Ancestral Sequence Reconstruction of Isopenicillin N Synthase

β-Lactam antibiotics account for over 65% of global antibiotic use and their discovery revolutionised treatment of infectious disease. The bicyclic core of these molecules is formed by the enzyme isopenicillin N synthase (IPNS), which catalyses the oxidative double ring closure of substrate tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN). This thesis presents research into the evolutionary origins of IPNS, and potential broader uses which exploit its broad substrate tolerance. Ancestral sequence reconstruction is used to predict the structures of eight ancestral proteins ranging from approximately 900 million to 4 billion years old. These proteins are modelled and key differences between extant and ancestral IPNS homologs are explored. The ancestral proteins are then “resurrected” by bacterial expression, and two ancestors are found to turn over ACV into IPN. The ages of several of these ancestors predate the Great Oxygenation Event, where the abundance of molecular oxygen in the Earth’s atmosphere dramatically increased. It was therefore hypothesised that IPNS may have performed a different role in the past, before it had access to its O2 cofactor. Preliminary screening for alternate activity begins with the replacement of the Fe(II) centre with other metals and exposure of an alternate substrate. Finally, work begins towards the synthesis of a tripeptide isostere of ACV, where the nitrogen of the cysteinyl-valine amide is replaced with carbon. Several potential routes to a cysteinyl-valine dipeptide isostere to be eventually coupled with protected L-α-aminoadipic acid are developed. We hypothesise that the IPNS ancestors purified in this study will be able to cyclise the structure into a cyclobutanone IPN homolog. This cyclobutanone homolog is the most basic of a class of β-lactam resistance inhibitors and could be used to enzymatically build a library of potential inhibitors for screening against resistant bacteria