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-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-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 alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-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)-beta-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-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically.
Van Lanen
Steven G
SG
Division of Pharmaceutical Sciences, University of Wisconsin National Cooperative Drug Discovery Group, Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Lin
Shuangjun
S
Dorrestein
Pieter C
PC
Kelleher
Neil L
NL
Shen
Ben
B
D000227:Adenine Nucleotides / Q000378:metabolism*; D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D053281:Enediynes / Q000737:chemistry; D005810:Multigene Family; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010453:Peptide Synthases / Q000737:chemistry / Q000502:physiology*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D017354:Point Mutation; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014443:Tyrosine / Q000138:chemical synthesis / Q000378:metabolism*
D000227:Adenine Nucleotides; D000617:Aminoglycosides; D000903:Antibiotics, Antineoplastic; D001426:Bacterial Proteins; D053281:Enediynes; C058024:C 1027; D014443:Tyrosine; D010649:Phenylalanine; D009713:Nucleotidyltransferases; D010453:Peptide Synthases