Studies of glucose-6-phosphate dehydrogenase
Creators: name: Zakaria, Nurul Aili
Publisher: name: The Australian National University
Year: 2016
Subjects: Glucose 6-phosphate dehydrogenase
G6PD
Directed evolution
Tetrazolium salt WST-8
Colorimetric screening assay OR Colourimetric screening assay
Enzyme-coupling reaction

description: Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucolactone with the reduction of NADP+ to NADPH. It is the first component of the pentose phosphate pathway (PPP) that is a central and important biochemical pathway. The PPP functions to produce building block compounds and intermediates for energy production. The first three enzymes of the PPP are referred to as the oxidative phase and produce NADPH that is crucial for dealing with oxygen stress, especially in people with G6PD deficiency. There are tests for G6PD deficiency, but those used in the field are not reliable. The existing assays for G6PD use the NADPH it generates to reduce a dye and produce a colour change, which can be observed with the naked eye and can be used to measure the kinetic parameters of the purified G6PD in the laboratory. Unfortunately, it suffers from a number of problems when used with crude preparations under field conditions. The equilibrium constant for the G6PD reaction is close to 1 so that the G6PD substrate will only be depleted if subsequent enzymes in the PPP are present. The extent of colour change will also depend upon the activity of the next two enzymes in the PPP and these may vary from person to person. The mutant forms of G6PD may be unstable, and may gives rise to false positive results during the assays. Hence, sensitive and reliable assays are needed. Here, we report an improvised colorimetric method for G6PD detection using tetrazolium salt, WST-8. The method used enzyme-coupling reaction of G6PD with another two downstream enzymes in the PPP. Addition of large excess of the two enzymes decreased the response time and increases the sensitivity of the test. Consequently, this enables detection of G6PD enzyme activity at low substrate concentrations compared with an assay of NADPH absorbance at 340 nm. The methods also generate NADPH eight times faster than reaction with G6PD alone. This makes the test more sensitive and predictive, which is also ideal for industrial applications. We also extend the application of the colorimetric WST-8 assay for screening of recombinant libraries in a directed evolution study for improved stability. The screening protocols were modified to colony agar-blotting using filter paper as primary screening and 96 well plates for secondary screening. The orange coloured formazans produced by the reaction, make it suitable and reliable for high-throughput qualitative and quantitative assays for dehydrogenases. With only four rounds of evolution, the method successfully identified promising variants with far improved thermal and chemical (urea) stability. Studies characterizing the properties and understanding the effect of mutations on variants are also discussed. The mutations responsible for improved stability were found located at the large structural NADP+ domain that is important for integrity and stability of the structure. The results also showed that the mutations brought changes in the oligomeric forms of G6PD. The thermostable variants were found to form higher order oligomers compared to the native enzyme. The correlation between enzyme stability and oligomerisation was also studied briefly.