Streptomyces toyocaensis produces A47934, a teicoplanin-like type-IV glycopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. A47934 differs from the type-I vancomycin glycopeptides, which possess a tricyclic peptide backbone, by the presence of an additional ring closure between the aromatic amino acids 1 and 3. To elucidate the order of crosslinking reactions, P450 mono-oxygenase-inactivation mutants (DeltastaF, DeltastaG, DeltastaH, and DeltastaJ) of the A47934 producer were generated, and the accumulated intermediates were analyzed. Thus, the formation of each crosslink could unambiguously be assigned to a specific oxygenase. The structure of the released intermediates from the wild-type nonribosomal peptide synthetase assembly line facilitated the determination of the cyclization order. Unexpectedly, the additional ring closure in A47934, catalyzed by StaG, is the second oxygenase reaction.