The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and gamma-peptide, but, curiously, no enzyme synthesizing alpha-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of alpha-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.