scholarly journals Cysteine-10 on 17β-Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Lyubomir G. Nashev ◽  
Atanas G. Atanasov ◽  
Michael E. Baker ◽  
Alex Odermatt
Keyword(s):  
Structure Function ◽  
Enzyme Inhibitors ◽  
3D Structure ◽  
Hydroxysteroid Dehydrogenase ◽  
Binding Pocket ◽  
Cysteine Residue ◽  
Kinetic Properties ◽  
Binding Region ◽  
Cofactor Binding

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the conversion of estrone to the potent estrogen estradiol. 17β-HSD1 is highly expressed in breast and ovary tissues and represents a prognostic marker for the tumor progression and survival of patients with breast cancer and other estrogen-dependent tumors. Therefore, the enzyme is considered a promising drug target against estrogen-dependent cancers. For the development of novel inhibitors, an improved understanding of the structure-function relationships is essential. In the present study, we examined the role of a cysteine residue, Cys10, in the Rossmann-fold NADPH binding region, for 17β-HSD1 function and tested the sensitivity towards sulfhydryl modifying chemicals. 3D structure modeling revealed important interactions of Cys10with residues involved in the stabilization of amino acids of the NADPH binding pocket. Analysis of enzyme activity revealed that 17β-HSD1 was irreversibly inhibited by the sulfhydryl modifying agents N-ethylmaleimide (NEM) and dithiocarbamates. Preincubation with increasing concentrations of NADPH protected 17β-HSD1 from inhibition by these chemicals. Cys10Ser mutant 17β-HSD1 was partially protected from inhibition by NEM and dithiocarbamates, emphasizing the importance of Cys10in the cofactor binding region. Substitution of Cys10with serine resulted in a decreased protein half-life, without significantly altering kinetic properties. Despite the fact that Cys10on 17β-HSD1 seems to have limited potential as a target for new enzyme inhibitors, the present study provides new insight into the structure-function relationships of this enzyme.

scholarly journals Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis

1994 ◽  
Vol 300 (1) ◽  
pp. 7-13 ◽  
Author(s):  
J M Candy ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Binding Motif ◽  
Sequence Motif ◽  
Cofactor Binding

Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.

scholarly journals Perspective on the Structural Basis for Human Aldo-Keto Reductase 1B10 Inhibition

Metabolites ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 865
Author(s):  
Francesc Xavier Ruiz ◽  
Xavier Parés ◽  
Jaume Farrés
Keyword(s):  
Enzyme Inhibitors ◽  
Binding Pocket ◽  
Anion Binding ◽  
Structural Basis ◽  
Inhibitor Binding ◽  
Reversible Inhibitors ◽  
Alternative Conformation ◽  
Variable Loops ◽  
Cancer Types

Human aldo-keto reductase 1B10 (AKR1B10) is overexpressed in many cancer types and is involved in chemoresistance. This makes AKR1B10 to be an interesting drug target and thus many enzyme inhibitors have been investigated. High-resolution crystallographic structures of AKR1B10 with various reversible inhibitors were deeply analyzed and compared to those of analogous complexes with aldose reductase (AR). In both enzymes, the active site included an anion-binding pocket and, in some cases, inhibitor binding caused the opening of a transient specificity pocket. Different structural conformers were revealed upon inhibitor binding, emphasizing the importance of the highly variable loops, which participate in the transient opening of additional binding subpockets. Two key differences between AKR1B10 and AR were observed regarding the role of external loops in inhibitor binding. The first corresponded to the alternative conformation of Trp112 (Trp111 in AR). The second difference dealt with loop A mobility, which defined a larger and more loosely packed subpocket in AKR1B10. From this analysis, the general features that a selective AKR1B10 inhibitor should comply with are the following: an anchoring moiety to the anion-binding pocket, keeping Trp112 in its native conformation (AKR1B10-like), and not opening the specificity pocket in AR.

S95C substitution in CuZn-SOD of Ipomoea carnea: impact on the structure, function and stability

2016 ◽  
Vol 12 (10) ◽  
pp. 3017-3031 ◽  
Author(s):  
Panchanand Mishra ◽  
Suresh Satpati ◽  
Sudhira Kumar Baral ◽  
Anshuman Dixit ◽  
Surendra Chandra Sabat
Keyword(s):  
Superoxide Dismutase ◽  
Structure Function ◽  
Superoxide Radicals ◽  
Kinetic Properties ◽  
Ipomoea Carnea ◽  
The Stability

Superoxide dismutase (SOD) is a unique homo-dimeric enzyme that can scavenge toxic superoxide radicals by dismutation reaction. This study analysed the role of Cys in modulating the stability and kinetic properties of IcSOD.

scholarly journals Novel xylan degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205

2020 ◽  
Author(s):  
Javier A. Linares-Pastén ◽  
Johan Sebastian Hero ◽  
José Horacio Pisa ◽  
Cristina Teixeira ◽  
Margareta Nyman ◽  
...  
Keyword(s):  
3D Structure ◽  
Extracellular Enzyme ◽  
Binding Pocket ◽  
Human Gastrointestinal Tract ◽  
Intracellular Location ◽  
Inflammatory Conditions ◽  
Hydrolysis Products ◽  
Encoding Genes ◽  
Hydrolysis Of

AbstractPrevotella copri DSM18205 is a bacterium, classified under Bacteroidetes that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers in P. copri, which currently are unexplored. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight GH-encoding genes, respectively. Three of the eight gene products were successfully produced in Escherichia coli: One monomeric two-domain extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two dimeric single module enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 subfamily 1 enzyme (37 kDa). The GH43_12 enzyme was hydrolysing arabinofuranose residues from different substrates, and a model of the 3D-structure revealed a single arabinose binding pocket. The GH10 (1) and GH43_1 are cleaving the xylan backbone. Hydrolysis products of GH10 (1) were DP2-4, and seven subsites (−3 to +4) were predicted in the 3D-model of the GH10 active site. GH43_1 mainly produced xylose (in line with its intracellular location). Based on our results we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolyses mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular β-xylosidase, catalysing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalysing removal of arabinose-residues on xylan polymers.

ZUR BEDEUTUNG DES HYPOPHYSÄREN LUTEINISIERUNGSHORMONS FÜR SCHWANGERSCHAFT, GEBURT UND LACTATION BEI DER RATTE

1970 ◽  
Vol 65 (3_Suppl) ◽  
pp. S5-S32 ◽  
Author(s):  
K. Loewit
Keyword(s):  
Luteinizing Hormone ◽  
Early Pregnancy ◽  
Hydroxysteroid Dehydrogenase ◽  
The State ◽  
Termination Of Pregnancy ◽  
Progesterone Level ◽  
Corpora Lutea ◽  
Regulatory Properties ◽  
Duration Of Pregnancy

ABSTRACT The role of luteinizing hormone (LH) for the maintenance of pregnancy, parturition and lactation was investigated by immunological and histochemical methods in the rat. Neutralisation of endogenous rat-LH with Rabbit-Anti-Bovine-LH-Serum (selective hypophysectomy) from days 7-12 of pregnancy resulted in reabsorption of the foetuses and the reappearance of strong 20α-hydroxysteroid-dehydrogenase (20α-OHSD) activity in the corpora lutea (CL) of pregnancy, which normally show no such activity at that time. This effect could be prevented in part by concurrent pregnenolone administration and fully by progesterone, but was not influenced by oestrogen or prolactin. It is concluded that in early pregnancy LH is the main luteotrophic hormone in the rat even though prolactin might act synergistically with it. Antiserum treatment after the 12th day of gestation had no influence on the state or duration of pregnancy or on parturition. LH-injections during the first half of pregnancy had no luteolytic effects i. e. they did not activate 20α-OHSD activity. After day 16 they advanced the reappearance of the enzyme, but delayed parturition or resulted in stillbirths. Neither LH nor antiserum seemed to alter lactation. Since progesterone prevented both the termination of pregnancy and the recurrence of 20α-OHSD activity, it should have some regulatory properties on the enzyme. It is discussed whether the gonadotrophin-dependent progesterone level could regulate the 20α-OHSD activity rather than result from it.

The role of hepatic 11[b]-hydroxysteroid dehydrogenase type 1 in cholesterol homeostasis

2013 ◽  
pp. 1-1
Author(s):  
Kajal Manwani ◽  
Tak Y Man ◽  
Christopher J Kenyon ◽  
Ruth Andrew ◽  
Karen E Chapman ◽  
...  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Cholesterol Homeostasis

scholarly journals Engineering subtilisin proteases that specifically degrade active RAS

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yingwei Chen ◽  
Eric A. Toth ◽  
Biao Ruan ◽  
Eun Jung Choi ◽  
Richard Simmerman ◽  
...  
Keyword(s):  
Active Form ◽  
High Specificity ◽  
Kinetic Properties ◽  
Target Sequence ◽  
Reporter System ◽  
Human Cell Culture ◽  
Conserved Sequence ◽  
Cofactor Binding ◽  
Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

scholarly journals Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure–Function Map

2021 ◽  
Vol 89 (1-2) ◽  
pp. 103-116
Author(s):  
Julie Baker Phillips ◽  
David H. Ardell
Keyword(s):  
Structure Function ◽  
Metal Ion ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Heterologous Gene ◽  
Multigene Families ◽  
Ion Binding ◽  
Trna Genes ◽  
Structural Factors ◽  
Trna Structure

AbstractThe evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

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