Incomplete citric acid cycle obliges aminolevulinic acid synthesis via the C5 pathway in a methylotroph

Abstract

~~The enzymic activities of the citric acid cycle and the connected pathway of 5-aminolevulinic acid (ALA) formation in the methylotroph Methyfophifus rnethyfotrophus (strain AS1) have been studied. The organism has the enzymes required for conversion of pyruvate to 2-oxoglutarate. Of these, isocitrate dehydrogenase is unusual because of its preference 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. Furthermore, alternative routes of 2-oxoglutarate oxidation to succinate are undetectable. The enzymes of the glyoxylate cycle are also absent. This suggests that the cycle in M. methylotrophus has no catabolic role and is purely biosynthetic. We also show that M. rnethyfotrophus uses the C, pathway of ALA formation. Cell-free extracts can convert glutamate to ALA in an ATP-, NADPH-and tRNA-dependent manner via the intermediate formation of Glu-tRNAG'" and glutamate 1-semialdehyde. Consistent with the absence of a detectable route by which it could synthesize succinate, M. rnethyfotrophus cannot generate ALA from succinyl-CoA and glycine, the pathway found in mammalian cells and yeast.

The biosynthesis of periplasmic electron transport proteins in methylotrophic bacteria

“…The first involves the condensation of succinyl coenzyme A and glycine, which is catalysed by ALA synthase, and this pathway operates in P. denitrifcans (Page & Ferguson, 1994). In the alternative C, pathwa ALA is formed from glutamate, with glutamyl tRNA'lU as the first intermediate ; as might be predicted, this pathway occurs in the obligate methylotroph M. metlylotrophus, which cannot make succinate via the TCA cycle since it lacks 2-oxoglutarate dehydrogenase (Lloyd et al, 1993).…”

Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis.

“…The availability of cloned genes and antibodies will greatly aid the elucidation of the molecular basis underlying the effects of various environmental stimuli (e.g., hormones, heme, and light) on ALA synthesis. The availability and the subsequent expression of the Arabidopsis HEMA and GSA7 genes will permit the establishment of an in vitro GIu-ALA conversion assay with purified enzymes (Chen et al, 1990;Lloyd et al, 1993). This might prove useful in screening for inhibitors of ALA formation; these inhibitors may have potential as herbicides.…”

“…Assays for Glu-tRNA reductase (GIuTR) activity were modified from the method of Chen et al (1990) as described by Lloyd et al (1993) by using precharged E. coli 14C-GIu-tRNA as substrate and converting the produced GSA to ALA by the action of added E. coli GSA 2,l-aminomutase (GSA-AM).…”

A point mutation in Euglena gracilis chloroplast tRNA(Glu) uncouples protein and chlorophyll biosynthesis.

“…ALA formed was purified as described (18,20). GluTR activity was completely abolished when precharged tRNA was preincubated with RNase A at 1.7 ug/ml for 15 min at 30°C before the addition of extract.…”