A cooperative network of molecular 'hot spots' highlights the complexity of LH3 collagen glycosyltransferase activities

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 applications. SIGNIFICANCE STATEMENT Multifunctional collagen lysyl hydroxylase-glycosyltransferase LH3 is essential for biosynthesis and maturation of collagen molecules and has strong implications in rare developmental diseases as well as in metastatic spreading of solid tumors. LH3 is the only human enzyme capable of carrying out two different glycosyltransferase reaction mechanisms within the same catalytic site to produce glucosyl-galactosyl-hydroxylysine, the most ubiquitous glycosylation pattern found in nature. This work elucidates the molecular networks enabling the LH3 glycosyltransferase reactions, highlighting a highly cooperative network of amino acid residues that concertedly ensure collagen glycosylation, and suggesting that the LH3 molecular organization may be the progenitor of more specialized glycosyltransferases. In addition, this work presents the first two small molecule scaffolds capable of modulating LH3 glycosyltransferase activities.