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https://www.readbyqxmd.com/read/28193327/cloning-expression-and-biochemical-characterization-of-a-novel-nadp-dependent-7%C3%AE-hydroxysteroid-dehydrogenase-from-clostridium-difficile-and-its-application-for-the-oxidation-of-bile-acids
#1
Daniel Bakonyi, Werner Hummel
A gene encoding a novel 7α-specific NADP(+)-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition...
April 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28128053/the-%C3%AE-sheet-core-is-the-favored-candidate-of-engineering-sdr-for-enhancing-thermostability-but-not-for-activity
#2
Deshuai Lou, Jun Tan, Liancai Zhu, Shunlin Ji, Bochu Wang
7α-Hydroxysteroid dehydrogenases (7α-HSDHs) can stereoselectively catalyze steroids, aromatic α-ketoesters, and benzaldehyde analogues playing a critical role in the biotransformation and poor thermostability that hinders their biomedical and industrial applications. Based on the three-dimensional structure of 7α-HSDH from Clostridium absonum (CA 7α-HSDH), recently reported program MAESTRO was used to compute the ΔΔG and predict the single-point mutants that could enhance its thermostability. Four mutants were selected and verified experimentally...
January 26, 2017: Protein and Peptide Letters
https://www.readbyqxmd.com/read/28116898/engineering-7%C3%AE-hydroxysteroid-dehydrogenase-for-enhanced-ursodeoxycholic-acid-production-by-multiobjective-directed-evolution
#3
Ming-Min Zheng, Ke-Cai Chen, Ru-Feng Wang, Hao Li, Chun-Xiu Li, Jian-He Xu
Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH...
February 3, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/27577999/triclosan-resistome-from-metagenome-reveals-diverse-enoyl-acyl-carrier-protein-reductases-and-selective-enrichment-of-triclosan-resistance-genes
#4
Raees Khan, Hyun Gi Kong, Yong-Hoon Jung, Jinhee Choi, Kwang-Yeol Baek, Eul Chul Hwang, Seon-Woo Lee
Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS...
2016: Scientific Reports
https://www.readbyqxmd.com/read/26961171/the-three-dimensional-structure-of-clostridium-absonum-7%C3%AE-hydroxysteroid-dehydrogenase-new-insights-into-the-conserved-arginines-for-nadp-h-recognition
#5
Deshuai Lou, Bochu Wang, Jun Tan, Liancai Zhu, Xiaoxi Cen, Qingzhi Ji, Yue Wang
7α-hydroxysteroid dehydrogenase (7α-HSDH) can catalyse the oxidation of C7 α-OH of the steroid nucleus in the bile acid metabolism. In the paper we determined the crystal structure of 7α-HSDH from Clostridium absonum (CA 7α-HSDH) complexed with taurochenodeoxycholic acid (TCDCA) and NADP(+) by X-ray diffraction, which, as a tetramer, possesses the typical α/β folding pattern. The four subunits of an asymmetric unit lie in the fact that there are the stable hydrophobic interactions between Q-axis-related subunits...
2016: Scientific Reports
https://www.readbyqxmd.com/read/26833240/radical-mediated-dehydrogenation-of-bile-acids-by-means-of-hydrogen-atom-transfer-to-triplet-carbonyls
#6
P Miro, M L Marin, M A Miranda
The aim of the present paper is to explore the potential of radical-mediated dehydrogenation of bile salts (BSs), which is reminiscent of the enzymatic action of hydroxysteroid dehydrogenase enzymes (HSDH). The concept has been demonstrated using triplet carbonyls that can be efficiently generated upon selective UVA-excitation. Hydrogen atom transfer (HAT) from BSs to triplet benzophenone (BP) derivatives gave rise to radicals, ultimately leading to reduction of the BP chromophore with concomitant formation of the oxo-analogs of the corresponding BSs...
March 7, 2016: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/26063505/analysis-of-loss-of-function-mutants-in-aspartate-kinase-and-homoserine-dehydrogenase-genes-points-to-complexity-in-the-regulation-of-aspartate-derived-amino-acid-contents
#7
Teresa J Clark, Yan Lu
Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays...
August 2015: Plant Physiology
https://www.readbyqxmd.com/read/25517115/bacterial-bile-metabolising-gene-abundance-in-crohn-s-ulcerative-colitis-and-type-2-diabetes-metagenomes
#8
Alain Labbé, Jorge G Ganopolsky, Christopher J Martoni, Satya Prakash, Mitchell L Jones
We performed an analysis to determine the importance of bile acid modification genes in the gut microbiome of inflammatory bowel disease and type 2 diabetic patients. We used publicly available metagenomic datasets from the Human Microbiome Project and the MetaHIT consortium, and determined the abundance of bile salt hydrolase gene (bsh), 7 alpha-dehydroxylase gene (adh) and 7-alpha hydroxysteroid dehydrogenase gene (hsdh) in fecal bacteria in diseased populations of Crohn's disease (CD), Ulcerative Colitis (UC) and Type 2 diabetes mellitus (T2DM)...
2014: PloS One
https://www.readbyqxmd.com/read/25131646/enzymatic-routes-for-the-synthesis-of-ursodeoxycholic-acid
#9
Thorsten Eggert, Daniel Bakonyi, Werner Hummel
Ursodeoxycholic acid, a secondary bile acid, is used as a drug for the treatment of various liver diseases, the optimal dose comprises the range of 8-10mg/kg/day. For industrial syntheses, the structural complexity of this bile acid requires the use of an appropriate starting material as well as the application of regio- and enantio-selective enzymes for its derivatization. Most strategies for the synthesis start from cholic acid or chenodeoxycholic acid. The latter requires the conversion of the hydroxyl group at C-7 from α- into β-position in order to obtain ursodeoxycholic acid...
December 10, 2014: Journal of Biotechnology
https://www.readbyqxmd.com/read/25081557/engineering-a-hydroxysteroid-dehydrogenase-to-improve-its-soluble-expression-for-the-asymmetric-reduction-of-cortisone-to-11%C3%AE-hydrocortisone
#10
Dalong Zhang, Rui Zhang, Jie Zhang, Liying Chen, Chunxia Zhao, Wenyue Dong, Qing Zhao, Qiaqing Wu, Dunming Zhu
11β-Hydrocortisone (11β-HC) is an important anti-inflammatory drug and intermediate for the synthesis of other steroids. One of the methods for the synthesis of 11β-HC is the asymmetric reduction of cortisone catalyzed by a highly regioselective and stereoselective 11β-hydroxysteroid dehydrogenase (11β-HSDH). However, this process has been prohibited by the poor soluble expression of the membrane-anchoring protein 11β-HSDH in prokaryotes. To overcome this obstacle, a mutant III-1G1 (Phe80Leu/Thr105Ser/Ala260Thr/Tyr274Stop) truncated at position 274 with improved yield of soluble protein was stepwise obtained from the 11β-HSDH from guinea pig by random mutagenesis combining with structural complementation assay and C-terminal truncating library screening...
November 2014: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/24810359/carboxyl-terminal-and-arg38-are-essential-for-activity-of-the-7%C3%AE-hydroxysteroid-dehydrogenase-from-clostridium-absonum
#11
Deshuai Lou, Bochu Wang, Jun Tan, Liancai Zhu
7α-hydroxysteroid dehydrogenase (7α-HSDH, EC 1.1.1.159), one of the short-chain dehydrogenase/reductase superfamily, catalyzes the dehydrogenation of C7 hydroxyl group of the steroid skeleton of bile acids. Clostridium absonum 7α-HSDH (Ca 7α-HSDH) was cloned and heterologously expressed in Escherichia coli. The function of carboxyterminal (C-terminal) and Arg38 of Ca 7α-HSDH was investigated through truncations and site-directed mutagenesis, respectively. When 2 and 6 amino acids of C-terminal were removed, the catalytic efficiency (k(cat)/K(m)) of Ca 7α-HSDH remained 19...
2014: Protein and Peptide Letters
https://www.readbyqxmd.com/read/24344690/rational-design-of-allosteric-regulation-of-homoserine-dehydrogenase-by-a-nonnatural-inhibitor-l-lysine
#12
Zhen Chen, Sugima Rappert, An-Ping Zeng
Allosteric proteins, which can sense different signals, are interesting biological parts for synthetic biology. In particular, the design of an artificial allosteric enzyme to sense an unnatural signal is both challenging and highly desired, for example, for a precise and dynamical control of fluxes of growth-essential but byproduct pathways in metabolic engineering of industrial microorganisms. In this work, we used homoserine dehydrogenase (HSDH) of Corynebacterium glutamicum, which is naturally allosterically regulated by threonine and isoleucine, as an example to demonstrate the feasibility of reengineering an allosteric enzyme to respond to an unnatural inhibitor L-lysine...
February 20, 2015: ACS Synthetic Biology
https://www.readbyqxmd.com/read/24006814/cypermethrin-induced-pathological-and-biochemical-changes-in-reproductive-organs-of-female-rats
#13
G K Sangha, Kamalpreet Kaur, K S Khera
Cypermethrin, a synthetic pyrethroid, has broad spectrum use in domestic agriculture, and veterinary applications due to its high bioefficacy, enhanced stability and low mammalian toxicity. The present investigation was performed to investigate the sub acute effects of cypermethrin (25EC) in female rats. Cypermethrin (25EC) at a dose of 50 mg kg(-1) body weight (1/5th LD50) was orally given to female rats for 4 weeks. Control rats received similar amount of ground nut oil. Significant decrease in ovarian weight (15...
January 2013: Journal of Environmental Biology
https://www.readbyqxmd.com/read/23928522/head-shaking-during-dix-hallpike-exam-increases-the-diagnostic-yield-of-posterior-semicircular-canal-bppv
#14
Daniel M Kaplan, Youval Slovik, Be Zion Joshua, Marc Puterman, Mordechai Kraus
OBJECTIVES: To examine whether shaking the head during the DH exam (HSDH) may improve diagnosis of posterior semicircular canal benign paroxysmal positional vertigo (pBPPV) in patients with a negative Dix-Hallpike (DH) examination. STUDY DESIGN: A prospective cohort clinical study on consecutive patients with dizziness, who were seen by 2 experienced neurotologists. Patients underwent a complete neurotology examination, including positioning testing with roll test and DH...
October 2013: Otology & Neurotology
https://www.readbyqxmd.com/read/23860761/handheld-shape-discrimination-hyperacuity-test-on-a-mobile-device-for-remote-monitoring-of-visual-function-in-maculopathy
#15
COMPARATIVE STUDY
Yi-Zhong Wang, Yu-Guang He, Gina Mitzel, Song Zhang, Mike Bartlett
PURPOSE: Frequency monitoring of age-related macular degeneration (AMD) and diabetic retinopathy (DR) is crucial for timely intervention. This study evaluated a handheld shape discrimination hyperacuity (hSDH) test iPhone app designed for visual function self-monitoring in patients with AMD and DR. METHODS: One hundred subjects (27 visually normal, 37 with AMD, and 36 with DR) were included based on clinical documentation and visual acuity of 20/100 or better. The hSDH test was implemented on the iOS platform...
August 2013: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/23772041/clostridium-scindens-a-human-gut-microbe-with-a-high-potential-to-convert-glucocorticoids-into-androgens
#16
Jason M Ridlon, Shigeo Ikegawa, João M P Alves, Biao Zhou, Akiko Kobayashi, Takashi Iida, Kuniko Mitamura, Genzoh Tanabe, Myrna Serrano, Ainee De Guzman, Patsy Cooper, Gregory A Buck, Phillip B Hylemon
Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, (1)H and (13)C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (≈ 1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage...
September 2013: Journal of Lipid Research
https://www.readbyqxmd.com/read/23729502/contribution-of-the-7%C3%AE-hydroxysteroid-dehydrogenase-from-ruminococcus-gnavus-n53-to-ursodeoxycholic-acid-formation-in-the-human-colon
#17
Ja-Young Lee, Hisashi Arai, Yusuke Nakamura, Satoru Fukiya, Masaru Wada, Atsushi Yokota
Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer...
November 2013: Journal of Lipid Research
https://www.readbyqxmd.com/read/23556418/regulatory-mechanisms-after-short-and-long-term-perturbed-lysine-biosynthesis-in-the-aspartate-pathway-the-need-for-isogenes-in-arabidopsis-thaliana
#18
Pieter Van Bochaute, Alexandre Novoa, Steven Ballet, Sven Erik Rognes, Geert Angenon
The aspartate-derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.2.4), homoserine dehydrogenase (HSDH; EC 1.1.1.3) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7), formerly referred to as dihydrodipicolinate synthase (DHDPS). They are encoded by isoenzyme families and it is not known why such families are evolutionarily maintained...
December 2013: Physiologia Plantarum
https://www.readbyqxmd.com/read/23330433/-3beta-hydroxysteroid-dehydrogenase-in-ependimocytes-of-brain-lateral-ventricle-lining-and-vascular-plexus-villi-in-rats-of-different-age
#19
B Ia Ryzhavskiĭ, O V Zadvornaia
Using the histochemical method, the activity of 3beta-hydroxysteroid dehydrogenase (HSDH) was studied in the brain of laboratory male albino rats of different age groups: 5-6 days (n = 6), 45-50 days (n = 12), and 6 months (n = 15). The quantitative assessment of reaction intensity was performed with the cytospectrophotometer. The results obtained indicate that the ependimocytes lining the brain lateral ventricles and covering the villi of their vascular plexuses are characterized by the presence of HSDH activity typical to that of steroid-producing cells...
2012: Morfologi︠a︡
https://www.readbyqxmd.com/read/23112234/modulating-the-function-of-human-serine-racemase-and-human-serine-dehydratase-by-protein-engineering
#20
Cyong-Yi Wang, Shan Chi Ku, Cheng-Chung Lee, Andrew H-J Wang
D-Serine is a co-agonist of N-methyl D-aspartate, a glutamate receptor, which is a major excitatory neurotransmitter receptor in the brain. Human serine racemase (hSR) and serine dehydratase (hSDH) are two important pyridoxal-5'-phosphate-dependent enzymes that synthesize and degrade D-serine, respectively. hSR and hSDH have significant sequence homology (28% identity) and are similar in their structural folds (root-mean-square deviation, 1.12 Å). Sequence alignment and structural comparison between hSR and hSDH reveal that S84 in hSR and A65 in hSDH play important roles in their respective enzyme activities...
November 2012: Protein Engineering, Design & Selection: PEDS
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