scholarly journals The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase

2018 ◽  
Vol 59 (6) ◽  
pp. 1005-1014 ◽  
Author(s):  
Saravanan Devendran ◽  
Sean M. Mythen ◽  
Jason M. Ridlon
Keyword(s):  
Escherichia Coli ◽  
Hydroxysteroid Dehydrogenase ◽  
Spectrophotometric Assay ◽  
Side Chain ◽  
Rna Seq ◽  
Ph Optimum ◽  
Absolute Requirement ◽  
Gut Microbe ◽  
Major Facilitator ◽  
Coupled Assay

Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11β-hydro­xyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli. The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min−1. Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.

INVESTIGATIONS INTO THE POSSIBLE EFFECTS OF NATURALLY OCCURRING STEROIDS ON BIOSYNTHESIS OF 16-ANDROSTENES AND ANDROGENS IN MICROSOMES OF BOAR TESTIS

1981 ◽  
Vol 88 (3) ◽  
pp. 409-418 ◽  
Author(s):  
G. M. COOKE ◽  
D. B. GOWER
Keyword(s):  
Microsomal Fraction ◽  
Hydroxysteroid Dehydrogenase ◽  
Side Chain ◽  
Biosynthetic Pathways ◽  
Michaelis Constant ◽  
Ph Optimum ◽  
Naturally Occurring ◽  
Chain Cleavage ◽  
High Concentrations

The possible interactions between naturally occurring steroids and three enzymes of the biosynthetic pathways of 16-androstenes and androgens in the boar testis were investigated. High concentrations (1 mmol/l) of several steroids reduced the production of 5,16-androstadien-3β-ol when a microsomal fraction of boar testis was incubated with pregnenolone at pH 7·0 (the pH optimum of this reaction was found to be 6·6). When some of these inhibitors were investigated in more detail using Lineweaver–Burk analyses, the apparent inhibition constants, Ki, increased in value with increasing concentrations of inhibitors. When testosterone was added to 5,16-androstadien-3β-ol synthetase assays, the apparent Ki for 0·1 μmol testosterone/l was 0·165 μmol/l whereas those for 1·0, 10·0 and 100·0 μmol testosterone/l were 1·65, 16·5 and 48·7 μmol/l respectively. The apparent Michaelis constant, Km, of the reaction was 0·6 μmol/l. Similar results were obtained when oestrone, 17α-hydroxyprogesterone and 4,16-androstadien-3-one were added as effectors. At physiological concentrations, these steroids would not affect the biosynthesis of 5,16-androstadien-3 β-ol in vivo. Similarly, both 5α-androst-16-en-3β-ol, quantitatively the most important 16-androstene in the boar testis and 5,16-androstadien-3β-ol were examined for their effects on the 17α-hydroxy-C21 -steroid, C-17,20 lyase (C-17,20 lyase) and 17β-hydroxysteroid dehydrogenase (17β-OHSDH) enzymes of androgen biosynthesis. Neither of these enzymes was affected by the 16-androstene steroids even at concentrations of 100 μmol/l, and the apparent Km values were 3·3 and 26·0 μmol/l for C-17,20 lyase and 17β-OHSDH respectively. This lack of interaction between these pathways implies that the high levels of 16-androstene steroids produced by the testis will not interfere with androgen production, and also that the two side-chain cleavage steps from C21 precursors to C19 steroids are catalysed by independent systems.

scholarly journals Binding Site of the Bridged Macrolides in the Escherichia coli Ribosome

2005 ◽  
Vol 49 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Liqun Xiong ◽  
Yakov Korkhin ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Tight Binding ◽  
Dimethyl Sulfate ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Side Chains ◽  
Alkyl Aryl ◽  
E Coli ◽  
Exit Tunnel

ABSTRACT Ketolides represent the latest group of macrolide antibiotics. Tight binding of ketolides to the ribosome appears to correlate with the presence of an extended alkyl-aryl side chain. Recently developed 6,11-bridged bicyclic ketolides extend the spectrum of platforms used to generate new potent macrolides with extended alkyl-aryl side chains. The purpose of the present study was to characterize the site of binding and the action of bridged macrolides in the ribosomes of Escherichia coli. All the bridged macrolides investigated efficiently protected A2058 and A2059 in domain V of 23S rRNA from modification by dimethyl sulfate and U2609 from modification by carbodiimide. In addition, bridged macrolides that carry extended alkyl-aryl side chains protruding from the 6,11 bridge protected A752 in helix 35 of domain II of 23S rRNA from modification by dimethyl sulfate. Bridged macrolides efficiently displaced erythromycin from the ribosome in a competition binding assay. The A2058G mutation in 23S rRNA conferred resistance to the bridged macrolides. The U2609C mutation, which renders E. coli resistant to the previously studied ketolides telithromycin and cethromycin, barely affected cell susceptibility to the bridged macrolides used in this study. The results of the biochemical and genetic studies indicate that in the E. coli ribosome, bridged macrolides bind in the nascent peptide exit tunnel at the site previously described for other macrolide antibiotics. The presence of the side chain promotes the formation of specific interactions with the helix 35 of 23S rRNA.

Characterization of squalene epoxidase activity from the dermatophyte Trichophyton rubrum and its inhibition by terbinafine and other antimycotic agents.

1996 ◽  
Vol 40 (2) ◽  
pp. 443-447 ◽  
Author(s):  
B Favre ◽  
N S Ryder
Keyword(s):  
Biochemical Characterization ◽  
Trichophyton Rubrum ◽  
Side Chain ◽  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
High Potency ◽  
Whole Cells ◽  
Absolute Requirement ◽  
Tertiary Amino

Squalene epoxidase (SE) is the primary target of the allylamine antimycotic agents terbinafine and naftifine and also of the thiocarbamates. Although all of these drugs are employed primarily in dermatological therapy, SE from dermatophyte fungi has not been previously investigated. We report here the biochemical characterization of SE activity from Trichophyton rubrum and the effects of terbinafine and other inhibitors. Microsomal SE activity from T. rubrum was not dependent on soluble cytoplasmic factors but had an absolute requirement for NADPH or NADH and was stimulated by flavin adenine dinucleotide. Kinetic analyses revealed that under optimal conditions the Km for squalene was 13 microM and its Vmax was 0.71 nmol/h/mg of protein. Terbinafine was the most potent inhibitor tested, with a 50% inhibitory concentration (IC50) of 15.8 nM. This inhibition was noncompetitive with regard to the substrate squalene. A structure-activity relationship study with some analogs of terbinafine indicated that the tertiary amino structure of terbinafine was crucial for its high potency, as well as the tert-alkyl side chain. Naftifine had a lower potency (IC50, 114.6 nM) than terbinafine. Inhibition was also demonstrated by the thiocarbamates tolciclate (IC50, 28.0 nM) and tolnaftate (IC50, 51.5 nM). Interestingly, the morpholine amorolfine also displayed a weak but significant effect (IC50, 30 microM). T. rubrum SE was only slightly more sensitive (approximately twofold) to terbinafine inhibition than was the Candida albicans enzyme. Therefore, this difference cannot fully explain the much higher susceptibility (> or = 100-fold) of dermatophytes than of yeasts to this drug. The sensitivity to terbinafine of ergosterol biosynthesis in whole cells of T. rubrum (IC50, 1.5 nM) is 10-fold higher than that of SE activity, suggesting that the drug accumulates in the fungus.

scholarly journals Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Kumari Sonal Choudhary ◽  
Julia A. Kleinmanns ◽  
Katherine Decker ◽  
Anand V. Sastry ◽  
Ye Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Target Gene ◽  
Target Genes ◽  
Rna Seq ◽  
Content Type ◽  
E Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

scholarly journals Competition between protons and substrate for binding to the major facilitator superfamily multidrug/H+ antiporter MdtM

2021 ◽  
Vol 2 ◽  
Author(s):  
Christopher J. Law
Keyword(s):  
Escherichia Coli ◽  
General Feature ◽  
Pathogenic Bacteria ◽  
Major Facilitator Superfamily ◽  
Electrochemical Gradient ◽  
Multidrug Efflux ◽  
Protein Fluorescence ◽  
Ph Values ◽  
Major Facilitator ◽  
Intrinsic Protein Fluorescence

Abstract Proton electrochemical gradient-driven multidrug efflux activity of representatives of the major facilitator superfamily (MFS) of secondary active transporters contributes to antimicrobial resistance of pathogenic bacteria. Integral to the mechanism of these transporters is a proposed competition between substrate and protons for the binding site of the protein. The current work investigated the competition between protons and antimicrobial substrate for binding to the Escherichia coli MFS multidrug/H+ antiporter MdtM by measuring the quench of intrinsic protein fluorescence upon titration of substrate tetraphenylphosphonium into a solution of purified MdtM over a range of pH values between pH 8.8 and 5.9. The results, which revealed that protons inhibit binding of substrate to MdtM in a competitive manner, are consistent with those reported in a study on the related MFS multidrug/H+ antiporter MdfA and provide further evidence that competition for binding between substrate and protons is a general feature of secondary multidrug efflux.

scholarly journals Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli

2001 ◽  
Vol 67 (4) ◽  
pp. 1601-1606 ◽  
Author(s):  
Mitsunori Ishiguro ◽  
Satoshi Kaneko ◽  
Atsushi Kuno ◽  
Yoshinori Koyama ◽  
Shigeki Yoshida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Side Chain ◽  
Catalytic Function ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACT The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable α-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r, 53,514). The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus brockianus was over 70%.Thermus sp. strain T2 α-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C forp-nitrophenyl-α-d-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal α-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea α-galactosidase I. The enzyme has only one Cys residue in the molecule.para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

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