Abstract
Kutznerides are hexadepsipeptide antifungal and antimicrobial agents containing O-methyl-L-serine in their very unique peptidic backbone. During kutznerides biosynthesis, this O-methylated amino-acid residue is proposed to result from the action of an adenylation (A) domain present in KtzH, which is interrupted by the S-adenosylmethionine-binding-containing part of a methyltransferase. In this study, we co-expressed recombinant KtzH(A 4 MA 4 T 4) with its MbtH-like protein partner KtzJ and demonstrated the requirement for KtzJ in producing soluble and active KtzH(A 4 MA 4 T 4). We demonstrated the specificity of KtzH(A 4 MA 4 T 4) toward L-Ser and showed the activity of the partial methyltransferase enzyme in O-methylation of L-Ser after its covalent attachment to the thiolation domain of KtzH(A 4 MA 4 T 4). The insights gained from this work may guide future study and development of engineered interrupted adenylation domains for combinatorial biosynthetic methodologies.
| Original language | English |
|---|---|
| Pages (from-to) | 59-64 |
| Number of pages | 6 |
| Journal | Journal of Antibiotics |
| Volume | 67 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 2014 |
Bibliographical note
Funding Information:This work was supported by a NSF CAREER Award (grant no. MCB-1149427) (to SG-T) as well as by startup funds from the University of Kentucky (to SG-T). We thank Finn P. Maloney and Dr Wenjing Chen for cloning of the KtzH and KtzJ constructs used in this study. We thank Dr Oleg V. Tsodikov for critical reading of the manuscript and insightful comments. We thank Dr Anders Broberg (Swedish University of Agricultural Sciences, Uppsala, Sweden) for the gift of the Kutzneria sp. 744 bacterial strain used to isolate genomic DNA. We thank Dr Michael G. Thomas (University of Wisconsin-Madison, WI, USA) for the gift of the BL21 (DE3)ybdZ::aac(3)IV bacterial strain. SG-T thanks Professor Christopher T. Walsh, not only an amazing mentor but also a continuous source of inspiration and support.
Funding
This work was supported by a NSF CAREER Award (grant no. MCB-1149427) (to SG-T) as well as by startup funds from the University of Kentucky (to SG-T). We thank Finn P. Maloney and Dr Wenjing Chen for cloning of the KtzH and KtzJ constructs used in this study. We thank Dr Oleg V. Tsodikov for critical reading of the manuscript and insightful comments. We thank Dr Anders Broberg (Swedish University of Agricultural Sciences, Uppsala, Sweden) for the gift of the Kutzneria sp. 744 bacterial strain used to isolate genomic DNA. We thank Dr Michael G. Thomas (University of Wisconsin-Madison, WI, USA) for the gift of the BL21 (DE3)ybdZ::aac(3)IV bacterial strain. SG-T thanks Professor Christopher T. Walsh, not only an amazing mentor but also a continuous source of inspiration and support.
| Funders | Funder number |
|---|---|
| National Science Foundation (NSF) | 1408798 |
Keywords
- Antifungal
- Antimicrobial
- Interrupted adenylation domain
- MbtH-like protein
- Methyltransferase
- Nonribosomal peptide
- Protein production and activation
ASJC Scopus subject areas
- Pharmacology
- Drug Discovery