<i>In vitro</i> characterization of nonribosomal peptide synthetase-dependent <i>O</i>-(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine
Yusuke Shikai, Seiji Kawai, Yohei Katsuyama, Yasuo Ohnishi
Abstract
-(2-hydrazineylideneacetyl)serine (HDA-Ser) attached to its CP domain from l-serine and HDA moiety-attached AzsN. The thioesterase AzsB hydrolyzes it to yield HDA-Ser, which appears to be converted to azaserine by oxidation. Bioinformatics analysis of the Cy domain of AzsO showed that it has a conserved DxxxxD motif; however, two conserved amino acid residues (Thr and Asp) important for heterocyclization are substituted for Asn. Site-directed mutagenesis of two Asp residues in the DxxxxD motif (D193 and D198) and two substituted Asn residues (N414 and N447) indicated that these four residues are important for ester bond synthesis. These results showed that the diazo ester of azasrine is synthesized by the stepwise oxidation of the HAA moiety and provided another strategy to biosynthesize the diazo group.