Post-translational α-Keto-β-amino Acids: Chemical Reactivity and Protein Engineering

Abstract

Natural products or secondary metabolites are heterogeneous group of complex chemical compounds that are ubiquitous in all life forms. Peptide natural products contain wide range of nonproteinogenic amino acids that affect their structure, proteolytic stability and bioactivity. Chemical diversity of these highly modified peptides translates to a spectrum of important biological functions such as potent antibiotic and cytotoxic activities. Biosynthesis of peptide natural products follow two orthogonal pathways involving either ribosomal or nonribosomal process. This work pivots on ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are found across all kingdoms of life. Nature has evolved remarkable RiPPs enzymology that introduces variety of posttranslational modifications (PTMs) on gene encoded precursor peptides. We describe the genome-guided discovery of a fundamentally new PTM that installs β-amino acids in ribosomal proteins. The new and synthetically challenging peptide backbone modification is catalysed by an uncharacterized subfamily of radical S-adenosyl L-methionine enzyme - splicease. Spliceotide, an as-yet unknown natural product, is encoded in the plp biosynthetic gene cluster found in cyanobacterium Pleurocapsa. Isotopic labelling studies revealed that the protein splicing involves an impressive carbon-carbon bond cleavage-reconnection at the XYG motif and net excision of a tyramine equivalent that produces an α-keto-β-amino moiety. It has been demonstrated that PlpXY splicease can incorporate a variety of β-amino acids into the genetically encoded precursor PlpA3 and protease inhibitor sequences. As follow-up to this study, the body of work featured in this thesis focuses on biotechnological applications of the protein splicing. Initial insights on splicease substrate recognition revealed that PTM is introduced independently of the cognate PlpA3 precursor leader peptide. The splicease modification is governed by the core peptide sequence that can be as short as 10 amino acids. The minimal core peptide was genetically fused to either C- or N- terminus, or inserted in internal loop regions of maltose binding protein and tested for splicease activity in heterologous coexpression. The protein splicing was exercised at different locations of five different target protein including fluorescent protein mCherry and dihydrofolate reductase. We leverage the protein splicing to develop a new bioorthogonal protein labelling technology that combines the aforementioned peptide motif-guided tyramine excision and bioorthogonal ɑ-ketoamide reactivity. Together this thesis provides a functional characterization of a novel RiPP enzymology found in cyanobacteria. The enzyme substrate tolerance studies have paved the way for heterologous production of proteins containing β-amino acids, which in turn enabled development of bioorthogonal protein labelling technology.