Escherichia coli L-aspartate-alpha-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry.
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.
Ramjee
M K
MK
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K.
Genschel
U
U
Abell
C
C
Smith
A G
AG
D000409:Alanine / Q000378:metabolism; D002262:Carboxy-Lyases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005784:Gene Amplification; D005816:Genetic Complementation Test; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D010205:Pantothenic Acid / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D010205:Pantothenic Acid; D002262:Carboxy-Lyases; C000623030:PanD protein, E coli; C019104:aspartate-alpha-decarboxylase; D005968:Glutamate Decarboxylase; D000409:Alanine