PT  - JOURNAL ARTICLE
AU  - Antonella Chiapparino
AU  - Francesca De Giorgi
AU  - Luigi Scietti
AU  - Silvia Faravelli
AU  - Tony Roscioli
AU  - Federico Forneris
TI  - A cooperative network of molecular “hot spots” highlights the complexity of LH3 collagen glycosyltransferase activities
AID  - 10.1101/841486
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 841486
4099  - http://biorxiv.org/content/early/2020/04/04/841486.short
4100  - http://biorxiv.org/content/early/2020/04/04/841486.full
AB  - Hydroxylysine glycosylations are collagen-specific post-translational modifications essential for maturation and homeostasis of fibrillar as well as non-fibrillar collagen molecules. Lysyl hydroxylase 3 (LH3) is the only human enzyme capable of performing two chemically-distinct collagen glycosyltransferase reactions using the same catalytic site: inverting beta-1,O-galactosylation of hydroxylysines and retaining alpha-1,2-glycosylation of galactosyl hydroxylysines.Here, we used structure-based mutagenesis to show that both glycosyltransferase activities are strongly dependent on a broad cooperative network of amino acid side chains which includes the first-shell environment of the glycosyltransferase catalytic site and shares features with both retaining and inverting enzymes. We identified critical “hot spots” leading to selective loss of inverting activity without affecting the retaining reaction. Finally, we present molecular structures of LH3 in complex with UDP-sugar analogs which provide the first structural templates for LH3 glycosyltransferase inhibitor development.Collectively, our data provide a comprehensive overview of the complex network of shapes, charges and interactions that enable LH3 glycosyltransferase activities, expanding the molecular framework for the manipulation of glycosyltransferase functions in biomedical and biotechnological applications.