Non-ribosomal peptide synthetases (NRPS) are modular enzymes that catalyse the formation of peptides ranging from those containing the standard proteinogenic amino acids through to peptides containing exclusively non-proteinogenic amino acids ^[1]. The adenylation domains of NRPS are responsible for the selection of building blocks that contribute to the final peptide sequence. The diversity of peptides biosynthesised by NRPS pathways includes the glycopeptide antibiotics (GPA) (e.g. teicoplanin) and has led to these being used extensively for medicinal purposes ^[1,2]. However, the rise in antimicrobial resistance has necessitated the modification of such antibiotics to overcome resistance. A novel pathway to alter the structure of peptides produced by an NRPS is modification of the peptide backbone through incorporation of esters in which the A-domain selectively accepts hydroxy acids over amino acids ^[2]. The Tcp9 protein of teicoplanin produced by Actinoplanes teichomyceticus presents an ideal candidate for the exploration of engineering the A-domain selectivity for acceptance of hydroxy acids^[3]. In this study, residues from known hydroxy/keto accepting A-domains were chosen as candidates for the creation of triple mutations in module 1 of Tcp9 protein. Here, we report the 2.00 Å and 2.18 Å resolution X-ray crystal structure of these P283M and P283A mutants. The P283M structure revealed a different confirmation of the methionine residue in comparison to the methionine observed in keto accepting A-domains. The methionine is positioned directly into the binding pocket of the substrate disrupting substrate binding. Sequence and structural alignments revealed replacement of tyrosine to phenylalanine residues at position 281 and 367 in keto accepting A-domains could potentially open the binding pocket for substrate binding. This study provides the structural basis for engineering A-domains that can selectively accept alcohol/keto substrates instead of amino acids which therefore help to overcome antibiotic resistance and for the generation of novel glycopeptide antibiotics.