Biochemical and structural studies of ribosomally synthesised and post-translationally modified peptide biosynthetic complexes

Zheng, Dazhong (2024) Biochemical and structural studies of ribosomally synthesised and post-translationally modified peptide biosynthetic complexes. PhD thesis, University of Glasgow.

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a prominent class of natural products that present a wide spectrum of chemical structures and bioactivities, spanning from anti-cancer to antimicrobial properties. They raised interest among the scientific community due to their intriguing biosynthesis pathways which utilise the ribosome to generate a peptidic scaffold that can be subsequently modified by biosynthetic enzymes to produce the mature natural product. Among the myriad of choices of RiPP classes, three of them were chosen to be studied: thioholgamide, thiomuracin and crocagin.

Recent studies have unveiled the biosynthetic step of thioholgamide (Tho), yet the formation mechanism of the AviCysMe is poorly understood. Further studies of its biosynthetic gene cluster’s enzymes have narrowed down candidates responsible for the macrocyclisation, namely ThoE and ThoF. In this study, our efforts were focused on gaining insight by solving their structure using X-ray crystallography. The atomic coordinates of ThoF were successfully solved while ThoE still remains mysterious. A homologue of thioholgamide, thiosparsoamide (Spa), presents an interesting alternative ring closure mechanism. Although Spa pathway contains all the genes necessary for the cyclisation reaction, it appears to utilise a distant gene, namely SpaKC, in combination with SpaF. Demonstrated to function as a dimer, cryoEM was employed to elucidate their relationship. Unfortunately, no complex was observed upon capturing the particles.

Thiomuracin (Tbt) is another RiPP class that employs multiple proteins to produce thiazole motifs. Azoline-forming natural products typically involved an YcaO protein (TbtG), but in Tbt pathway, one domain is missing, and the protein seems to work in close relationship with an Ocin-ThiF-like protein (TbtF). This pathway was investigated through different biophysical assays and capturing the threedimensional structure of the complex. A first comprehension of an heterocomplex atomic structure of TbtF/G was given. In addition, the crystal structure of TbtE was also solved, the protein responsible for the turnover of the thiazoline to its subsequent thiazole. Lastly, the biosynthetic steps were broken down here and chemical reaction insights of crocagin, an alkaloid motif containing new RiPP class. Its class-defining, tetracyclic-hexahydropyrrolo[2,3]indoline motif, product of tryptophan and tyrosine combination, was shown to be the product of a threeprotein association, namely CgnB/C/E. This intermediate is subsequently removed of its leader peptide, allowing further modification. Besides elucidating the biosynthesis of crocagin, a structural study of a tailoring enzyme in crocagin biosynthesis was also proposed. CgnL was involved in methylation reaction after the proteolysis of the leader peptide.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Due to copyright issues this thesis is not available for viewing.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Science and Engineering > School of Engineering
• Supervisor's Name: Koehnke, Professor Jesko
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84639
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 29 Oct 2024 10:16
• Last Modified: 30 Oct 2024 09:55
• Thesis DOI: 10.5525/gla.thesis.84639
• URI: https://theses.gla.ac.uk/id/eprint/84639