DltA (D-alanine-D-alanyl carrier protein ligase) and DltC (D-alanyl carrier protein) are involved in the D-alanylation of lipoteichoic acids of gram-positive bacteria. DltA catalyzes the adenylation of D-alanine and its subsequent transfer to the thiol group of the DltC 4′-phosphopantetheine cofactor. These reactions, which classify them into a superfamily of adenylate forming enzymes that also includes adenylation (A) domains of non-ribosomal peptide synthetases (NRPSs), involve large scale domain rearrangements. Both proteins were crystallized and the structure of DltA was solved, revealing Cys268 as a major determinant for D-alanine substrate specificity. The structure displayed a conformation relevant to the thiolation reaction. Modelling based on known A-domain structures suggests that these enzymes exist in a number of metastable states, whereby small changes in electrostatics can lead to reorganization of the two domains.