Structural and functional role of disulphide bonds and substrate binding residues of the human beta-galactoside alpha-2,3-sialyltransferase 1 (hST3Gal1).

Overexpression of hST3Gal1 leads to hypersialylation of cell-surface glycoconjugates, a cancer-associated condition that promotes cell growth, migration and invasion. Upregulation of this enzyme in ovarian cancer is linked to cancer progression and metastasis, contributing also to chemotherapy resistance. Strategies for preventing metastasis include the inhibition of hST3Gal1, which demands structure-based studies on its strict regioselectivity and substrate/donor preference. Herein we describe the contribution of various residues constituting donor CMP-Neu5Ac and acceptor Galβ1-3GalNAc-R binding sites to catalysis. Removal of hydrogen bonds and/or stacking interactions among substrates and residues Y191, Y230, N147, S148 and N170 affected the enzyme's activity to a different extent, revealing the fine control needed for an optimal catalytic performance. To gain further understanding of the correlation among structure, activity and stability, the in vitro role of hST3Gal1 disulphide bonds was analysed. As expected, disruption of the Glycosyltransferase family 29 (GT29) invariant bond C142-C281, as well as the ST3Gal1 subfamily conserved disulphide C61-C139 inactivates the enzyme. While disulphide C59-C64 is not essential for function, its absence reduces the activity (k) for donor and acceptor substrates to about 67 and 72%, respectively, and diminishes the enzyme's melting temperature (T) by 7 °C.