The post-translational modification of serine and threonine residues of nucleocytoplasmic proteins with 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) is a reversible process implicated in multiple cellular processes. The enzyme O-GlcNAcase catalyzes the cleavage of ␤-O-linked GlcNAc (O-GlcNAc) from modified proteins and is a member of the family 84 glycoside hydrolases. The family 20 ␤-hexosaminidases bear no apparent sequence similarity yet are functionally related to O-GlcNAcase because both enzymes cleave terminal GlcNAc residues from glycoconjugates. Lysosomal ␤-hexosaminidase is known to use substrate-assisted catalysis involving the 2-acetamido group of the substrate; however, the catalytic mechanism of human O-GlcNAcase is unknown. By using a series of 4-methylumbelliferyl 2-deoxy-2-N-fluoroacetyl-␤-D-glucopyranoside substrates, Taft-like linear free energy analyses of these enzymes indicates that O-GlcNAcase uses a catalytic mechanism involving anchimeric assistance. Humans have three genes encoding enzymes that cleave terminal N-acetylglucosamine residues from glycoconjugates. The first of these, O-GlcNAcase, 1 is a member of family 84 of glycoside hydrolases that includes enzymes from organisms as diverse as prokaryotic pathogens and humans (1, 2). 2 The substrates of O-GlcNAcase are post-translationally modified glycoproteins bearing the monosaccharide 2-acetamido-2-deoxy-␤-D-glucopyranoside linked to serine or threonine residues (O-GlcNAc) (3-5). This post-translational modification is abundant in mammalian cells (3) and is found on many cellular proteins having a wide range of vital cellular functions, including for example, transcription (6 -9), proteasomal degradation (10), and cellular signaling (11), and is also found on many structural proteins (12)(13)(14). Consistent with the abundance of O-GlcNAc on intracellular proteins, it appears to have roles in the etiology of several disease states, including type II diabetes (11, 15), Alzheimer (13,16,17), and cancer (18). Although O-GlcNAcase was likely isolated earlier on (19,20), an understanding of its biochemical role in acting to cleave O-GlcNAc off from modified serine and threonine residues of proteins waited some 20 years (21). More recently OGlcNAcase has been cloned (23), partially characterized (24), and suggested to have additional activity as a histone acetyltransferase (22). Little, however, is known about the catalytic mechanism of this enzyme.HEXA and HEXB are the two other genes that encode enzymes catalyzing the hydrolytic cleavage of terminal N-acetylglucosamine residues from glycoconjugates. The gene products of HEXA and HEXB predominantly yield two dimeric isozymes. The homodimeric isozyme, hexosaminidase B (␤␤), is composed of two ␤-subunits, and the heterodimeric isozyme, hexosaminidase A (␣␤), is composed of an ␣-and a ␤-subunit. Both of these enzymes are normally localized within the lysosome. The two subunits bear a high level of sequence identity, and both are members of family 20 of glycoside hydrolases. The dysfunction of...

The NagZ class of retaining exo-glucosaminidases play a critical role in peptidoglycan recycling in Gram-negative bacteria and the induction of resistance to beta-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-beta-d-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochemical characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to beta-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnology.

Orbitides are cyclic ribosomally synthesized and post-translationally modified peptides from plants; they consist of standard amino acids arranged in an unbroken chain of peptide bonds. These cyclic peptides are stable and range in size and topologies making them potential scaffolds for peptide drugs; some display valuable biological activities. Recently, two orbitides whose sequences were buried in those of seed storage albumin precursors were said to represent the first observable step in the evolution of larger and hydrophilic bicyclic peptides. Here, guided by transcriptome data, we investigated peptide extracts of 40 species specifically for the more hydrophobic orbitides and confirmed 44 peptides by tandem mass spectrome- This study has revealed what is likely to be a very large new family of orbitides, uniquely buried alongside albumin and processed by AEP.