A223 would normally be the highly conserved aspartate residue that is involved in the recognition of the α-amino group of an amino acid building block, but its replacement with a small, neutral amino acid allowed for preferential binding of PLA substrates. C318, which replaced an isoleucine, is thought to use its side chain to hydrogen bond to the PLA substrates. P313 is a conformationally strained residue that, when replaced with the native leucine, would provide a backbone NH that is within hydrogen bonding distance to the backbone carbonyl of residue 318, making it unavailable for interaction with the substrate. The L313P mutation was serendipitous in this study, but it highlighted the significance of the backbone atoms in substrate orientation and binding in the active site pocket.