scholarly journals Comparative enzymology of 11β-hydroxysteroid dehydrogenase type 1 from six species

2005 ◽  
Vol 35 (1) ◽  
pp. 89-101 ◽  
Author(s):  
Spyridon Arampatzis ◽  
Bert Kadereit ◽  
Daniela Schuster ◽  
Zoltan Balazs ◽  
Roberto A S Schweizer ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Animal Experiments ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Binding Pocket ◽  
Substrate Affinity ◽  
Drug Candidates ◽  
Species Specific

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), catalyzing the intracellular activation of cortisone to cortisol, is currently considered a promising target to treat patients with metabolic syndrome; hence, there is considerable interest in the development of selective inhibitors. For preclinical tests of such inhibitors, the characteristics of 11β-HSD1 from the commonly used species have to be known. Therefore, we determined differences in substrate affinity and inhibitor effects for 11β-HSD1 from six species. The differences in catalytic activities with cortisone and 11-dehydrocorticosterone were rather modest. Human, hamster and guinea-pig 11β-HSD1 displayed the highest catalytic efficiency in the oxoreduction of cortisone, while mouse and rat showed intermediate and dog the lowest activity. Murine 11β-HSD1 most efficiently reduced 11-dehydrocorticosterone, while the enzyme from dog showed lower activity than those from the other species. 7-Ketocholesterol (7KC) was stereospecifically converted to 7β-hydroxycholesterol by recombinant 11β-HSD1 from all species analyzed except hamster, which showed a slight preference for the formation of 7α-hydroxycholesterol. Importantly, guinea-pig and canine 11β-HSD1 displayed very low 7-oxoreductase activities. Furthermore, we demonstrate significant species-specific variability in the potency of various 11β-HSD1 inhibitors, including endogenous compounds, natural chemicals and pharmaceutical compounds. The results suggest significant differences in the three-dimensional organization of the hydrophobic substrate-binding pocket of 11β-HSD1, and they emphasize that species-specific variability must be considered in the interpretation of results obtained from different animal experiments. The assessment of such differences, by cell-based test systems, may help to choose the appropriate animal for safety and efficacy studies of novel potential drug candidates.

scholarly journals Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1882
Author(s):  
Wei Xia ◽  
Yingguo Bai ◽  
Pengjun Shi
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Substrate Binding ◽  
Enzymatic Saccharification ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Substrate Affinity ◽  
Hydrogen Bonding Networks

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

scholarly journals Comparative Enzymology of 11β-Hydroxysteroid Dehydrogenase Type 1 from Glucocorticoid Resistant (Guinea Pig)VersusSensitive (Human) Species

2002 ◽  
Vol 278 (3) ◽  
pp. 2030-2035 ◽  
Author(s):  
Naeem Shafqat ◽  
Björn Elleby ◽  
Stefan Svensson ◽  
Jawed Shafqat ◽  
Hans Jörnvall ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Hydroxysteroid Dehydrogenase ◽  
Human Species ◽  
Comparative Enzymology

scholarly journals The Crystal Structure of Guinea Pig 11β-Hydroxysteroid Dehydrogenase Type 1 Provides a Model for Enzyme-Lipid Bilayer Interactions

2004 ◽  
Vol 280 (5) ◽  
pp. 3789-3794 ◽  
Author(s):  
Derek Ogg ◽  
Björn Elleby ◽  
Carina Norström ◽  
Karin Stefansson ◽  
Lars Abrahmsén ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Guinea Pig ◽  
Lipid Bilayer ◽  
Hydroxysteroid Dehydrogenase

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

scholarly journals Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1

1994 ◽  
Vol 304 (1) ◽  
pp. 289-293 ◽  
Author(s):  
T J Puranen ◽  
M H Poutanen ◽  
H E Peltoketo ◽  
P T Vihko ◽  
R K Vihko
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor Development by Lentiviral Screening Based on Computational Modeling

Pharmacology ◽  
2018 ◽  
Vol 102 (3-4) ◽  
pp. 169-179
Author(s):  
Haifeng Liu ◽  
Lingyu Li ◽  
Chunlei Zhang ◽  
Hongzhi Li ◽  
Jieting Liu ◽  
...  
Keyword(s):  
Viral Vector ◽  
Three Dimensional ◽  
Hydroxysteroid Dehydrogenase ◽  
Gene Bank ◽  
Dimensional Structure ◽  
Drug Candidate ◽  
Lentiviral Transduction ◽  
Stable Manner

In this study, rat and human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have been cloned by lentiviral transduction and expressed by CHO-K1 cells. The results showed that recombinant plasmids contained R11bhsd1 or H11bhsd1 have been constructed, which is consistent with the gene bank respectively. A clone cell was selected with G418 and cultivated to express 11β-HSD1. 11β-HSD1 catalytic activity of rat and human were 99.5 and 98.7%, respectively, determined by scanning radiometer. And the cloned CHO-K1 cells expressed the protein of 11β-HSD1 in a long-term and stable manner, which makes it suitable for screening 11β-HSD1 inhibitor. The three-dimensional structure of 11β-HSD1 was used for studying the interaction between inhibitor and enzyme by the binding poses predicted by AutoDock and LeDock software. The docking results revealed that compound 8 forms 2 hydrogen bonds with the residues of Gly-216 and Ile-218 in 11β-HSD1, that is to say compound 8 maybe a good 11β-HSD1 inhibitor. Moreover, C57BL/6 mice with R11bHsd1 overexpression had a higher body weight, glucose, total cholesterol, and triglyceride levels compared to the mice treated with an empty viral vector. The results might provide a beneficial foundation for selecting inhibitors of 11β-HSD1 or for researching drug candidate mechanisms.

scholarly journals Serine 124 completes the Tyr, Lys and Ser triad responsible for the catalysis of human type 1 3beta-hydroxysteroid dehydrogenase

2004 ◽  
Vol 33 (1) ◽  
pp. 253-261 ◽  
Author(s):  
JL Thomas ◽  
WL Duax ◽  
A Addlagatta ◽  
LA Scaccia ◽  
KA Frizzell ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Three Dimensional ◽  
Homology Model ◽  
Hydroxysteroid Dehydrogenase ◽  
Catalytic Triad ◽  
Hydroxyl Group ◽  
Wild Type ◽  
Cofactor Binding ◽  
Human Type

Human 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17alpha-hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3beta-HSD reaction. Our three-dimensional homology model of 3beta-HSD shows that Tyr154 and Lys158 are oriented near the 3beta-hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3beta-HSD activity and has a K(m) value (83.6 microM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3beta-HSD activity (DHEA K(m)=11.2 microM; k(cat)=0.8 min(-1)) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K(m) value (27.6 microM) that is almost identical to wild-type. The K(m) value (4.3 microM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3beta-HSD and NADH as the activator for isomerase with K(m) values that are similar to wild-type. The 3beta-HSD activities of S123A and wild-type 3beta-HSD increase by 2.7-fold when the pH is raised from 7.4 to the optimal pH 9.7, but S124A exhibits very low residual 3beta-HSD activity that is pH-independent.These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3beta-HSD activity. Since there are 29 Ser residues in the primary structure of human 3beta-HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3beta-HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3beta-HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.

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