Full‐Length Human Collagen Lysyl Hydroxylases

Abstract

Procollagen lysyl hydroxylases and glycosyltransferases (LH, also known as procollagen lysyl‐2‐oxoglutarate dioxygenases (PLOD)) are essential biosynthesis enzymes present in all collagen‐containing organisms, from sponges to humans. Higher vertebrates present three separate PLOD genes encoding for distinct enzyme isoforms (LH1, LH2a/b, and LH3), sharing ∼70% amino acid sequence identity. The LH1 and LH2 isoforms exclusively display Fe 2+ , 2‐oxoglutarate‐dependent lysyl 5‐hydroxylase activity, whereas LH3 is a multifunctional enzyme, able to further catalyze the Mn 2+ ‐dependent β‐(1, O )‐galactosylation and the subsequent α‐(1,2)‐glucosylation of 5‐hydroxylysines. Despite exclusive selectivity for lysine residues within collagenous polypeptides, little is known about the specificity of LH enzymes for different amino acid sequences in different collagen types: LH1 and LH3 isoforms act on collagen triple‐helical regions, whereas the LH2 isoform specifically hydroxylates collagen telopeptides, yet no consensus sequences, nor minimum sequence lengths, have been proposed as requirements for catalysis. Available crystal structures of full‐length human LH3 show an elongated homodimeric quaternary structure, with three aligned domains constituting each enzyme's polypeptide: the N‐terminal glycosyltransferase (GT) domain, a central noncatalytic accessory (AC) domain, and a C‐terminal lysyl hydroxylase (LH) domain. Dimerization occurs in the C‐terminal domain, in proximity to the LH catalytic site. Dimerization is indeed essential for LH activity, but is dispensable for the glycosyltransferase activities of LH3.

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

“…Collagen hydroxylysine galactosylation is a key step in the complex post-translational modification pathway that leads to Glc-Gal-Hyl, the most abundant O-linked glycosylation found in the animal kingdom 12,13,16 . Following decades of intensive biochemical investigations addressing the enzymes involved in this process and their associated functions, the recent years have witnessed increasing efforts to produce a comprehensive molecular description of these reactions and the actors involved, with particular attention to multifunctional LH/PLOD lysyl hydroxylases-glucosyltransferases 17,34 . The lack of experimental structure data about GLT25D/COLGALT galactosyltransferases have limited our understanding of the molecular mechanisms of these glycosyltransferases, despite previous significant efforts 16,27,37 .…”

“…The path to the formation of Glc-Gal-Hyl starts with the formation of 5-hydroxylysine (Hyl) operated by collagen lysyl hydroxylases (LH, also known as procollagen, lysyl 2-oxoglutarate dioxygenases (PLOD)). The LH/PLOD enzyme family is characterized in humans by three different isoforms (LH1/PLOD1, LH2/PLOD2 and LH3/PLOD3) 11,17,18 . The LH reaction depends on the presence of tightly bound Fe 2+ in the C-terminal domain of LH/PLODs, which is essential for the electron transfer, and on ascorbate, that preserves the redox state of the metal ion [19][20][21] .…”

A Fe2+-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes

“…Among the different PTMs, lysine (Lys) hydroxylation is key for proper collagen fibril formation, thus defining the overall physiochemical properties of ECM ( Yamauchi and Sricholpech, 2012 ). The collagen lysyl hydroxylase (LH/PLOD) enzyme family comprises the three isoforms LH1/PLOD1, LH2/PLOD2 and LH3/PLOD3 (encoded by the procollagen-lysine, 2-oxoglutarate 5-dioxygenase ( PLOD ) genes) and is the sole enzyme capable of hydroxylating collagen Lys in humans ( Scietti and Forneris, 2020 ). These enzymes use Fe 2+ , 2-oxoglutarate (2-OG), ascorbate and O 2 to catalyze the addition of a hydroxyl group in position 5 of collagen Lys, yielding 5-hydroxylysine (Hyl) with the release of succinate and CO 2 ( Figure 1 ).…”

“…In particular, the crystal structure of the full-length LH3/PLOD3, the first of a human collagen lysyl hydroxylase, provided key insights on catalytic pockets. LH3/PLOD3 is a multifunctional enzyme capable of performing collagen lysine hydroxylation (as the other two isoforms) and glycosylation ( Scietti and Forneris, 2020 ; De Giorgi et al, 2021 ). Indeed, LH3/PLOD3 can additionally catalyze the galactosylation and further glucosylation of Hyl to form α-(1,2)-glucosyl-β-(1,O)-galactosyl-5-hydroxylysine in vitro .…”