Exploring and exploiting the enzymes involved in tambjamine YP1 natural product biosynthesis

Natural products are secondary metabolites produced by many different organisms such as bacteria, fungi and plants. These biologically active molecules have been widely exploited for medicinal purposes generating a large proportion of the drugs in clinical use today. Tambjamine YP1 is an antimicrobial, antimalarial and cytotoxic bipyrrole natural product produced by the anti-biofouling marine microorganism Pseudoalteromonas tunicata. Its biosynthetic pathway is encoded in an operon (the tam cluster) consisting of 19 genes, 10 of which have proposed biosynthetic functions involved in bipyrrole formation and attachment of a fatty amine tail. However, details of the biosynthesis of these moieties have not been elucidated. This thesis presents studies on the characterization of four of the recombinant enzymes isolated from Escherichia coli (TamA, TamD, TamF and TamH) and identifies their roles in the biosynthetic pathway of tambjamine YP1. Biosynthesis of the bipyrrole backbone is proposed to begin with the formation of the first pyrrole ring from L-proline, a common route to pyrrole biosynthesis in natural products. In contrast, the second pyrrole ring is assembled in an unusual linear manner by two didomain enzymes, TamF and TamD. Sequence analysis of TamF suggests it is a didomain fusion composed of a chain length factor (CLF) and a ketosynthase (KS). Similarly, TamD is also an unusual didomain fusion of an acyl carrier protein (ACP) and an α-oxoamine synthase (AOS). Together TamF and TamD produce the first bipyrrole intermediate in the tambjamine YP1 biosynthetic pathway, 4-hydroxy-2,2’-bipyrrole 5-methanol (HBM). Mass spectrometry (MS) analysis shows that TamF can be loaded with a cysteine-bound, β-ketopyrrole thioester and the TamD ACP domain can be converted to the malonyl-ACP form. TamF then catalyzes the Claisen-like condensation between the malonyl-TamD ACP and its β-ketopyrrole substrate to elongate the chain by two carbons. The resulting TamD ACP-bound diketopyrrole product is a substrate for the TamD AOS domain that catalyzes a second Claisen-like condensation with L-serine to release the product, HBM. This work defines a role for TamF and TamD in HBM biosynthesis and presents the first in vitro production and detection of this key bipyrrole intermediate by high performance liquid chromatography (HPLC) and MS analysis. Attached to the tambjamine YP1 bipyrrole backbone is a fatty amine tail predicted to be derived from a C12 fatty acid. Previously, this moiety was proposed to be activated as a CoA thioester by a fatty acid CoA ligase (FACL) from outside the tam cluster. However, sequence analysis of TamA, an enzyme in the tam cluster with no previously assigned function, suggested that it is an unusual didomain fusion of a fatty acid AMP ligase (FAAL) with a Cterminal ACP domain that could carry out this function. Surprisingly, MS analysis of recombinant TamA revealed the presence of bound C11 and C12 fatty acid adenylates, which can be transferred to the ACP domain upon attachment of a 4’-phosphopantetheine (4’-PP) to a conserved serine residue. An in trans acyl-transfer assay was developed using the recombinant TamA ACP domain and was used to screen fatty acid substrates with variable chain lengths (C2-C16). It was found that TamA can efficiently transfer several fatty acids (C6- C13) with the best substrate being the C12 acid. Contrary to the previous hypothesis, MS analysis showed that the C12 acylated TamA ACP domain acts as a substrate for TamH, a downstream enzyme in the pathway. This interesting didomain fusion contains thioester reductase (TR) and transaminase (TA) domains which work sequentially to produce the C12 amine from the TamA ACP-bound fatty acid thioester. This amine product can subsequently combine with an HBM-derived aldehyde to generate the mature tambjamine YP1 natural product. Furthermore, preliminary data on the use of TamA for the production of the recently discovered class of N-acyl histidine amides from Legionella pneumophila is presented. In summary, key enzymes from the tambjamine YP1 pathway have been functionally characterized. As well as defining their roles in the pathway, this work also has potential impact on understanding the biosynthesis of other pyrroles (e.g. prodiginines) and polyketides. This work lays the foundations for engineering of the Tam proteins to produce novel tambjamine derivatives for biological testing