Abstract
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.
Highlights
The biosynthetic gene cluster for C-1027 was previously cloned and sequenced, and analysis of the open reading frames (ORFs)4 provided genetic evidence to adequately propose a mechanism of biosynthesis, transport, resistance, and regulation of C-1027 [5]
A minimal nonribosomal peptide synthetase (NRPS) module consists of three domains, an adenylation domain that sequentially selects, activates, and loads an amino acid to the 4Ј-phosphopantetheine prosthetic group of a peptidyl carrier protein (PCP) domain and a condensation domain that catalyzes the formation of an amide bond
Organization of NRPS within C-1027 Biosynthetic Gene Cluster—The gene cluster for C-1027 biosynthesis contains three ORFs with sequence homology to condensation (SgcC5), adenylation (SgcC1), and PCP (SgcC2) domains found in modular NRPSs
Summary
Chemicals and Instrumentation—If not mentioned, chemicals and instruments used were identical to that previously reported [11]. NdeI/HindIII fragment was isolated and cloned into pET28a to yield pBS1038 The former construct produces SgcC1 as a C-terminal His6-tagged protein, the latter construct produces SgcC1 as a C- and N-terminal His6-tagged protein, both of which have the 338 amino acids at the N terminus of SgcC1 deleted. Amino Acid-dependent ATP-[32P]PPi Exchange Assays—Assessment of the SgcC1 adenylation enzyme substrate specificity was performed as described previously [11]. The steady-state kinetic parameters for SgcC1 were determined with activity assays carried out at 30 °C in 100 mM TrisHCl (pH 9.0), 5 mM MgCl2, 0.1 mM EDTA, 5 mM ATP, 1.0 M [32P]PPi, and varied co-substrate as follows: 25–1600 M for 3-chloro--tyrosine, 3-hydroxy--tyrosine, and -phenylalanine, 5–370 M for (R)--tyrosine, and 0.5–200 M for (S)-tyrosine.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
