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.