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Volume 272, Number 14, Issue of April 4, 1997 pp. 9210-9214
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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D-Alanyl-D-Lactate and D-Alanyl-D-Alanine Synthesis by D-Alanyl-D-Alanine Ligase from Vancomycin-resistant Leuconostoc mesenteroides
EFFECTS OF A PHENYLALANINE 261 TO TYROSINE MUTATION

(Received for publication, October 31, 1996, and in revised form, January 24, 1997)

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to ~90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr²¹⁶, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe²⁶¹. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to ~90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The TyrPhe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.

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