Studying non-covalent enzyme carbohydrate interactions by STD NMR

Brecker, Lothar; Schwarz, Alexandra; Goedl, Christiane; Kratzer, Regina; Tyl, Catrin; Nidetzky, Bernd

doi:10.1016/j.carres.2007.12.023

Cited by 11 publications

(15 citation statements)

References 37 publications

(76 reference statements)

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“…In plants substrate is turned into the C-3-branched sugar UDP-apiose (b) as well as to UDP-galacturonic acid (UDP-GalUA) (c). [16][17][18][19][20] Obtained binding pattern results are correlated to recently solved crystal structure of hUXS1A and docking studies of UDP-GlcUA, 10 providing a better understanding of substrate speci-city of this enzyme and may give useful information in designing various mutants. [4][5][6][7][8][9] different tissues, which may lead to e.g.…”

Section: Introductionmentioning

confidence: 84%

Saturation transfer difference NMR to study substrate and product binding to human UDP-xylose synthase (hUXS1A) during catalytic event

et al. 2015

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The human form of UDP-xylose synthase (hUXS1A) is studied with respect to its substrate and co-enzyme binding in binary and ternary complexes using saturation transfer difference (STD) NMR and in situ NMR. Obtained binding pattern results are correlated to the recently solved crystal structure of hUXS1A and docking studies of UDP-GlcUA, providing a better understanding of substrate specificity of this enzyme and may give useful information in mutant designing. In unproductive binary complexes UDPsaccharide aglycone moieties show strong STD effects with the protein. In contrast, pyranoside rings (Glc, GlcUA, Gal) indicate less interaction with the hUXS1A active site, which enables the required ring distortion of the pyranoside ring in UDP-GlcUA. In productive ternary complexes UDP-GlcUA possesses reasonable binding, while produced UDP-Xyl shows smaller STD responses and does not efficiently compete with the substrate for binding at the active site. STD NMR derived binding studies of NAD + demonstrate tight interaction between co-factor and hUXS1A. Higher magnetization of NAD + in the presence of enzymatic product is observed and suggests increased contact with groups on the protein. Furthermore, binding studies of substrate analogues having the same stereochemistry as the investigated UDP-saccharides and a small aglycone residue indicate a different mode of action, not guided by the anchor groups.

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Section: Introductionmentioning

confidence: 84%

Saturation transfer difference NMR to study substrate and product binding to human UDP-xylose synthase (hUXS1A) during catalytic event

et al. 2015

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“…Dabei geht es um die strukturelle Änderung dieser Liganden während der Reaktion. Aus den Daten lassen sich kinetische und mechanistische Modelle der enzymatischen Umsetzungen erstellen und konkretisieren 6. So wird untersucht, wie das Enzym Stärke‐Phosphorylase aus Corynebacterium callunae das Produkt Glucosyl‐1‐phosphat bindet.…”

Section: Strukturdetails Und Bindungen: Noe Und Std‐nmrunclassified

Protein‐Ligand‐Wechselwirkungen

Brecker¹,

Husa²

2013

Nachr Chem

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“…Application with catalytically active enzymes and truly converted substrates instead of inhibitors is challenging, as the required high enzyme concentration leads to rapid depletion of the substrate and the disappearance of its signal. Reaction temperatures below the optimum for the enzyme allows studying an enzyme with its natural substrates 14. 15 A 1000‐fold decrease of the catalytic efficiency of two glycoside hydrolases (to the μ M −1 s −1 range) allowed a detailed and very insightful characterisation of substrate binding in a slow but still catalytically competent system 14.…”

Section: Introductionmentioning

confidence: 99%

“…Reaction temperatures below the optimum for the enzyme allows studying an enzyme with its natural substrates 14. 15 A 1000‐fold decrease of the catalytic efficiency of two glycoside hydrolases (to the μ M −1 s −1 range) allowed a detailed and very insightful characterisation of substrate binding in a slow but still catalytically competent system 14. Although these methods alleviate the problem of substrate consumption, STD‐NMR is mostly limited to quasi‐stationary systems.…”

Section: Introductionmentioning

confidence: 99%

STD‐NMR‐Based Protein Engineering of the Unique Arylpropionate‐Racemase AMDase G74C

et al. 2015

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Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.

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Studying non-covalent enzyme carbohydrate interactions by STD NMR

Cited by 11 publications

References 37 publications

Saturation transfer difference NMR to study substrate and product binding to human UDP-xylose synthase (hUXS1A) during catalytic event

Saturation transfer difference NMR to study substrate and product binding to human UDP-xylose synthase (hUXS1A) during catalytic event

Protein‐Ligand‐Wechselwirkungen

STD‐NMR‐Based Protein Engineering of the Unique Arylpropionate‐Racemase AMDase G74C

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