Alazopeptin biosynthesis includes a characteristic peptide-formation machinery that uses a hydrolase (AzpM) to synthesize a dipeptide composed of two molecules of 6-diazo-5-oxo-l-norleucine (DON). Detailed analyses using a DON analogue, azaserine, show that AzpM catalyzes the condensation of two DON molecules tethered to a carrier protein and the subsequent hydrolysis. Enzymatic synthesis of alazopeptin analogues composed of azaserine was also established.
During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/β hydrolase AzpM synthesizes the DON-DON dipeptide using DON tethered to the carrier protein AzpF (DON-AzpF). However, whether AzpM catalyzes the condensation of DON-AzpF with DON or DON-AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS). We found that AzpM catalyzed the condensation between AZS-AzpF and DON-AzpF, but not between AZS-AzpF and DON. Possible reaction intermediates, DON-DON-AzpF and AZS-AZS-AzpF, were also detected during AzpM-catalyzed dipeptide formation from DON-AzpF and AZS-AzpF, respectively. From these results, we concluded that AzpM catalyzed the condensation of the two molecules of DON-AzpF and subsequent hydrolysis to produce DON-DON. Thus, AzpM is an unprecedented α/β hydrolase that catalyzes dipeptide synthesis from two molecules of a carrier protein-tethered amino acid.
Conflict of interest
The authors declare no conflict of interest.
Data Availability Statement
Research data are not shared.
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
|cbic202100700-sup-0001-misc_information.pdf317.7 KB||Supporting Information|
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
- 1, , J. Ind. Microbiol. Biotechnol. 2016, 43, 155–176.
- 2, , Acc. Chem. Res. 2017, 50, 1566–1576.
- 3, , Angew. Chem. Int. Ed. 2017, 56, 3770–3821;
- 4, , , Nat. Prod. Rep. 2012, 29, 1074–1098.
- 5, , , , Nat. Prod. Rep. 2020, 37, 355–379.
- 6, , , , Chem. Biol. 2002, 9, 103–112.
- 7, , , , Biochemistry 2005, 44, 2770–2780.
- 8, , , Proc. Natl. Acad. Sci. USA 2005, 102, 10111–10116.
- 9, , , , , , , , , Chem. Biol. 2006, 13, 1183–1191.
- 10, , , J. Am. Chem. Soc. 2006, 128, 3900–3901.
- 11, , , Biochemistry 2000, 39, 15522–15530.
- 12, , , , , , Angew. Chem. Int. Ed. 2020, 59, 21535–21540;
- 13, , , , , , Angew. Chem. Int. Ed. 2021, 60, 10319–10325;
- 14, , , , , J. Am. Chem. Soc. 1956, 78, 3075–3077.
- 15, , , , , , , , Antibiot. Annu. 1956, 730–735.
- 16, , , , , , , Nature 1954, 173, 72–73.
- 17, , in: Mech. Action (Eds.: D. Gottlieb, P. D. Shaw), Springer, Heidelberg, 1967, pp. 481–493.
- 18, , J. Nat. Prod. 2020, 6, 1990–1997.
- 19, , , Chem. Biol. 2007, 14, 13–22.
- 20, , , , , , , , , Nature 2019, 565, 112–117.
- 21, , , FEBS J. 2009, 276, 1641–1653.
- 22, , , , , Curr. Opin. Chem. Biol. 2013, 17, 537–545.
- 23, , , , , , , , , , , , Nucleic Acids Res. 2021, 49, D412–D419.
- 24, , ACS Catal. 2015, 5, 6153–6176.