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The Escherichia coli panD gene, encoding l-aspartate-alpha-decarboxylase, was
cloned by PCR, and shown to complement a panD mutant defective in beta-alanine
biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is
synthesized initially as an inactive proenzyme (the pi-protein), which is
proteolytically cleaved at a specific X-Ser bond to produce a beta-subunit with
XOH at its C-terminus and an alpha-subunit with a pyruvoyl group at its
N-terminus, derived from the serine. The recombinant enzyme, as purified, is a
tetramer, and comprises principally the unprocessed pi-subunit (of 13.8 kDa),
with a small proportion of the alpha- and beta-subunits (11 kDa and 2.8 kDa
respectively). Incubation of the purified enzyme at elevated temperatures for
several hours results in further processing. Using fluorescein
thiosemicarbazide, the completely processed enzyme was shown to contain three
pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the
N-terminus of some of the alpha-subunits was confirmed by electrospray mass
spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for
aspartate decarboxylase was established and used to determine the Km and kcat
for l-aspartate as 151+/-16 microM and 0.57 s-1 respectively. ESMS was also used
to observe substrate and product adducts trapped on the pyruvoyl group by sodium
cyanoborohydride treatment.
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