Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum |
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Authors: | Mike S. M. Jetten Anthony J. Sinskey |
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Affiliation: | (1) Department of Microbiology and Enzymology, Kluyver Laboratory for Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands;(2) Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 02139 Cambridge, MA, USA |
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Abstract: | Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an 4 subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO2, but no other -ketoacids were used as substrate. The cation Mn2+ was required for full activity, but could be substituted by Mg2+, Co2+, Ni2+ and Ca2+. Monovalent ions like Na+, K+ or NH4+ were not required for activity. The enzyme was inhibited by Cu2+, Zn2+, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a Km for OAA of 2.1 mM and a Km of 1.2 mM for Mn2+. The Vmax was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent kcat of 311 s–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase |
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Keywords: | lysine biosynthesis aspartate-derived amino acids oxaloacetate decarboxylase pyruvate kinase Corynebacterium glutamicum |
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