13 12 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 Macromolecular Crystallography Group Beamline I04 Understanding the biochemistry of gut bacteria Biochemistry – Chemistry – Genetics – Structural Biology – Life Sciences & Biotech Lipids are vital components of all cells, forming the main component of cell membranes. Many different lipids are found in membranes, with a wide range of functions beyond their primary role in cellular compartmentalisation. Inositol is a carbocyclic sugar that is a key player in eukaryotic cells and forms the polar head group of inositol lipids. Inositol lipids are not major components of eukaryotic cell membranes, but they play important roles in cell division and signalling between cells. While inositol lipids are widely distributed across eukaryotes, little is known of their role and structure in bacteria. An international team of researchers focused on understanding how different lipids are synthesised in bacteria investigated how the dominant gut microbe Bacteroides thetaiotaomicron makes inositol lipids. B. thetaiotaomicron is an important member of the human gut microbiome and can use a wide range of dietary carbohydrates as carbon sources. The team is interested in how these bacteria interact with the host through various signalling molecules, including lipids. For this research, they used a range of techniques, including lipid analysis and biochemical characterisation of enzymes combined with structural biology. Diamond’s I04 Macromolecular Crystallography beamline is a versatile facility for macromolecular crystallography, with the ability to focus the beam on very small crystals and access different X-ray energies. The ability to adjust the beam size to the sample size in order to optimize signal to noise, combined with a very stable beam, allowed the researchers to obtain very high quality data that permitted structure solution and the generation of a high resolution model of one of the key enzymes in this pathway, the myo-inosotol phosphate synthase. After identifying the genes responsible for B. thetaiotaomicron inositol lipid, the researchers deleted each of these from the genome to see their effect on the production of the inositol lipids. They identified the different lipids produced in these genomic deletion strains using mass spectrometry. These results allowed them to determine the order in which different components of the final inositol lipid are combined. B. thetaiotaomicron bacteria with genomic deletions in the inositol lipid synthesis genes are less able to survive in their host. Although we do not know exactly why this is the case, it may be due to changes in the cell membrane and associated protective polysaccharide capsule around the cells that makes the cells more susceptible to antimicrobial compounds produced by the host, or change how the cells are seen by the host’s immune system. Changes in the composition of the gut microbiome can affect host health. By developing an understanding of the biochemistry of bacteria in the gut, we can gain knowledge of the roles they play in various diseases, such as inflammatory bowel diseases. The role of inositol lipids in eukaryotic signalling and cellular homeostasis is well established, and the fact that they are produced by important members of the human microbiome has implications for communication between these bacteria and their hosts. As we learn more about the lipids made by bacteria and how they are made, we can investigate how they use them to manipulate their host to survive and thrive. Related publication: Heaver, SL, et al. Characterization of inositol lipid metabolism in gut- associated Bacteroidetes. Nature Microbiology 7 , 986–1000 (2022). DOI: 10.1038/s41564-022-01152-6 Funding acknowledgement: Max Planck Society BBSRC, grants BB/V001620/1 and BB/V00168X/1 NIH grant R24GM137782-01. Corresponding authors: Jon Marles-Wright, Newcastle University, Jon
[email protected] Ruth Ley, Max Planck Institute for Developmental Biology,
[email protected] Structure of the B. thetaiotaomicron myo-inositol phosphate synthase enzyme. The enzyme adopts a structure with four identical subunits arranged as a dimer of dimers. Two subunits are shown with their molecular surfaces, and two with secondary structure elements displayed as tubes and arrows for alpha helices and beta-strands respectively. The NAD + cofactor, that participates in the isomerisation reaction to produce inositol-3-phosphate from glucose-6-phosphate, is highlighted in the structure and shown as sticks buried in the active site of the structure. Macromolecular Crystallography Group Beamline I03 Characterisation of the enzymatic degradation of PET plastic as found in plastic bottles Earth Sciences & Environment – Biotechnology – Pollution – Catalysis – Chemistry – Structural Biology – Materials Science – Engineering & Technology – Biophysics – Polymer Science – Life Sciences & Biotech Plastic products may have revolutionised the world, but their poor degradability is causing major pollution problems. Poly(ethylene terephthalate) (PET) is one of the most abundant plastics used in polyester textiles and in packaging for food and drinks. PET’s water-repellent properties make it a good choice for drinks bottles. However, they alsomake discarded PET bottles highly resistant to breaking down in the natural environment. Remaining intact for hundreds of years, PET waste will accumulate unless we can find a way to deal with it. A possible solution presented itself in 2018, when an international team of researchers announced the discovery of a bacterium with the amazing ability to use plastic as an energy source. Ideonella sakaiensis, initially found feeding on waste from an industrial PET recycling facility in Japan, degrades PET from plastic bottles into its building blocks. Central to this ability is the bacterium’s production of a PET-digesting enzyme called PETase. PETase is a mesophilic enzyme, meaning that it is only stable at moderate temperatures (30°C). However, PET degradation is better at higher temperatures. Therefore, a more thermostable enzyme is desirable. The team’s ongoing research has produced and identified PETase variants, including a double amino acid mutant (mutating residue 159 from tryptophan to histidine and 238 from serine to phenylalanine) with improved PET degradation relative to the wild-type PETase. In this work, they used Diamond’s I03 beamline to examine the structural basis of the double mutant's enhanced PET degradation in greater detail. The 1.45 Å resolution data set they collected for the double mutant allowed them to determine its atomic protein structure. They also used differential scanning calorimetry to determine the melting temperatures of the enzymes and high-performance liquid chromatography to quantify the products in biochemical assays. Their results show that the double mutant PETase narrows the active site cleft and has a 10°C higher melting temperature than the wild-type enzyme. Furthermore, while the activity of the wild-type enzymes drops after 48 hours, the double mutant PETase is active at 40°C for more than a week. However, the wild-type enzyme is more active at 30°C than the double mutant and is also more active at 40°C within the first two days. The past decade has seen incredible progress in identifying, characterising and engineering PET hydrolases, including the PETase from Ideonella sakaiensis featured in this study. However, engineering the enzyme is just one of the tools in the toolkit for improving efficiency. Reaction optimisation and process design tuned to the characteristics of the waste stream may also prove critical in developing an efficient enzymatic recycling process. Strategies for implementing and scaling-up enzymatic recycling technologies are on the horizon. Related publication: Erickson, E. et al. Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation. ChemSusChem 15 , (2022). DOI: 10.1002/cssc.202101932 Funding acknowledgement: Research England E3 funding Corresponding authors: Dr Michael Zahn, University of Portsmouth,
[email protected] Prof Andy Pickford, University of Portsmouth,
[email protected] Artist's interpretation of a plastic PET bottle degradation by enzyme PETase.