Carbohydrate-active enzymes

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Authors: ^^^Stephen Withers^^^ and ^^^Spencer Williams^^^
Responsible Curator: ^^^Spencer Williams^^^

Individual monosaccharide units have the potential to be joined together to form oligo- and polysaccharides, with the glycosidic linkage occurring between the anomeric position of one sugar with the hydroxyl group of another. Owing to the many hydroxy groups on each sugar, the potential for two possible anomeric configurations, and the possibility of different ring sizes (pyranose and furanose are the most common), there is a combinatorially-large number of structures possible. Further, carbohydrates can be linked to other, non-carbohydrate molecules to generate a wide range of glycoconjugates. Reflecting this structural diversity, there is a large diversity of enzymes involved in the biosynthesis, modification, binding and catabolism of carbohydrates.

The CAZy classification

The Carbohydrate Active EnZyme (CAZy) classification is a sequence-based classification of enzymes that are active on carbohydrate structures (see [1, 2, 3] for key reviews). The creation of a family requires at least one biochemically-characterized member, and is based on the concept that sequence defines protein structure, and protein structure defines function. Generally, but not exclusively, functional properties often extend to other members of the family, and provides a framework upon which to base testable hypotheses of enzyme structure and function.

Glycoside hydrolases (GH)

Strictly speaking, the term 'glycoside hydrolase' or 'glycosidase' refers to enzymes that catalyze the hydrolytic cleavage of the glycosidic bond to give the carbohydrate hemiacetal. However, it is found that sequence-based classification methods often group in enzymes that have non-hydrolytic activities into the same families as hydrolytic enzymes.

Transglycosidases: Sequence analysis classifies transglycosidases with retaining glycoside hydrolases. According to all available evidencetransglycosidases and glycoside hydrolases use the same mechanism, except that a sugar or some other group, rather than water, acts as the nucleophile.

Phosphorylases: Sequence similarly classifies many, but all (see Glycosyltransferases, below) phosphorylases with retaining and inverting glycoside hydrolases. Enzymatic cleavage of the bond between two sugars or between a sugar and another group by reaction with phosphate is termed phosphorolysis, and yields the sugar-1-phosphate, and the reaction is reversible, allowing synthesis of glycosidic linkages form sugar-1-phosphates. Again, GH-like phosphorylases share mechanistic similarities with glycoside hydrolases.

Alpha-glucan lyases: An unusual group of enzymes has been found within family GH31 termed alpha-glucan lyases that degrade starch via an elimination mechanism, rather than via hydrolysis, forming an unsaturated (enol) product that tautomerises to its keto form, 1,5-anhydro fructose. Again, there are mechanistic similarities between alpha-glucan lyases and glycoside hydrolases.

NAD-dependent glycoside hydrolases: Another unusual group of enzymes use an NAD-cofactor to hydrolyze through a mechanism involving a redox reaction. These enzymes are found within familes GH4 and GH109.

Seminal publications on GH classification: [4, 5, 6, 7].

Key reviews on GH and related mechanisms (e.g. GH4 enzymes and GH31 lyases): [8, 9].

Polysaccharide lyases (PL)

Polysaccharide lyases (PLs) cleave uronic acid-containing polysaccharides via a β-elimination mechanism to generate an unsaturated hexenuronic acid residue and a new reducing end at the point of cleavage. These enzymes are distinct from alpha-glucan lyases, which are classified within the GH modules, as described above.

Focussed publication on the PL classification: [10].

Reviews of PL mechanisms: [9, 10, 11].

Auxiliary activities (AA)

The original CAZy classification scheme aimed to describe the families of enzymes that cleave or build complex carbohydrates and the associated carbohydrate binding modules. The discovery that members of families CBM33 and family GH61 are lytic polysaccharide monooxygenases (LPMO), gave impetus to a reclassification of these families into a new category, the "Auxiliary Activities" [12]. The auxiliary activities are familes of catalytic proteins that are potentially involved in plant cell degradation through an ability to help the original GH, PL and CE enzymes gain access to the carbohydrates comprising the plant cell wall. Currently, the AA families contain redox active enzymes.

Introduction of the AA classification: [12].

Glycosyltransferases (GT)

The principal enzymes that catalyze glycoside synthesis are glycosyltransferases. Glycosyltransferases utilize various sugar-1-phosphate derivatives including:

Sugar mono- or diphosphonucleotides, in which case the resulting enzymes are referred to as Leloir glycosyltransferases.
Non-nucleotide carbohydrate donors including various sugar polyprenol mono- or diphosphates, in which case the enzymes are referred to as non-Leloir glycosyltransferases.
Sugar-1-phosphates and -pyrophosphates, in which case the enzymes are referred to as phosphorylases or pyrophosphorylases. Only some of such enzymes are classified as GTs on the basis of sequence (e.g. glycogen phosphorylase of GT35), others are classified as GHs.

Seminal publications on GT classification: [13, 14].

Key GT review: [15].

References

All Medline abstracts: PubMed