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Pyruvate carboxylase from Corynebacterium glutamicum : purification and characterization

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Abstract

Pyruvate carboxylase of Corynebacterium glutamicum serves as anaplerotic enzyme when cells are growing on carbohydrates and plays an important role in the industrial production of metabolites derived from the tricarboxylic acid cycle, such as l-glutamate or l-lysine. Previous studies suggested that the enzyme from C. glutamicum is very labile, as activity could only be measured in permeabilized cells, but not in cell-free extracts. In this study, we established conditions allowing activity measurements in cell-free extracts of C. glutamicum and purification of the enzyme by avidin affinity chromatography and gel filtration. Using a coupled enzymatic assay with malate dehydrogenase, Vmax values between 20 and 25 μmol min−1 mg−1 were measured for purified pyruvate carboxylase corresponding to turnover numbers of 160 – 200 s−1 for the tetrameric enzyme. The concentration dependency for pyruvate and ATP followed Michaelis-Menten kinetics with Km values of 3.76 ± 0.72 mM and 0.61 ± 0.13 mM, respectively. For bicarbonate, concentrations ≥5 mM were required to obtain activity and half-maximal rates were found at 13.25 ± 4.88 mM. ADP and aspartate inhibited PCx activity with apparent Ki values of 1.5 mM and 9.3 mM, respectively. Acetyl-CoA had a weak inhibitory effect, but only at low concentrations up to 50 μM. The results presented here enable further detailed biochemical and structural studies of this enzyme.

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  1. IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, 52425, Jülich, Germany

    Maike Kortmann, Meike Baumgart & Michael Bott

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  1. Maike Kortmann
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  2. Meike Baumgart
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Correspondence to Michael Bott.

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Kortmann, M., Baumgart, M. & Bott, M. Pyruvate carboxylase from Corynebacterium glutamicum : purification and characterization . Appl Microbiol Biotechnol 103, 6571–6580 (2019). https://doi.org/10.1007/s00253-019-09982-x

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