Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027

C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-β- phenylalanine moiety that is derived from L-α-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-β-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any α-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[ ³²P]PP_i exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated β-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-β-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic β-amino acids such as β-phenylalanine, 3-chloro-β-tyrosine, and 3-hydroxy-β-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all α-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-β-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-β-tyrosyl-S-SgcC2 thioester substrate, and represents the first β-amino acid-specific adenylation enzyme characterized biochemically.