Characterization of a β‐N‐acetylhexosaminidase and a β‐N‐acetylglucosaminidase/β‐glucosidase from Cellulomonas fimi

Abstract

Most enzymes catalyzing the hydrolysis of terminal b-N-acetylglucosaminide linkages belong to families 3 and 20 of the glycoside hydrolases ([1,2] and the glycoside hydrolases database at URL http://afmb.cnrs-mrs. fr/CAZY/). Members of the two families greatly differ in structure, enzyme mechanism, substrate specificity, and physiologic function (for a review see [3] and references cited therein). The enzymes in family 20 are designated as N-acetylhexosaminidases (EC 3.2.1.52) because they hydrolyze b-N-acetylgalactosaminides and b-N-acetylglucosaminides, with about a four-fold greater activity on the latter ([1], and references cited therein). b-N-Acetylglucosaminidases (EC 3.2.1.52) in family 3 are much more specific for the gluco-configuration, The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular b-N-acetylglucosaminidases, a family 20 b-N-acetylhexosaminidase (Hex20), and a novel family 3-b-N-acetylglucosaminidase ⁄ b-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: k cat ⁄ K m values at 25°C were 0.066 s )1 AEmm )1 and 0.076 s Abbreviations DNP-2FGlc, 2¢,4¢-dinitrophenyl 2-deoxy-2-fluoro-b-D-glucopyranoside; Dp, degree of polarization;

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

“…For GH3 enzymes, it has been found that the 2-acetamido group of the substrate is not essential for cleavage of the glycosidic bond, consistent with these enzymes using an enzymic nucleophile. 19,27 The X-ray structure of NagZ from Vibrio cholerae (VcNagZ) in complex with its GlcNAc product (PDB entry: 1Y65) corroborates this finding, clearly showing that the 2-acetamido group of bound GlcNAc is not positioned to participate directly in CAO bond cleavage. Crystallographic studies of GH20 human HexA 25 and HexB, 24 and GH84 bacterial homologues of human O-GlcNAcase from Bacteroides thetaiotaomicron (BtGH84) 22 and Clostridium perfringens 26 have revealed that these families of enzymes bind the 2-acetamido group in a strikingly different manner.…”

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

“…This modified motif is also present in the only other known glycoside phosphorylase in the GH3 family, Nag3 31,32 . In order to confirm that the activity detected was indeed associated with this gene it was subcloned into a pET expression vector and heterologously expressed and purified.…”

“…In both cases the interaction at that position is between an amide and a hydroxyl, but the directionality of the interaction is inverted; a beautiful example of specificity swapping. Indeed, it had previously been speculated that the broadened substrate specificity seen for Nag3, which also prefers glucoside to N-acetylhexosaminide substrates, was due to the substitutions seen in the modified sequence motif 31 :…”

“…BLASTX query of these against the CAZy database revealed a novel GH3 ORF (hereafter referred to as bglP) located within the overlapping region of the two fosmids. Based on amino acid sequence analysis (Figure 5), bglP was predicted to encode a β-glucosidase/Nacetylglucosaminidase (NagZ), from a hexosaminidase subgroup of GH3 characterized by the sequence motif 33 K-H-(FI)-P-G-(HL)-G-X 4 -D-(ST)-H. However, the motif found in bglP had substitutions at two residues (underlined):This modified motif is also present in the only other known glycoside phosphorylase in the GH3 family, Nag3 31,32 . In order to confirm that the activity detected was indeed associated with this gene it was subcloned into a pET expression vector and heterologously expressed and purified.…”

“…These enzymes use a β-inverting mechanism in which phosphate attacks directly on the sugar anomeric center, forming α-glucose-1-phosphate (αG1P) (Figure 1B). The third enzyme, the β-glucosidase/N-acetylglucosaminidase Nag3 from Cellulomonas fimi 31,32 , belongs to CAZY family GH3 and uses a β-retaining mechanism involving a covalent α-glycosyl-enzyme intermediate that is then attacked by phosphate, forming a β-glucose-1-phosphate (βG1P) (or βGlcNAc-1-phosphate) product ( Figure 1C).As shown in Figure 2A all three enzymes cleave DNPGlc in the presence of phosphate, as can be observed by monitoring the increase in absorbance at 400 nm. Importantly, all three enzymes are only minimally active in the absence of phosphate.…”