Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a key enzyme in the production of vital antibiotics, including penicillins and cephalosporins. A recent study led by Professor Chris Schofield from the Ineos Oxford Institute for Antimicrobial Research (IOI) uses cutting-edge structural techniques to better understand how this enzyme changes shape as it mediates the formation of the β-lactam ring, a component of these drugs that is essential for their ability to kill bacteria.

Penicillins were some of the first antibiotics discovered and remain among the most widely prescribed drugs to treat a diverse range of bacterial infections. They belong to a class of antibiotics called β-lactams, which all have a characteristic β-lactam ring in their chemical structure that is important for their antibacterial activity. During the synthesis of these drugs, the β-lactam ring is assembled by a protein catalyst (enzyme) called isopenicillin N synthase (IPNS). The reaction mediated by this enzyme starts with a linear molecule (the linear tripeptide L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine, or ACV), which in the presence of iron (Fe) and dioxygen (O₂) is turned into a ring-containing structure. While the β-lactam ring is essential for the killing activity of these antibiotics, how IPNS is able to form this structure is still being elucidated.

Previously, IOI researchers were part of an international consortium that used time-resolved crystallography – an experimental technique used by scientists to directly visualise the structure of molecules – to analyse the shape of IPNS at different stages of antibiotic synthesis. They showed that when IPNS, already bound to Fe and ACV (in a complex called IPNS:Fe:ACV), reacts with O₂, this leads to changes in the overall shape of the enzyme. Notably, these changes in shape of IPNS occur not only at the place where IPNS binds the ACV substrate (called the active site), but are propagated to structurally remote regions. These include changes to the structure of two helix-shaped regions on the surface of IPNS, called α3 and α10, whereas other helix regions do not seem to experience shape alterations. However, how exactly these conformational changes happen remained unclear.

Now, in a paper published in the Journal of Biological Chemistry, Patrick Rabe and colleagues use a variety of spectroscopy techniques – including ¹⁹F NMR (nuclear magnetic resonance) and EPR (electron paramagnetic resonance) – to characterise how IPNS changes shape during catalysis in even greater detail. These techniques, which measure how molecules interact with electromagnetic radiation, can be used to understand how the positions of different regions of the protein change relative to one another. Importantly, whereas the previous crystallography studies only provide snapshots of the structure of enzymes at specific points during their reactions, ¹⁹F NMR and EPR can be used to study the dynamic changes in shape of proteins in solution, which better represents the natural environment where these reactions take place.

To enable these studies on IPNS dynamics, the authors first generated variants of the protein that could be tagged by different labels located in the helical regions. These labelled variants were then used to investigate how binding of Fe, ACV and NO (which serves as a surrogate for O₂) induce shape changes in IPNS in solution. The results showed that once IPNS binds Fe, ACV and NO, the α3 and α10 helices are more flexible than another helix-shaped region, termed α6 helix, confirming that relatively small changes at the active site of the enzyme can affect the protein shape far away from this location. In addition to the spectroscopy experiments, the authors carried out crystallographic studies to directly visualize the structure of different labelled forms of IPNS. These studies provided further evidence for shape changes involving some, but not all, of the helical regions of IPNS. Collectively, the spectroscopic and crystallographic studies not only provide important information on how IPNS works during the synthesis of antibiotics but may also be useful for guiding future studies aimed at engineering this and other enzymes, to modify their activity or the reactions that they can perform.

Professor Christopher Schofield, who supervised the project, highlighted that: “The reaction catalysed by IPNS to make the cyclic structure of the penicillins is unique as far as we know – as synthetic chemists have not yet been able to reproduce it. These studies suggest that this may be because motions extending through large regions of IPNS are involved in converting the linear peptide into the cyclic penicillin structure. The results have implications for catalyst design and for understanding why mutations away from the active site of enzymes, including those involved in antimicrobial resistance, may have profound effects on catalysis.”

Dr. Patrick Rabe, who led the work, added that: “Pioneering work in the past focused mainly on active site changes during catalysis. Advances in cutting-edge technologies now allow for the analysis of changes of the global protein structure tuning these fascinating biological reactions. Inspired by these and future findings, we will hopefully be able to use this knowledge to alter reaction pathways, which may enable the design of potential new drugs for therapeutic benefit to combat antimicrobial resistance”.

Read the full paper: Rabe, P., Walla, C.C., Goodyear, N.K. et al. Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase. J Biol Chem https://doi.org/10.1016/j.jbc.2022.102249 (2022).