In conclusion, we have unequivocally established that A. baumannii ACICU KpsS1 is a PseT enzyme. To the best of our knowledge, this is the first example of dedicated PseT activity in vitro. Additionally, as a result of the functional studies on the Im9-KpsS1 enzyme, a Pse5Ac7Ac-a2,6-PNP-b-DGlcp disaccharide 15 was synthesised in a tandem two-pot enzymatic process using seven enzymes, confirming that KpsS1 is also a retaining PseT. Although full elucidation of the nature of this retaining mechanism will likely require structural and biochemical studies using Psebased probes, this study confirms that KpsS1 defines a new GT family (GTXXX) which includes homologous proteins with conserved sequence motifs that are early candidates for mechanistic exploration.