A Divergent Substrate-Binding Loop within the Pro-oncogenic Protein Anterior Gradient-2 Forms a Docking Site for Reptin

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21) and, surprisingly, diffracted to a higher resolution of 1.63 Å than previously deposited Thal structures (P64; ~2.2 Å) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.

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Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding

Life Sciences ◽

10.1016/j.lfs.2009.06.005 ◽

2009 ◽

Vol 85 (7-8) ◽

pp. 303-308 ◽

Cited By ~ 5

Author(s):

Takeshi Miura ◽

Toru Nishinaka ◽

Tomoyuki Terada

Keyword(s):

Substrate Binding ◽

Coenzyme Binding ◽

Loop Region ◽

Binding Loop

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Molecular dynamics-based investigation of InhA substrate binding loop for diverse biological activity of direct InhA inhibitors

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2015.1118410 ◽

2016 ◽

Vol 34 (11) ◽

pp. 2434-2452 ◽

Cited By ~ 6

Author(s):

Vivek Kumar ◽

M. Elizabeth Sobhia

Keyword(s):

Molecular Dynamics ◽

Biological Activity ◽

Substrate Binding ◽

Binding Loop ◽

Diverse Biological Activity

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Imidazole- and Benzimidazole-Based Inhibitors of the Kinase IspE: Targeting the Substrate-Binding Site and the Triphosphate-Binding Loop of the ATP Site

European Journal of Organic Chemistry ◽

10.1002/ejoc.201201467 ◽

2013 ◽

Vol 2013 (6) ◽

pp. 1068-1079 ◽

Cited By ~ 12

Author(s):

Paolo Mombelli ◽

Camille Le Chapelain ◽

Noah Munzinger ◽

Evelyne Joliat ◽

Boris Illarionov ◽

...

Keyword(s):

Binding Site ◽

Substrate Binding ◽

Substrate Binding Site ◽

Binding Loop

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Characterisation of the flexibility of substrate binding loop in the binding of direct InhA inhibitors

International Journal of Computational Biology and Drug Design ◽

10.1504/ijcbdd.2013.056795 ◽

2013 ◽

Vol 6 (4) ◽

pp. 318 ◽

Cited By ~ 6

Author(s):

Vivek Kumar ◽

M. Elizabeth Sobhia

Keyword(s):

Substrate Binding ◽

Binding Loop

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Insights into subtle conformational differences in the substrate-binding loop of fungal 17β-hydroxysteroid dehydrogenase: a combined structural and kinetic approach

Biochemical Journal ◽

10.1042/bj20110567 ◽

2011 ◽

Vol 441 (1) ◽

pp. 151-160 ◽

Cited By ~ 8

Author(s):

Alberto Cassetta ◽

Ivet Krastanova ◽

Katja Kristan ◽

Mojca Brunskole Švegelj ◽

Doriano Lamba ◽

...

Keyword(s):

Substrate Binding ◽

Hydroxysteroid Dehydrogenase ◽

Crystallographic Analysis ◽

Ternary Complexes ◽

Stacking Interactions ◽

Kinetic Experiment ◽

Steroidal Compound ◽

Steroid Binding ◽

Binding Loop ◽

Structural Aspects

The 17β-HSD (17β-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17β-hydroxy counterparts. 17β-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17β-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17β-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17β-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17β-HSDcl [IC50 2.8 μM; at 100 μM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π–π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme–coenzyme–steroid ternary complexes.

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