D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized byd d-alanine-d d-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL fromYersinia pestis Yersinia pestis (YpDDL) were determined at 1.7-2.5 angstrom resolution: apo, AMP-bound, ADP-bound, adenosine 5-(,-imido)triphosphate-bound, andd d-alanyl-d d-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the -loop) and loop 4, constitute the binding sites for twod d-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the -loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both thed d-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops.