Significant discovery of vitamin B6 biosynthesis enzyme

A simple explanation for a complex biochemical synthesis leading to effective drug design

A paper published in Nature Chemical Biology is the culmination of more than 10 years of research, for a team led by Dr Ivo Tews of Southampton University, into the structure of proteins involved in the biosynthesis of vitamin B6 in plants and bacteria. Pyridoxal 5′-phosphate (PLP) is an active form of vitamin B6; the PLP-synthase complex, which consists of the enzymes Pdx1 and Pdx2, is found in all domains of life. The published work focuses on a Pdx1 enzyme from Arabidopsis thaliana, and furthers our understanding of how the protein catalyses the synthesis of vitamin B6. X-ray crystallography and spectroscopy allowed the discovery of previously unknown intermediate stages of the process, which offers up a new target for drugs designed to combat diseases such as malaria and TB by affecting the synthesis of a vitamin that is essential for all organisms.

     
 
Figure 1: The electron density map and model of the I320 intermediate structuregenerated in the synthesis of vitamin B6.
 
Ten years of research
Over the course of ten years of research, the team experimented with enzymes from more than ten different organisms. Whilst the proteins in all of the organisms are very similar, and can be expected to work in similar ways, small changes in them make a big difference to the crystals produced. During the experiments, researchers added a substrate to the crystallised proteins, so that the resulting reactions happened in crystal form. They determined that the Pdx1 enzyme from the plant Arabidopsis thaliana was the most suitable candidate for their experiments.
 
Throughout their research, the team has worked with Cornell University, Diamond Light Source and ESRF. Their work at Diamond involved X-ray crystallography and spectroscopy on the I24 beamline. X-ray crystallography was used to show the different shapes the enzyme takes on during the catalysis of vitamin B6 biosynthesis, and allowed the team to uncover a previously unknown intermediate stage - I320. Given that this process takes place in a wide range of organisms, including those that cause human diseases, unlocking this information gives drug designers a new target, and could lead to more effective treatments for diseases like malaria and TB.
 
Multi-crystal analysis to assess beam damage
One of the techniques described in the paper is a multi-crystal analysis that was used to assess damage to the target crystals, caused by exposure to the X-ray beam. The team wanted to investigate whether damage to the crystals was leading to protein changes. They collected 50 partial datasets using very brief exposures to the X-ray beam to ensure any X-ray induced damage would be minimal. These datasets were then merged together to form a single composite dataset, which could then be used to determine a low dose structure for I320.
 
Diamond PhD programme
The research at Diamond was carried out in part by PhD student Matthew Rodrigues. Dr Rodrigues’ PhD was co-funded and co-supervised by Dr Gwyndaf Evans from Diamond and Dr Ivo Tews from University of Southampton. Diamond partners with degree-awarding institutions to offer a large PhD programme - there are currently over 70 PhD students - as part of its commitment to developing skilled researchers. Each year Diamond issues a call for PhD project proposals, which are peer-reviewed, by a panel selected by the board of directors, to ensure that the most exciting projects are funded. These opportunities are advertised here and by the partner institution. Diamond’s status as a world-leading facility makes this a very good opportunity for students, who have access to experts on the beamline staff throughout. By virtue of being here for the duration of a studentship Dr Rodrigues was able to gain wide range of experience encompassing the basics of micro-crystal positioning through to the process of complex data analyses and dealing with challenging samples. Diamond’s PhD students are also encouraged to attend conferences and present their work, and are given poster and presentation training in preparation, as well as a full range of non-academic support from Student Engagement Officer Kristina Penman. 

 

To find out more about the I24 beamline, or to discuss potential experimental applications, please contact Principal Beamline Scientist Dr Robin Owen: robin.owen@diamond.ac.uk. In order to discover more about the PhD programme at Diamond, please see here or contact diamond.students@diamond.ac.uk.

Related publication:

Rodrigues MJ et al. Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis. Nature Chemical Biology, advance online publication (2017). DOI: 10.1038/nCHeMBIO.2273