Carbohydrate Esterase Family 9

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Author: ^^^Alex Anderson^^^
Responsible Curator: ^^^Michael Suits^^^

Carbohydrate Esterase Family 9
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	not known
CAZy DB link
https://www.cazy.org/CE9.html

Substrate specificities

CE family 9 esterases catalyze the deacetylation of N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate. This reaction has been demonstrated to be important for both amino sugar metabolism and peptidoglycan cell wall recycling in bacteria [1]. Experimental substrate specificity profiles for two CE9 enzymes demonstrated that they are active on other structurally similar amino sugar phosphates, such as N-acetyl- galactosamine-6-phosphate and N-acetyl-mannosamine-6-phosphate, although their reported affinities are 40-fold and 6-fold lower, respectively [2].

Kinetics and Mechanism

Removal of the acetate group by CE9 enzymes is proposed to be carried out by nucleophilic attack of the acetate carbon by a metal-bound hydroxide ion [3]. A proton is donated to the amine leaving group by a catalytic acid residue, and the tetrahedral transition state is stabilized either by the interaction of a second metal ion with the polarized carbonyl oxygen [3], or by a catalytic base residue where a second metal ion is absent from the active site, although the latter has not been experimentally demonstrated.

Catalytic Residues

The precise mechanism of catalysis has yet to be elucidated for CE9, although several conserved features in the active sites of resolved CE9 members suggest they play an important role in their function. In Bacillus subtilis NagA, Thermotoga maritima NagA, and Mycobacterium smegmatis NagA, four histidine residues are responsible for coordination of the metal cofactor(s), along with a glutamate in B. subtilis and T. maritima, and an aspartic acid in M. smegmatis [3, 4, 5]. The Escherichia coli NagA appears to have a glutamine, gluatamate, asparagine and an aspartate as the coordination enviroment, although this structure crystallized as the apoenzyme, and so this configuration is uncertain [5]. In all structures, a strictly conserved aspartic acid residue is then thought to serve as a base to activate a water molecule, and then as an acid to protonate the leaving amine [3].

Three-dimensional structures

The resolved structures of CE9 enzymes demonstrate variability in their organization and metal binding. For example, Vibrio cholerae NagA and B. subtilis NagA form dimers in their biologically relevant assemblies [3, 4], while E. coli NagA forms a tetramer [5]. Additionally, these same enzymes appear to contain a Ni²⁺ ion [4], two Fe²⁺ ions [3], and a Zn²⁺ ion [5] in their active sites, respectively. All resolved CE9 enzymes contain a distorted (β/α)₈ fold containing the active site, and a small β-sheet domain comprising residues from both the N- and C-termini.

Family Firsts

First characterized

The E. coli N-acetylglucosamine-6-phosphate deacetylase NagA was the first CE9 enzyme to have its activity demonstrated [6].

First 3-D structure

The first structure of a CE9 enzyme published was the B. subtilis NagA, containing a two-Fe²⁺ catalytic center [3].

First mechanistic insight

The structure of the B. subtilis NagA enzyme was reported with a bound N-acetylglucosamine-6-phosphate molecule and provided evidence for the proposed metal-dependent catalytic mechanism [3].

References

All Medline abstracts: PubMed