Background and aims:
The glyoxalase pathway is responsible for the detoxification of reactive dicarbonyls that are generated as a by-product of cellular metabolism. Glyoxalase 1 (GLO1) is the rate-limiting enzyme of the glyoxalase pathway. Reduced activity of GLO1 leading to dicarbonyl stress is associated with accelerated ageing and age-related dysfunction. Despite the increased burden of AGEs, in ageing animals there is a reduction in GLO1 activity. At the same time, the expression of Glo1 mRNA is unaltered. We hypothesise that post-translational modification of GLO1 is increased with age and contributes to reduced GLO1 activity. We have previously used in silico modelling to show that phosphorylation of Threonine-107 (T107), a key residue of an exo-loop adjacent to but not within the catalytic domain, potentially induces a change in the structure of GLO1, including altered accessibility and binding of substrate to the catalytic domain.
Methods and Results:
We produced and purified several GLO1 T107 mutant proteins. Ni-NTA affinity-purified GLO1 T107 variants demonstrated varied catalytic-activity compared to wild-type GLO1. We confirm that selective mutation at T107 of GLO1 alters the structure of GLO1, as demonstrated by Small Angle X-ray Scattering (SAXS), and consistent with our in silico modelling. In particular, phospho-mimetic residues at position 107 displayed significant differences in protein dimensions and activity when compared to wild-type GLO1. Additionally, a synthetically generated phosphorylated T107 purified protein variant indicates that phosphorylation at T107 does alter the activity of GLO1 compared to wild-type unphosphorylated GLO1.
Conclusion:
These findings support the critical nature of the residue at position 107 and its modification in maintaining GLO1 structure and thereby function. These results have interesting implications for the role of GLO1 activity in diabetes, cancer, age-related decline and in ageing per se.