B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

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enzymes measured in any tissue. Similar methods could be applied for the identification
of additional cDNAs involved in cobalamin metabolism.
To determine whether the bovine and human cDNAs that we identified encode
ATRs, the cDNAs were cloned and over expressed in E. coli. Cell extracts from the
expression strains were then used for biochemical studies. Both the soluble and insoluble
fractions of the crude cell extracts of bovine and human expression strains were found to
have ATR activity. This suggests that the cDNAs we identified encode ATR enzymes.
Additional studies were used to show that the human cDNA could function as an ATR
in vivo. An S. enterica ATR mutant transformed with a plasmid-encoded source of the
human ATR was used for growth studies on 1,2-propanediol supplemented with HOCbl.
From these experiments, we showed that the human ATR restored wild-type phenotype
to an ATR-deficient S. enterica strain proving that the human ATR can function
physiologically in a heterologous host.
Previous studies have shown that patients with cblB methylmalonic aciduria lack
ATR activity (Fenton and Rosenberg 1981). To determine if mutations in the ATR
underlie cblB methylmalonic aciduria, expression of this enzyme was monitored in
normal and cblB mutant fibroblast cells. Western blot analysis using antibodies specific
for the ATR showed that expression of this protein was altered in fibroblast cells from
patients with cblB when compared to control cells. Additional studies conducted
concurrently in another lab showed that the ATR gene (MMAB) corresponded to the cblB
complementation group of methylmalonic aciduria (Dobson et al. 2002).
Biochemical Characterization of the Human Adenosyltransferase
In Chapter 3, we investigated the biochemical properties of the human ATR.
Dobson et al. previously analyzed the MMAB gene and identified two amino acid