Rapid O2-evolving technologies for time-resolved spectroscopic & crystallographic applications

Enzymes such as chlorite dismutase and manganese catalase, which produce rapid bursts of oxygen upon substrate addition, offer a powerful tool for mechanistic investigations of oxygen-dependent enzymatic reactions.

These enzymes can quickly oxygenate an anoxic environment, allowing for the almost immediate and simultaneous initiation of oxygen-dependent processes, which is particularly valuable when studying oxygenase enzymes involved in important physiological reactions.

This approach addresses the challenge of limited oxygen availability in mechanistic studies by providing a controlled and rapid means of oxygen delivery, enabling precise synchronization of reaction initiation.

Consequently, this opens up novel opportunities for time-resolved analysis of reaction intermediates and helps to overcome experimental limitations posed by slow or uneven diffusion from existing methods.

I aim to develop novel methodologies which make use of these enzyme systems to facilitate spectroscopic and crystallographic study of oxygenases.

I will specifically be collaborating with the teams working at beam lines I24 and VMXi at Diamond Light Source synchrotron in Harwell campus, hoping to adapt and develop their new drop-on-demand sample delivery systems to investigate enzymatic reaction mechanisms in-crystallo.

By using micro-crystals to enable rapid oxygen diffusion into the crystals, I aim to conduct time-resolved crystallographic studies to enable identification of novel reaction intermediates. Through this project, I will specifically aim to use Renilla reniformis luciferase (RLuc) as a model oxygenase enzyme.

RLuc is an oxygen-dependant a light-emitting enzyme, used ubiquitously in biochemistry as a reporter molecule.

However, despite its widespread use, mechanistic understanding of the enzyme is lacking, and the emission characteristics are sub-optimal for in-vivo study.

By studying RLuc using this novel rapid-oxygen generating system, I hope to advance our structural and mechanistic knowledge of this remarkable enzyme, identifying proposed intermediates during catalysis and uncovering potential protein dynamics involved in the light-emitting process.

These findings will serve as a foundation for directed protein engineering enabling optimisation of the enzyme for in-vivo reporting functions.

This project therefore falls within the EPSRC Chemical biology and biological chemistry research area through aiming to create a novel technology which will enable time-resolved mechanistic study of oxygenases, specifically including RLuc.

This research has significant potential impact as oxygenases serve a multitude of physiologically important roles (including the hypoxia response and DNA repair) and are thus central in many human diseases (such as pulmonary hypertension and cancer).

By advancing the available technology enabling study of these enzymes, the search for drugs and treatments against these diseases can be further facilitated.