Molecular structure and enzymatic mechanism of the human collagen hydroxylysine galactosyltransferase GLT25D1/COLGALT1
M De Marco, Sristi Raj, Luigi Scietti, Daiana Mattoteia, Stefano Liberi, Elisabetta Moroni, Alberta Pinnola, Alice Vetrano, Claudio Iacobucci, Carlo Santambrogio, Giorgio Colombo, Federico Forneris
Abstract
During collagen biosynthesis, lysine residues undergo extensive post-translational modifications through the alternate action of two distinct metal ion-dependent enzyme families (i.e., LH/PLODs and GLT25D/COLGALT), ultimately producing the highly conserved α-(1,2)-glucosyl-β-(1,O)-galactosyl-5-hydroxylysine pattern. Malfunctions in these enzymes are linked to developmental pathologies and extracellular matrix alterations associated to enhanced aggressiveness of solid tumors. Here, we characterized human GLT25D1/COLGALT1, revealing an elongated head-to-head homodimeric assembly. Each monomer encompasses two domains (named GT1 and GT2), both unexpectedly capable of binding metal ion cofactors and UDP-α-galactose donor substrates, resulting in four candidate catalytic sites per dimer. We identify the catalytic site in GT2, featuring an unusual Glu-Asp-Asp motif critical for Mn2+ binding, ruling out direct catalytic roles for the GT1 domain, but showing that in this domain the unexpectedly bound Ca2+ and UDP-α-galactose cofactors are critical for folding stability. Dimerization, albeit not essential for GLT25D1/COLGALT1 activity, provides a critical molecular contact site for multi-enzyme assembly interactions with partner multifunctional LH/PLOD lysyl hydroxylase-glycosyltransferase enzymes. Hydroxylysine galactosylation is essential for collagen maturation. This work reveals the molecular structure of human GLT25D1, its catalytic residues, and a noncatalytic structural domain stabilized by metal ion and donor substrate binding.