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Working on the aerobic degradation of cholesterol in
the early 1950s, Thressa set out to produce precursors of steroid hormones and cortisone. Unfortunately, cholesterol
was degraded completely by the microorganism, so she
could not detect any of the desired intermediate products
to be used for hormone production. In the course of
this unsuccessful endeavor, however, Thressa was able
to identify cholesterol oxidase, an enzyme that oxidizes
cholesterol. This enzyme was later used clinically for
the measurement of cholesterol levels in blood.
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In the mid-1950s, Thressa returned to a subject more
closely related to her graduate work at Berkeley: the
study of metabolic systems under strictly anaerobic,
oxygen-free conditions. She found an example of anaerobic
metabolism in the
fermentation of lysine, an amino
acid. Lysine was broken down into smaller molecules
(acetate, butyrate, and ammonia) in the absence of
oxygen, and this degradation was catalyzed by cell-free
extracts of C. sticklandii , an
anaerobic bacterium she
had isolated from San Francisco Bay mud. By looking into
which chemical bonds were cleaved in lysine labeled with
the radioactive tracer 14C and how the broken pieces
were distributed among the radioactive products, Thressa
demonstrated that this fermentation process involved
two independent metabolic pathways. They turned out to
be dependent upon the presence of vitamin
B12 .
She also examined several other cases of amino acid
metabolism. Among them, the reduction of glycine into
acetate and ammonia was especially important, because
this proved to be an energy-conserving, rather than
an energy-consuming, process, linked to the formation
of ATP (Adenosine
triphosphate), the universal carrier of energy in cells.
While conducting her research on amino acid metabolism,
Thressa re-visited the question of how methane gas is
produced in oxygen-free conditions. In fact, the production
of methane is one of the most commonly observed phenomena
in nature. Cows, sheep, and other cud-chewing animals
known as ruminants often expel this gas from their mouths.
Methane gas is also produced in the black mud of ponds,
lakes, and marshes where organic material is undergoing
decay. By the time Thressa came back to the study of methane
biosynthesis, this subject had attracted much attention
from biochemists because of its relationship to another
topicthe function of vitamin B12
in living systems. |
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Thressa
in her laboratory, 1953. |
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The structure
of vitamin B12
coenzyme and its biochemical function.
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Vitamin B12 was
first isolated in 1948 in a crystalline form. This
triggered an intense investigation of its structure
by X-ray crystallographers, including Dorothy
Hodgkin,
a British scientist who finally elucidated the complete
structure of this red, cobalt-containing substance
in 1955. As well as this structural study, there was
also an increasing effort to obtain a large amount
of vitamin B12 from natural sources for therapeutic
and experimental uses. In the early 1950s, it was found
that the sludge remaining in the fermentation tanks
of sewage disposal plants had unusually high amounts
of vitamin B12 compounds,
and that the final fermentation process was carried
out almost exclusively by methane-producing, anaerobic
bacteria.
In the late 1950s, by studying the formation
of methane by Methanosarcina barkeri and other
anaerobic bacteria, Thressa demonstrated that vitamin
B12 is involved in the methane-producing process. Furthermore,
she explained that the free form of vitamin B12 can
function as a methyl group carrier, and that its
coenzyme
forms serve as hydrogen carriers. This knowledge provided
the basis for much of the current understanding of methane
biosynthesis. In 1958, another breakthrough in the study
of vitamin B12 was made by Horace
A. Barker, Thressa's
mentor, who discovered the biologically active forms
(coenzyme forms) of B12 vitamins while working on the
anaerobic metabolism of glutamate. Coenzymes are non-protein
molecules that help the catalytic function of enzymes.
In the end, Thressa and her co-workers discovered 5
of the 12 known vitamin B12 -dependent enzymes, some
of which, as coenzyme forms, function in lysine fermentation.
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