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bioRxiv · 10.1101/2024.06.21.600124

Molecular Structure and Enzymatic Mechanism of the Human Collagen Hydroxylysine Galactosyltransferase GLT25D1/COLGALT1

De Marco, M.·Rai, S. R.·Scietti, L.·Mattoteia, D.·Liberi, S.·Pinnola, A.·Forneris, F.
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Abstract

During biosynthesis, collagen lysine residues undergo extensive post-translational modifications essential for the stability and functions of collagen supramolecular assemblies. In the endoplasmic reticulum, two distinct metal ion dependent enzyme families (i.e., multifunctional lysyl hydroxylases-glucosyltransferases LH/PLODs and galactosyltransferases GLT25D/COLGALT) alternatively operate on collagen lysine side chains ultimately generating the highly conserved -(1,2)-glucosyl-{beta}-(1,O)-galactosyl-5-hydroxylysine pattern. Malfunctions in the collagen lysine post-translational modification machinery is linked to multiple developmental pathologies as well as extracellular matrix alterations causing enhanced cellular proliferation and invasiveness of several solid tumors, prompting for an in-depth characterization of LH/PLOD and GLT25D/COLGALT enzyme families. Here, we present an integrative molecular study of GLT25D1/COLGALT, highlighting an elongated head-to-head homodimeric assembly characterized by an N-terminal segment of each monomer wrapping around its dimerization partner. Each monomer encompasses two Rossman fold-type domains (GT1 and GT2) separated by an extended linker. Both domains were found capable of binding Mn2+ cofactors and UDP--galactose donor substrates, resulting in four candidate catalytic sites per dimer. Site-directed mutagenesis and biochemical studies identify the C-terminal GT2 domain as the functional GLT25D1/COLGALT1 catalytic site, highlighting an unprecedented Glu-Asp-Asp motif critical for metal ion binding, and suggesting structural roles for the N-terminal GT1 essential for correct quaternary structure assembly. Conversely, dimerization was not a requirement for GLT25D1/COLGALT1 enzymatic activity in vitro, suggesting that the elongated enzyme homodimer assembly, resembling that of LH/PLOD binding partners, could represent a functional hallmark for correct recognition and successful processing of collagen lysine residues.

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De Marco, M., Rai, S. R., Scietti, L., Mattoteia, D., Liberi, S., Pinnola, A., Forneris, F.. 2024-06-22. Molecular Structure and Enzymatic Mechanism of the Human Collagen Hydroxylysine Galactosyltransferase GLT25D1/COLGALT1. https://doi.org/10.1101/2024.06.21.600124

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