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Article

d-Alanine:d-Alanine Ligase: Phosphonate and Phosphinate Intermediates with Wild Type and the Y216F Mutant^†^,^‡

Chang Fan,^§ Il-Seon Park, Christopher T. Walsh, and James R. Knox*^§

Department of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269-3125, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Biochemistry, 1997, 36 (9), pp 2531–2538

DOI: 10.1021/bi962431t

Publication Date (Web): March 4, 1997

†

This work was supported in part by NIH Grant AI-34330 to J.R.K.

‡

Atomic coordinates for WT/2 and Y216F/1 have been deposited in the Brookhaven Protein Data Bank under Accession Numbers 1IOV and 1IOW, respectively.

The University of Connecticut.

Harvard Medical School.

Author for correspondence: Email, knox@uconnvm.uconn.edu; telephone, 860-486-3133; FAX, 860-486-4745.

Abstract

The crystallographic structure of the d-alanine:d-alanine ligase of the ddlB gene of Escherichia coli complexed with a d-Ala-d-α-hydroxybutyrate phosphonate and the structure of the Y216F mutant ligase complexed with a d-Ala-d-Ala phosphinate have been determined to 2.2 and 1.9 Å resolution, respectively, and refined to R factors of 0.156 and 0.158. In each complex the inhibitor has reacted with ATP to produce ADP and a tight-binding phosphorylated transition state intermediate. Comparison of these two structures with the known crystal structure of the phosphinate intermediate of the wild-type ligase shows no major conformational changes, but B factors indicate differences in mobility of loops covering the binding site. The weaker inhibition of the Y216F mutant by both inhibitors is thought to be due in part to the loss of an interloop hydrogen bond. A similar mechanism may account for poor inhibition of VanA, the homologous d-Ala:d-lactate ligase produced by vancomycin-resistant enterococci.