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

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 alpha-(1,2)-glucosyl-beta-(1,2)-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 (GT1 and GT2), both unexpectedly capable of binding metal ion cofactors and UDP-alpha-galactose donor substrates, resulting in four candidate catalytic sites per dimer. We identified 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 the unexpectedly bound Ca2+ and UDP-alpha-galactose cofactors are critical for folding stability. Dimerization was not essential for GLT25D1/COLGALT1 activity, but rather a hallmark for multi-enzyme assembly interactions and/or collagen substrate recognition.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Alice Vetrano

LAB HEAD: Claudio Iacobucci

PROVIDER: PXD061021 | Pride | 2025-03-05

REPOSITORIES: Pride

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			Action	DRS
	LH3_GLT_8_GB4_1_2907.d.zip	Other
	LH3_GLT_8_GB4_1_2907_6.1.331.mgf	Mgf
	LH3_GLT_8_GB4_1_2907_6.1.331.zhrm	Other
	LH3_W158_R_9_GB6_1_2911.d.zip	Other
	LH3_W158_R_9_GB6_1_2911_6.1.331.mgf	Mgf

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