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Partial purification and specificity studies of the d-glutamate-adding and d-alanyl-d-alanine-adding enzymes from Escherichia coli K12

Authors

  • Catherine MICHAUD,

    1. Unité Associée du Centre National de la Recherche Scientifique, Biochimie Moléculaire et Cellulaire, Université de Paris-Sud, Orsay
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  • Didier BLANOT,

    Corresponding author
    1. Unité Associée du Centre National de la Recherche Scientifique, Biochimie Moléculaire et Cellulaire, Université de Paris-Sud, Orsay
      Correspondence to D. Blanot, Unité Associée 04/1131 du CNRS, Bâtiment 432, Université de Paris-Sud, F-91405 Orsay Cedex, France
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  • Bernard FLOURET,

    1. Unité Associée du Centre National de la Recherche Scientifique, Biochimie Moléculaire et Cellulaire, Université de Paris-Sud, Orsay
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  • Jean VAN HEIJENOORT

    1. Unité Associée du Centre National de la Recherche Scientifique, Biochimie Moléculaire et Cellulaire, Université de Paris-Sud, Orsay
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Correspondence to D. Blanot, Unité Associée 04/1131 du CNRS, Bâtiment 432, Université de Paris-Sud, F-91405 Orsay Cedex, France

Abstract

The d-glutamate-adding and d-alanyl-d-alanine-adding enzymes from Escherichia coli were partially purified by fast protein liquid chromatography on an anion exchanger. Their relative molecular masses, determined by gel filtration on Superose 12, were 54000 ± 2000 and 51000 ± 2000, respectively. In order to investigate the specificity of these ligases, several compounds derived from their respective nucleotide substrates were prepared. In the case of the d-Glu-adding enzyme, DDP-MurNAc-l-Ala (DDP = dihydrouridine 5′-diphosphate) and P1-MurNAc-l-Ala were substrates of the reaction. In the case of the d-Ala-d-Ala-adding enzyme, only DDP-MurNAc-l-Ala-d-Glu(-meso-A2pm) was a subsrate; P1-MurNAc-l-Ala-d-Glu(-meso-A2pm) was neither a substrate nor an inhibitor. Concerning the amino acid site of the d-Glu-adding enzyme, even closely related analogues of d-glutamate hardly inhibited the reaction.

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