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

2011 ◽  
Vol 441 (1) ◽  
pp. 151-160 ◽  
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.

scholarly journals Mixed Ligand Complexes of Transition Metal Ions with Selective Bio-Ligands in Surfactant-Water Mixtures

2019 ◽  
Vol 8 (2S11) ◽  
pp. 3182-3190
Keyword(s):  
Hydrogen Bonding ◽  
Transition Metal ◽  
Metal Ions ◽  
Chemical Speciation ◽  
Transition Metal Ions ◽  
Ternary Complexes ◽  
Mixed Ligand ◽  
Stacking Interactions ◽  
Micellar Media ◽  
Chelate Effect

Chemical speciation of ternary complexes of L-arginine and L-aspartic acid with essential transition metal ions was studied pH metrically. The following MLX, MLXH and ML2X ternary species are detected and reported in this paper. The existence of different ternary species is established from modeling studies using the computer program MINIQUAD75. The relative concentrations (M: L: X=1:2:2, 1:2:4, 1:4:2) and stabilities of the ternary species are compared with those of binary species. The extra stability associated with the ternary complexes is attributed to factors such as charge neutralization, chelate effect, stacking interactions and hydrogen bonding. Trend in variation of stability constants with the change in the mole fraction of the surfactant in various micellar media is explained on the basis of electrostatic and non-electrostatic forces. Distribution diagrams in relation to pH and plausible structures were presented.

scholarly journals Not Cleaving the His-tag of Thal Results in More Tightly Packed and Better-Diffracting Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1135
Author(s):  
Ann-Christin Moritzer ◽  
Tina Prior ◽  
Hartmut H. Niemann
Keyword(s):  
Active Site ◽  
Flavin Adenine Dinucleotide ◽  
Substrate Binding ◽  
Environmentally Friendly ◽  
Space Group ◽  
Cofactor Binding ◽  
Closed Conformation ◽  
His Tag ◽  
Halide Salts ◽  
Binding Loop

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.

Crystallographic analysis of substrate binding and catalysis in dihydrolipoyl transacetylase (E2p)

Biochemistry ◽  
1993 ◽  
Vol 32 (15) ◽  
pp. 3887-3901 ◽  
Author(s):  
Andrea Mattevi ◽  
Galya Obmolova ◽  
Kor H. Kalk ◽  
Alex Teplyakov ◽  
Wim G. J. Hol
Keyword(s):  
Substrate Binding ◽  
Crystallographic Analysis

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

2010 ◽  
Vol 404 (3) ◽  
pp. 418-438 ◽  
Author(s):  
Magdalena M. Maslon ◽  
Roman Hrstka ◽  
Borek Vojtesek ◽  
Ted R. Hupp
Keyword(s):  
Substrate Binding ◽  
Docking Site ◽  
Oncogenic Protein ◽  
Binding Loop

3β-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN THE HUMAN FOETAL ADRENAL CORTEX

1965 ◽  
Vol 48 (3) ◽  
pp. 423-428 ◽  
Author(s):  
M. Niemi ◽  
A. H. Baillie
Keyword(s):  
Dehydrogenase Activity ◽  
Adrenal Cortex ◽  
Substrate Binding ◽  
Hydroxysteroid Dehydrogenase ◽  
List Type ◽  
Histochemical Reaction ◽  
Enzyme Substrate ◽  
Human Male ◽  
Foetal Life ◽  
Free Steroid

ABSTRACT 3β-Hydroxysteroid dehydrogenase activity was studied histochemically in the adrenal cortex of ten human male foetuses, ranging in crownrump length from 3.0 cm to 18.3 cm, with the following steroids: 3β-hydroxy-pregn-5-en-20-one (pregnenolone). 3β,17α-dihydroxy-pregn-5-en-20-one (17α-hydroxypregnenolone). 3β-hydroxy-androst-5-en-17-one (DHA). 3β,17β-dihydroxy-androst-5-ene (androstenediol). 3β-sulphoxy-pregn-5-en-20-one (pregnenolone sulphate). 3β-sulphoxy-17α-hydroxy-pregn-5-en-20-one (17α-hydroxy-pregnenolone sulphate) 3β-sulphoxy-androst-5-en-17-one (DHAsulphate). 3β-hydroxy-5α-androstan-17-one (epiandrosterone). After incubation with pregnenolone, 17α-hydroxypregnenolone, DHA and androstenediol a positive histochemical reaction was obtained in the inner part of the »definitive« cortex and throughout the foetal cortex of all adrenals studied. Initially very weak, the reaction became strongly positive about the twelfth week of foetal life. Pregnenolone sulphate and 17α-hydroxypregnenolong sulphate also gave a histochemical reaction in all the adrenals investigated, but DHA sulphate differed significantly from the free steroid by giving a very poor reaction. Formazan deposition followed incubation with epiandrosterone in all adrenals used and this may imply that a δ5 configuration is not necessary for enzyme-substrate binding.

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