Glycopeptide antibiotics (GPAs) are non ribosomal peptides rich in modifications: introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assemblyline. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platforni (X-domain) interacting with three to four modifying Cytochrome P450-(P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding" site on the X-domain and how the order of the eyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyB(tei) and OxyA(tei). We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-linked peptide. On the basis of steady state kinetics of the OxyB,Lcatalyzed reaction, we propose- a model for P450 recruitment and peptide modification that involves continuous association/dissociation of the P450 enzymes with the NRPS,:followecl by specific recognition of the peptide cyclizatiOn state'fiby "the P450 (scanning). This "leads to an induced conformational change that enhances the affinity of the enzyme/substrate complex and initiates catalysis; product release then occurs, with the product itself becoming the substrate for the second-enzyme in the pathway. This model rationalizes our experimental findings for this complex enzyme cascade and provides insights into the orchestration of the-sequential peptide tailoring reactions" on the terminal NRPS moditle in GPA biosynthesis.