Biosynthetic studies of secondary metabolites by mass spectrometry

Abstract

Mass spectrometry is a powerful technique that can be used at every stage of natural product studies, from discovery and structural characterization of new compounds to biosynthetic enzyme identification and manipulation. The goal of this dissertation is to illustrate the utility of mass spectrometry in natural product studies through two examples: I) K-26, a hypotensive phosphonopeptide metabolite, and II) apoptolidin, an anticancer natural product of polyketide origin. The potent angiotensin converting enzyme inhibitor K-26 is a naturally occurring N-acetylated tripeptide containing isoleucine, tyrosine, and the non-proteinogenic amino acid (R)-1-amino-2-(4-hydroxyphenyl)ethyl phosphonic acid (AHEP). Structure-activity relationship studies (Chapter II) have shown that phosphonyl substitution is the critical determinant of activity in the K-26 molecule. Extremely low production levels of K-26 (less than 10 micrograms per L) required development of a highly sensitive method of detection. Isotopically labeled precursor incorporation experiments were designed to investigate the biosynthetic origin of AHEP and isotopic enrichment was calculated based on selected reaction monitoring (SRM) experiments (Chapter III). We have unambiguously identified tyrosine as an immediate precursor of AHEP and AHEP itself as a discrete precursor of K-26. After establishing the basic building blocks of K-26 and AHEP, biochemical experiments were undertaken in order to identify the genes and enzymes responsible for K-26 biosynthesis. Specifically, attempts were made to isolate the N acetyltransferase involved in the biosynthesis of K-26 (Chapter IV). An SRM based method was developed for detecting acetylation of dAcK26 and we proceeded with cell lysis and fractionation of the organism’s proteome. Several acetyltransferases were identified and their activity was evaluated. Apoptolidin is an anticancer agent produced by Nocardiopsis sp that induces cell death by a mitochondrial dependent apoptotic pathway. The sugars that are appended on the aglycone core seem to significantly increase the cytotoxicity of the molecule. Chemical glycosylation can be extremely challenging due to issues of protecting groups and stereoselectivity. We proposed using the glycosylating machinery of the organism to prepare analogues of apoptolidin by supplementing the producing organism with synthetic aglycone analogues and detecting glycosylation by mass spectrometry (Chapter V).