Studies of carbon metabolism by the facultative methylotroph Arthrobacter 2B2
The research has involved a study of the enzymology of carbon assimilation in the facultative methylotroph Arthrobacter 2B2, utilising methylamine and choline as a sole source of carbon. When growing on methylamine the organism uses the ribulose monophosphate cycle of formaldehyde fixation as the carbon assimilating pathway. The variant of the cycle used is that involving Embden-Meyerhof cleavage, coupled with transaldolase/transketolase re-arrangement. It is the most energetically favourable of the 4 possible variants but is rare amongst methylotrophs, the only other organism in which it has been reported to be of physiological significance is Arthrobacter Pl (Levering et al. ,1982 ) Two key enzymes of the ribulose mono phosphate cycle, hexulose phosphate synthase and phosphohexuloisomerase were purified and characterised. Many of the properties of the purified hexulose phosphate synthase from Arthrobacter 2B2 resembled those of similar enzymes purified from other methylotrophs. However, it differs from previously purified enzymes in having a molecular weight of 155000, consisting of two identical subunits. The specific activity of the purified enzyme was very low compared to those reported for similar enzymes from other organisms. The phosphohexuloisomerase from Arthrobacter 2B2 was different to those previously studied in other organisms, in being a trimeric protein of total molecular weight 108000, constructed of three identical subunits. The final specific activity of the purified enzyme was very low compared to those reported for similar enzymes from other organisms. When growing on methylamine as a sole source of carbon, the organism synthesises hydroxypyruvate reductase. This enzyme was purified and found to be strictly NADPH specific, and therefore, different from hydroxypyruvate reductases reported in other organisms. IX When growing on choline as sole source of carbon the organism synthesisE;s enzymes of the ribulose monophosphate cycle and the serine pathway indicating the possibility that choline metabolism proceeds in a similar manner to that described in Arthrobacter P1 (Levering rt al., 19B1 b). However, crude extracts of choline-grown Arthrobacter 2B2 were shown to contain high specific activities of formaldehyde dehydrogenase, which were not detected in extracts of the organism grown on Methylamine or glucose. This suggests that formaldehyde produced by the metabolism of choline, is oxidised by formaldehyde dehydrogenase. Crude extracts of choline grown Arthrobacter 2B2 contained high activities of NADPH-linked and NADH/NADPH-linked hydroxypyruvate reductase. Both enzymes were purified and their characteristics tested. Batch cul til res of cho1 ine-grown cells produced the two enzymes at different stages of growth, the NADH/NADPH-linked enzyme being produced during early-to mid-exponential phases, and the NADPH-linked enzyme during late exponential and stationary phases. This effect was studied further in chemostat continuous culture. From these studies it has been proposed that the oxidation of choline is not completed in one uninterrupted sequence, but that an intermediate of the catabolic pathway is allowed to build up during the early stages of batch growth. If this intermediate repressed the production of the NADPH-linked enzyme, this could provide an explanation for its delayed appearance during the growth cycle. However, under conditions of strict carbon limitation, such as would be present at very low dilution rates in continuous culture, the intermediate would itself be degraded thus lifting the repression of the synthesis of the NADPH-linked enzyme. A study was also made of the formaldehyde dehydrogenase of Methylophilus methylotrophus. This enzyme was purified, and its activity was demonstrated to be thiol-dependent. The thiol specificity was not limited to GSH, as cysteine was also active in this capacity.