Human OGA binds substrates in a conserved peptide recognition groove

Abstract

Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.

NagZ is required for beta-lactamase expression and full pathogenicity in Xanthomonas campestris pv. campestris str. 17

“…PNP-GlcNAc is an artificial chromophoric substrate for bGlcNAcase, such as the O-GlcNAcases in metazoans [26]; and the glucosaminidases and chitobiases in bacteria [6,22]. In this work we showed that the inactivation of NagZ xc decreased about 40% of the activity for pNP-GlcNAc.…”

Structure of a bacterial putative acetyltransferase defines the fold of the human O -GlcNAcase C-terminal domain

“…Bacterial homologues have previously been used to gain insights into the structure, mechanism and substrate recognition of the metazoan OGA-GH84 catalytic domain [30,32,34]. Of particular interest is a GH84 from the marine bacterium Oceanicola granulosus , Og OGA, which was recently crystallized [34], as it shows higher sequence identity to hOGA when compared with other bacterial OGA homologues, with sequence conservation extending beyond the catalytic core revealing a conserved peptide-binding groove [34]. Strikingly, close inspection of the Og OGA genomic location reveals an open reading frame coding for a predicted AT [25,38] immediately downstream of the Og OGA gene (figure 1 a ).…”

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

“…For OGA, our knowledge of how this enzyme recognizes substrates is still less understood. Structures of bacterial homologues of hOGA have been determined (32,46,47), and a conserved peptide substrate binding groove has been proposed (47); however, no detailed kinetic studies evaluating hOGA activity on O-GlcNAc-modified proteins have been performed. To gain insight into the processing of protein substrates by these two enzymes, we performed a series of kinetic studies examining both hOGT and hOGA activity on five structurally diverse proteins that can be recombinantly expressed at high levels within E. coli both in an O-GlcNAc-modified form and in their unmodified state.…”