Dry plant matter (biomass) is an abundantly available raw material for the production of biofuels. The principal carbohydrate polymer it contains, cellulose, is packed with glucose units that can be fermented into bioethanol - a sustainable liquid fuel. These polymers are difficult to break down chemically, but we get a helping hand from the natural enzymes that have evolved to do the job. Widely found enzymes, lytic polysaccharide monooxygenases (LPMOs), are major contributors to natural carbon recycling and are now used in commercial bioethanol production. However, questions remain around how these enzymes survive the powerful chemistry they wield. In work recently published in the Journal of the American Chemical Society, researchers from the University of Manchester, Novozymes, Graz University of Technology, the University of York and Diamond Light Source, used a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, electron paramagnetic resonance spectroscopy and High Energy Resolution Fluorescence Detection X-ray Absorption Spectroscopy (HERFD−XAS) to investigate how these oxidative enzymes protect themselves from harmful side reactions. Their results show that short-lived molecules produced during the breakdown of polysaccharides provide a built-in defence and repair mechanism
Low carbon fuels will play an important role in the decarbonisation of the transport sector. Sustainably produced biomass is a renewable, low carbon energy source that can be used to produce liquid and gaseous fuels to replace fossil fuels. However, 'first generation' biofuels produced from easy-to-digest edible crops such as sugarcane or corn starch require valuable arable land and can drive up food prices.
Making use of more difficult-to-digest sources such as plant stems and cardboard waste to produce 'second generation' biofuels is an attractive option.
Prof Paul Walton from the University of York says,
Fermenting things like grass or leaves, or waste paper, would give us a really good source of sustainable fuel, it's the Holy Grail of biofuels. The problem is breaking it down, but nature has solved that, and enzymes found in fungi or bacteria are being used commercially to make the bioethanol people are pumping into their petrol tanks. So understanding how these enzymes work, and how we can make them more efficient, is important.
To break down biomass, the enzymes have to cleave the bonds between glucose molecules, and an important question is how the enzymes themselves avoid being destroyed in the process. Understanding that involves catching a glimpse of a reaction that takes place in milliseconds.
Prof Walton continues,
During the reaction with the biomass, the active site of the LPMO enzymes - the part that does the work - is damaged, and that happens on a timescale of about 50 milliseconds. Our experiments used enzymes made and studied by our colleagues at the University of Manchester, led by Dr Derren Heyes and Prof Anthony Green. And Abbey Telfer, who is a PhD student between Diamond and the University of York, captured that moment of the reaction using a freeze quench technique. It literally stops the reaction in its tracks, so we can see what's happening in these tiny windows of time.
Once they had their frozen samples, the team analysed them using a variety of techniques. At Diamond's I20-Scanning beamline, they used HERFD−XANES to examine the active site of the enzyme.
Their results show a previously unseen, short-lived intermediate step in the reaction, and its role in defending the enzyme against oxidative damage. As well as demonstrating the protective mechanisms built-in to biological systems, this study has practical implications for maximising the efficiency of oxidative catalysts. Achieving optimal catalyst performance will require careful balancing of oxidant concentration and reducing equivalents.
For Prof Walton, this study is part of a larger project investigating LPMOs and related enzymes, particularly in their use for biomass degradation, but also the role they play in plant pathogens. The team will now continue their research at Diamond to study how these enzymes associate with their substrates - the target material - and how they are activated by its arrival.
To find out more about the I20-Scanning beamline or discuss potential applications, please contact the Science Group Leader, Sofia Diaz-Moreno: sofia.diaz-moreno@diamond.ac.uk
Zhao J et al. Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase. Journal of the American Chemical Society (2023). DOI:10.1021/jacs.3c06607.
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