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:
Consistent with our in silico modelling, we confirm that selective mutation at T107 of GLO1 changes the structure of GLO1, as demonstrated by Small Angle X-ray Scattering (SAXS) and the macromolecular and microfocus beamlines at the Australian Synchrotron (ANSTO, Melbourne, VIC, Australia). In particular, a phospho-mimetic mutant T107D-GLO1, shows an increase in protein dimensions when compared to wild-type GLO1. Ni-NTA affinity purified T107D-GLO1 and T107E-GLO1 also demonstrate little or no catalytic-activity compared to wild-type GLO1. Other mutants including T107A and T107P also show reduced activity, while T107V retains the activity of wild-type GLO1, but cannot be phosphorylated/de-activated.
Conclusion:
These findings support the critical nature of residue at position 107 in maintaining GLO1 structure and function and may have implications for age-related decline in GLO1 activity and ageing per se.
Key words:
Glyoxalase 1; GLO1; Post-translational modification; Phosphorylation; Structure