purely biosynthetic. We also show that M. methylotrophus uses t he C, pathway of ALA formation. Cell-free extracts can convert glutama te to ALA in an ATP-, NADPH- and tRNA-dependent manner via the interme diate formation of Glu-tRNA(Glu) and glutamate l-semialdehyde. Consist ent with the absence of a detectable route by which it could synthesiz e succinate, M. methylotrophus cannot generate ALA from succinyl-CoA a nd glycine, the pathway found in mammalian cells and yeast.