Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants

Biochemistry
Naniye CetinbaşDavid J Vocadlo

Abstract

O-GlcNAcase is a family 84 beta-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of beta-O-linked 2-acetamido-2-deoxy-d-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants of human O-GlcNAcase, D174A and D175A, were generated since these residues are highly conserved among family 84 glycoside hydrolases. Structure-reactivity studies of the D174A mutant enzyme reveals severely impaired catalytic activity across a broad range of substrates alongside a pH-activity profile consistent with deletion of a key catalytic residue. The D175A mutant enzyme shows a significant decrease in catalytic efficiency with substrates bearing poor leaving groups (up to 3000-fold), while for substates bearing good leading groups the difference is much smaller (7-fold). This mutant enzyme also cleaves thioglycosides with essentially the same catalytic efficiency as the wild-type enzyme. As well, addition of azide as an exogenous nucleophile increases the...Continue Reading

References

Sep 1, 1967·The Biochemical Journal·G LoweA Williams
Jun 1, 1996·The Biochemical Journal·B Henrissat, A Bairoch
Aug 1, 1997·European Journal of Biochemistry·S ArmingG Kreil
Sep 5, 2001·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·J A Hanover
Dec 8, 2005·Journal of the American Chemical Society·Matthew S MacauleyDavid J Vocadlo

❮ Previous
Next ❯

Citations

Sep 4, 2012·Journal of Chemical Information and Modeling·Nelson Alberto N de AlencarVicent Moliner
Jul 1, 2008·Nature Chemical Biology·Scott A YuzwaDavid J Vocadlo
Aug 19, 2011·Nature Reviews. Cancer·Chad Slawson, Gerald W Hart
Mar 28, 2006·Nature Structural & Molecular Biology·Rebecca J DennisGideon J Davies
Apr 14, 2011·Physical Chemistry Chemical Physics : PCCP·Andrea BottoniMatteo Calvaresi
Jul 1, 2008·The Journal of Biological Chemistry·Chutikarn ButkinareeGerald W Hart
Aug 3, 2010·Biochemistry. Biokhimii︠a︡·Jing LiPeng Wang
Feb 11, 2011·Bioscience, Biotechnology, and Biochemistry·Mitsutaka OgawaKazuo Kamemura
Apr 1, 2015·The Journal of Cell Biology·Michelle R Bond, John A Hanover
Aug 4, 2009·Biochimica Et Biophysica Acta·Carlos Martinez-FleitesGideon J Davies
Oct 17, 2012·Chemical Biology & Drug Design·Paulo Robson M SousaCláudio Nahum Alves
Oct 19, 2006·Biochemical and Biophysical Research Communications·Thomas N LeeRobert J Konrad
Jul 22, 2014·Critical Reviews in Biochemistry and Molecular Biology·Eun J KimJohn A Hanover
Dec 19, 2012·Chemical Society Reviews·Partha S BanerjeeJin Won Cho
May 6, 2016·Biochemistry·Matthew G AlteenTracey M Gloster
Jan 6, 2017·ACS Chemical Biology·Matthew WorthJiaoyang Jiang
Mar 21, 2017·Nature Structural & Molecular Biology·Baobin LiJiaoyang Jiang
Mar 28, 2017·Nature Chemical Biology·Christian RothGideon J Davies
Mar 28, 2017·Nature Chemical Biology·Nathaniel L ElsenDaniel J Klein
May 5, 2017·Nature Structural & Molecular Biology·Jennifer J Kohler
Mar 30, 2017·Biochimica Et Biophysica Acta. General Subjects·Kristýna Slámová, Pavla Bojarová
Mar 7, 2017·Biochimica Et Biophysica Acta. General Subjects·Hideki NakanishiXiao-Dong Gao
May 6, 2017·Chembiochem : a European Journal of Chemical Biology·Eun Ju Kim
Mar 2, 2011·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Eun J Kim
Jun 17, 2010·Biotechnology and Bioengineering·Thu V Vuong, David B Wilson
Dec 23, 2009·FEBS Letters·Helge C Dorfmueller, Daan M F van Aalten
Nov 1, 2013·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Yann FardiniTarik Issad
Jul 30, 2014·MedChemComm·Andrea C Rodriguez, Jennifer J Kohler
Feb 26, 2020·The Journal of Biological Chemistry·Villo MuhaDaan M F van Aalten
Oct 23, 2014·The Journal of Biological Chemistry·Jana AlonsoDaan M F van Aalten

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.