Elevation of Global O-GlcNAc Levels in 3T3-L1 Adipocytes by Selective Inhibition of O-GlcNAcase Does Not Induce Insulin Resistance

Macauley, Matthew; Bubb, Abigail K.; Martinez-Fleites, C.; Davies, G.J.; Vocadlo, David J.

doi:10.1074/jbc.m804525200

Cited by 108 publications

(139 citation statements)

References 52 publications

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“…2(E)] and compared this complex with that of BtGH84 bound to PUGNAc. 31 The electron density maps clearly reveal the presence of N-butyrylPUGNAc bound within the active site pocket of BtGH84 [ Fig. 2(E)].…”

Section: Resultsmentioning

confidence: 99%

“…Rearrangement of this loop also moves the general acid/base residue, Asp243, a remarkable 7.5 Å away from its normal position, making it unable to interact with the inhibitor. In the Xray structure of BtGH84 bound to PUGNAc, Asp243 sits in the same position as the unliganded enzyme, 22,31 and forms a hydrogen-bond to the carbamate nitrogen of the N-phenylcarbamate moiety of PUGNAc. Although this interaction is lost in the N-butyryl-PUGNAc complex, it is interesting to note that the N-phenylcarbamate moiety of N-butyryl-PUGNAc is flipped 180 so that its carbonyl oxygen can instead accept a hydrogen bond from the imidazole of His443.…”

Section: Resultsmentioning

confidence: 99%

“…The four carbon acyl group of N-butyryl-PUGNAc was bound within this pocket; however, its presence appears partly responsible for displacing the inhibitor from being seated deeply within the active site as seen for the BtGH84-PUGNAc complex. 31 This alternate binding mode induces a number of gross structural changes throughout the active site region of the enzyme as compared to the structure of the BtGH84-PUGNAc complex.…”

Section: Resultsmentioning

confidence: 99%

“…The inhibitor is bound via hydrogenbonds (dashed lines), including one between the carbonyl oxygen of the N-phenylcarbamate extension and His433, which appears to order this moiety relative to the flexible binding observed within VcNagZ DSC . F: Superposition of N-butyrylPUGNAc and PUGNAc bound 31 complexes of BtGH84 reveals large structural distortions and displacement of N-butyrylPUGNAc within the active site. Blue electron densities are maximum-likelihood weighted 2F obs À F calc syntheses contoured at 0.24 e/Å 3 .…”

Section: Resultsmentioning

confidence: 99%

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

Balcewich

Stubbs

et al. 2009

Protein Science

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NagZ is an exo-N-acetyl-b-glucosaminidase, found within Gram-negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N-acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC b-lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo-N-acetyl-b-glucosaminidases: O-GlcNAcase and the b-hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring-group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2-N-acyl derivatives of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram-negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N-valeryl-PUGNAc, the most selective known inhibitor of NagZ over both the human b-hexosaminidases and O-GlcNAcase. The selectivity stems from the five-carbon acyl chain of N-valeryl-PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O-GlcNAcase homologue bound to a related inhibitor N-butyryl-PUGNAc, which bears a four-carbon chain and is selective for both NagZ and O-GlcNAcase over the human b-hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O-GlcNAcase homologue. A comparison of these complexes, and with the human b-hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2-N-acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated b-lactam resistance.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Balcewich

Stubbs

et al. 2009

Protein Science

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“…Competitive inhibition of OGA glycosidase activity using a GlcNAc analogue O-(2-acetoamido-2 deoxy-Dglucopyranosylidene)-amino-N-phenylcaramate (PUGNAc) results in elevation of global O-GlcNAc levels, and suppresses insulin-stimulated glucose uptake and Akt phosphorylation in 3T3-L1 adipocytes 42) . However, such insulin-desensitizing effects are not observed when 3T3-L1 adipocytes are treated with 1,2-dideoxy-2'-propyl-α-D-glucopyranoso-[2,1-D]-Δ2'-thiazoline (NButGT), a more potent and selective OGA inhibitor 43) , and similar results were also obtained in a rodent model 44) , indicating that non-targeted globally elevated O-GlcNAc levels do not independently cause the development of insulin resistance 43,44) .…”

Section: Ogamentioning

confidence: 99%

Effects of exercise on the hexosamine biosynthetic pathway and glycosylation

Shirato

Kizaki

Ohno

et al. 2012

JPFSM

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Minor amounts of glucose incorporated into the cells is metabolized via the hexosamine biosynthetic pathway, resulting in the production of uridine-5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is utilized as a donor substrate for the N-and O-linked glycosylation of extracellular and membrane proteins in the Golgi apparatus, or for the O-linked GlcNAc modification (O-GlcNAcylation) of intracellular proteins in the cytosol. In particular, O-GlcNAcylation, the addition of GlcNAc to serine/threonine residues, is reversibly regulated by the enzymatic activities of O-GlcNAc transferase (OGT) and O-GlcNAcase. Since OGT activity is sensitive to the intracellular UDP-GlcNAc level, flux via the hexosamine pathway influences O-GlcNAcylation. Actually, excess flux via the hexosamine pathway and resultant increased O-GlcNAcylation are associated with several pathophysiological conditions, including insulin resistance. To date, there are several studies addressing the effects of exercise on the hexosamine pathway and O-GlcNAcylation. Thus, this short review focuses on the relationships among exercise, the hexosamine pathway, O-GlcNAcylation, and insulin resistance.

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Carbohydrases

Brumer

2010

Encyclopedia of Catalysis

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The enzymes that catalyze the synthesis and degradation of oligo‐ and polysaccharides are among the most important in the biosphere, because of the key role they play in the global carbon cycle. Thanks to a long and rich history of scientific study, the catalytic mechanisms of the glycoside hydrolases and transglycosylases are generally well understood in terms of enzyme‐substrate interactions and transition state structure. Recent structure‐function studies of polysaccharide lyases have also begun to unravel the secrets of catalysis in these distinct classes of carbohydrate‐active enzymes. Built upon a firm understanding of enzyme structure and mechanism, an impressive number of technological applications highlight the prowess of these biological catalysts.

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Elevation of Global O-GlcNAc Levels in 3T3-L1 Adipocytes by Selective Inhibition of O-GlcNAcase Does Not Induce Insulin Resistance

Cited by 108 publications

References 52 publications

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

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

Effects of exercise on the hexosamine biosynthetic pathway and glycosylation

Carbohydrases

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