Saprotrophic fungi and phytopathogens cause crops to decay and spread diseases in plants by breaking down organic matter. They do this by using enzymes such as galactose oxidase and glyoxal oxidase. In trees the fungi use enzymes to break through lignin and reach the glucose in cellulose, producing hydrogen peroxide as they do this. In leaves they aim for the veins.
Phytopathogens decay crops by degrading cell walls to reach the polysaccharides surrounding the plant liquids. These organisms can devastate crops once they take hold, sometimes causing losses of 30 – 50 per cent. This can have a dramatic effect on humans, damaging the economy and resulting in famines in developing countries and causing the price of food to rise for consumers. There is further value in understanding the processes that enzymes in fungi and phytopathogens use to break down plants.
The enzyme class galactose 6-oxidase/ glyoxal oxidase was first discovered in the late 1950s. Very little is understood about them so they present more opportunities to explore their range and phylogenetic diversity.
The function of two enzymes from the class, colletotrichum graminicola (CgrAlcOx) and c.gloeosporiodes (CglAlcOx), were examined by DeLu (Tyler) Yin et al. to observe their biocatalytic reactions. Collectotrichum was chosen because it is responsible for around 6 per cent of annual yield loss through blight in anthracnose leaf and rot in the stalks of maize.
The enzymes were shown to catalyse aliphatic alcohols very well, producing hydrogen peroxide H2O2 but could not oxidase galactose and galactosides. For example, CgrAlcOx had a minimal impact on galactose, raffinose and xyloglucan as well as glucose, xylose lactose and arabinose but it oxidized glycerol significantly.
The researchers used a Bruker EMX electron paramagnetic resonance instrument at 155 K and 9.3 GHz in combination with the Easyspin 4.0.0 software to identify what was happening. They analysed the EPR spectra produced by CgrAlcOx and CglAlcOx samples with and without glycerol. These samples had initially been incubated with ethylenediaminetetraacetic acid (EDTA), a chemical good for attracting metals, to remove any weak connections to copper, an element in the structures. High frequency microwave radiation in the EMX instrument can activate unpaired orbitals to encourage them to produce characteristic patterns of radiation. The instrument detects any unpaired electrons. For example, these could be H, OH or HO2. The EMX identifies and studies them to provide more information about what is happening in the reaction.
Using EPR, the crystal structures of CgrAlcOx and CglAlcOx had one mononuclear copper site. This coordinating ligand in the enzymes played an active role in the reactions with the aliphatic alcohols. Tests included investigating the resting states of the enzymes under EPR.
The CgrAlcOx was found to have a 3D structure with two domains and b-sheets and connecting loops.
- DeLu (Tyler) Yin et al. Structure–function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family. Nature Communications 2015; 6:10197 doi:10.1038/ncomms10197
- Andrew J. Baron et al. Structure and Mechanism of Galactose Oxidase. The Journal of Biological Chemistry, 1994, Vol. 269, No. 40, Issue of Oct 7, pp. 25095-25105
- Royal Society of Chemistry on EDTA http://www.rsc.org/chemistryworld/podcast/CIIEcompounds/transcripts/EDTA.asp
- Natural History of the Fungi information on saprotrophs http://website.nbm-mnb.ca/mycologywebpages/NaturalHistoryOfFungi/Saprotrophs.html
- Britannica on nature and importance of plant diseases: http://www.britannica.com/science/plant-disease