Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases

Abstract

1 Glycosyltransferases (GTs) are prevalent across the tree of life and regulate nearly all aspects of 2 cellular functions by catalyzing synthesis of glycosidic linkages between diverse donor and 3 acceptor substrates. Despite the availability of GT sequences from diverse organisms, the 4 evolutionary basis for their complex and diverse modes of catalytic and regulatory functions 5 remain enigmatic. Here, based on deep mining of over half a million GT-A fold sequences from 6 diverse organisms, we define a minimal core component shared among functionally diverse 7 enzymes. We find that variations in the common core and the emergence of hypervariable loops 8 extending from the core contributed to the evolution of catalytic and functional diversity. We 9 provide a phylogenetic framework relating diverse GT-A fold families for the first time and show 10 that inverting and retaining mechanisms emerged multiple times independently during the course 11 of evolution. We identify conserved modes of donor and acceptor recognition in evolutionarily 12 divergent families and pinpoint the sequence and structural features for functional specialization. 13Using the evolutionary information encoded in primary sequences, we trained a machine learning 14 classifier to predict donor specificity with nearly 88% accuracy and deployed it for the annotation 15 of understudied GTs in five model organisms. Our studies provide an evolutionary framework for 16 investigating the complex relationships connecting GT-A fold sequence, structure, function and 17 regulation. 18

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

“…The use of unique acceptor binding loop insertions in GT-B fold fucosyltransferases is highly reminiscent of hypervariable substrate binding loops in the evolution of GT-A fold glycosyltransferases (32,102,103). In contrast to the two Rossmann domains in GT-B fold enzymes, GT-A fold glycosyltransferases have a single, conserved Rossmann fold core and employ proximal residues for donor interactions, while more divergent extended loop insertions are free to rapidly evolve and diversify novel acceptor specificities (32,102,103). This rapid evolution of the loop insertions is constrained by the necessity of positioning the acceptor nucleophile for attack and access to a catalytic base for nucleophile activation relative to the C1 of the sugar donor.…”

“…While a general understanding of GT-A fold glycosyltransferase evolution is emerging (32,102,103), a parallel understanding of GT-B fold enzymes has lagged behind largely because of their more complex domain architectures and fewer determined protein structures in complex with substrate analogs. However, evolutionary parallels between the two protein fold classes are now evident and further insights into selective substrate recognition among the broader collection of GT-B glycosyltransferases will expand our understanding of how diverse glycan structures are elaborated in biological systems.…”

A phylogenetic view and functional annotation of the animal β1,3-glycosyltransferases of the GT31 CAZy family

“…However, in the 3D structure, the G residue of motif III is located far from the active site. The fourth conserved motif GxxYxxS (motif IV) found in all GT31 (Figure 3) constitutes the C-terminus part of a flexible G-loop, and the conserved glycine residue is involved in donor binding (Taujale et al 2020), and the first G residue of motif IV is located near the active site. In addition, the conserved aromatic aa residues in GT31 motifs II, III and IV were found to be well aligned in the MFNG structures (PDB 2J0A and 2J0B with UDP and Mn) suggesting their structural importance.…”

“…The last conserved motif (motif V) (E/D)DVxxGx(W/C) is located at the C-terminus part of the GT31, except for the CH sequences. This conserved motif together with the DxD motif is involved in catalytic functions of GT-A fold inverting GTs (Taujale et al 2020). The MFNG structure (PDB 2J0B) was solved with UDP but without the substrate.…”

Promiscuity and specificity of eukaryotic glycosyltransferases

“…New high-throughput experimental capabilities, including modular GTase expression systems [ 18 ], improved inference methods for determining enzymatic activity from mass spectrometry [ 11 , 19 , 20 ], and standardized glycan bioinformatic resources [ 21 , 22 , 23 ], are poised to yield comprehensive data on GTase substrate preferences. However, at present the most reliable data on GTases come from detailed studies of individual enzymes.…”