Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases

Abstract

Glycosyltransferases (GTs) are prevalent across the tree of life and regulate nearly all aspects of cellular functions. The evolutionary basis for their complex and diverse modes of catalytic functions remain enigmatic. Here, based on deep mining of over half million GT-A fold sequences, we define a minimal core component shared among functionally diverse enzymes. We find that variations in the common core and emergence of hypervariable loops extending from the core contributed to GT-A diversity. We provide a phylogenetic framework relating diverse GT-A fold families for the first time and show that inverting and retaining mechanisms emerged multiple times independently during evolution. Using evolutionary information encoded in primary sequences, we trained a machine learning classifier to predict donor specificity with nearly 90% accuracy and deployed it for the annotation of understudied GTs. Our studies provide an evolutionary framework for investigating complex relationships connecting GT-A fold sequence, structure, function and regulation.

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites

“…These transferases are highly divergent, with residues in the core GT-A fold sharing sequence identities of <17% ( Table S2 ). Despite the low degree of sequence conservation, the core structural elements of the GT-A folds were conserved, with the largest differences arising from loop insertions into the GT-A fold core ( 37 ). This set of GT-A fold glycosyltransferases display donor substrate diversity (enzymes employing UDP-GlcNAc, UDP-GalNAc, UDP-Glc, UDP-GlcA, UDP-Xyl, or GDP-Man sugar donors) as well as varied catalytic mechanisms that include metal-dependent and metal-independent inverting and metal-dependent retaining enzymes ( 38 ) ( Table S2 ).…”

“…The only significant sequence conservation in the donor binding site is found in the D x D motif of metal-dependent GT-A fold enzymes ( 37 ). The first residue in the D x D motif is either an Asp or Glu, which interacts with the hydroxyl groups of the donor sugar to position the substrate ( 37 , 39 ). The “x” residue is usually an acidic residue (Asp or Glu) or a small aliphatic.…”

“…S3 ). Prior evolutionary studies have identified four conserved landmark features among GT-A fold enzymes ( 37 ): the D x D motif for metal cation interactions, a “Glycine-rich” loop facing the acceptor and donor sugar site at the N-terminal end of the conserved β7 strand (β8 in B3GNT2, Fig. S1 ), an “xED” motif at the beginning of helix F (α10 in B3GNT2) harboring the catalytic base, and a “C-His” residue that coordinates with the metal ion.…”

“…S1 ), an “xED” motif at the beginning of helix F (α10 in B3GNT2) harboring the catalytic base, and a “C-His” residue that coordinates with the metal ion. In addition, this prior work identified three positions in the Rossmann fold core where hypervariable loops (HV1, HV2, and HV3) were potentially inserted to provide acceptor binding subsites ( 37 ). We anticipated a similar positioning of key structural features in the B3GNT2 active site.…”

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.…”