scholarly journals Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

2002 ◽  
Vol 68 (7) ◽  
pp. 3279-3286 ◽  
Author(s):  
Sandra Trott ◽  
Sibylle Bürger ◽  
Carsten Calaminus ◽  
Andreas Stolz
Keyword(s):  
Nitrile Hydratase ◽  
Rhodococcus Erythropolis ◽  
Nitrogen Sources ◽  
Whole Cells ◽  
E Coli ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Amide Hydrolysis ◽  
Rate Limiting ◽  
Amidase Gene

ABSTRACT The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, α-chlorophenylacetamide, and α-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.

scholarly journals Utilization of gluconate by Escherichia coli. Uptake of d-gluconate by a mutant impaired in gluconate kinase activity and by membrane vesicles derived therefrom*

1974 ◽  
Vol 140 (2) ◽  
pp. 193-203 ◽  
Author(s):  
J. M. Pouysségur ◽  
Pelin Faik ◽  
H. L. Kornberg
Keyword(s):  
Escherichia Coli ◽  
Kinase Activity ◽  
Membrane Vesicles ◽  
Dry Mass ◽  
Coli Strain ◽  
Whole Cells ◽  
E Coli ◽  
Phosphogluconate Dehydrogenase ◽  
Rate Limiting Step ◽  
Rate Limiting

1. From Escherichia coli strain K2.1.5c.8.9, which is devoid of 6-phosphogluconate dehydrogenase (gnd) and 6-phosphogluconate dehydratase (edd) activities, a mutant R6 was isolated that was tolerant to gluconate though still edd-, gnd-. 2. Measurements of the fate of labelled gluconate, of the conversion of gluconate into 6-phosphogluconate, and of the induction of gluconate kinase by the two organisms show that, although both inducibly form a gluconate-transport system, strain R6 is impaired in its ability to convert the gluconate thus taken up into 6-phosphogluconate; it was therefore used for study of the kinetics and energetics of gluconate uptake. 3. Suspensions of strain R6 induced for gluconate uptake took up this substrate via a ‘high affinity’ transport process, with Km about 10μm and Vmax. about 25nmol/min per mg dry mass; a ‘low affinity’ system demonstrated to occur in certain E. coli mutants was not induced under the conditions used in this work. 4. The uptake of gluconate was inhibited by lack of oxygen and by inhibitors of electron transport; such inhibitors also promoted the efflux of gluconate taken up. 5. Membrane vesicles prepared from strain R6 also manifested these properties when incubated with suitable electron donors, at rates similar to those observed with whole cells. 6. The results indicate that the active transport of gluconate into the cells is the rate-limiting step in gluconate utilization by E. coli, and that the mechanism of this process can be validly studied with membrane vesicles.

scholarly journals The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

2000 ◽  
Vol 66 (4) ◽  
pp. 1429-1434 ◽  
Author(s):  
Rainer Russ ◽  
Jörg Rau ◽  
Andreas Stolz
Keyword(s):  
Azo Dyes ◽  
Reduction Rate ◽  
Constitutive Expression ◽  
Azo Compounds ◽  
Flavin Reductase ◽  
Whole Cells ◽  
E Coli ◽  
Cell Extracts

ABSTRACT A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in theSphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-typeSphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.

scholarly journals Transcription closed and open complex formation coordinate expression of genes with a shared promoter region

2019 ◽  
Vol 16 (161) ◽  
pp. 20190507
Author(s):  
Antti Häkkinen ◽  
Samuel M. D. Oliveira ◽  
Ramakanth Neeli-Venkata ◽  
Andre S. Ribeiro
Keyword(s):  
Transcription Initiation ◽  
Regulatory Elements ◽  
Promoter Elements ◽  
E Coli ◽  
Rate Limiting Step ◽  
Expression Of Genes ◽  
Active Transcription ◽  
Rate Limiting ◽  
Kinetics Of

Many genes are spaced closely, allowing coordination without explicit control through shared regulatory elements and molecular interactions. We study the dynamics of a stochastic model of a gene-pair in a head-to-head configuration, sharing promoter elements, which accounts for the rate-limiting steps in transcription initiation. We find that only in specific regions of the parameter space of the rate-limiting steps is orderly coexpression exhibited, suggesting that successful cooperation between closely spaced genes requires the coevolution of compatible rate-limiting step configuration. The model predictions are validated using in vivo single-cell, single-RNA measurements of the dynamics of pairs of genes sharing promoter elements. Our results suggest that, in E. coli , the kinetics of the rate-limiting steps in active transcription can play a central role in shaping the dynamics of gene-pairs sharing promoter elements.

scholarly journals Characterization of a Carbonyl Reductase from Rhodococcus erythropolis WZ010 and Its Variant Y54F for Asymmetric Synthesis of (S)-N-Boc-3-Hydroxypiperidine

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3117 ◽  
Author(s):  
Xiangxian Ying ◽  
Jie Zhang ◽  
Can Wang ◽  
Meijuan Huang ◽  
Yuting Ji ◽  
...  
Keyword(s):  
Asymmetric Reduction ◽  
Rhodococcus Erythropolis ◽  
Catalytic Efficiency ◽  
Carbonyl Reductase ◽  
Specific Enzyme ◽  
Practical Applications ◽  
Whole Cells ◽  
E Coli ◽  
Irreversible Reduction

The recombinant carbonyl reductase from Rhodococcus erythropolis WZ010 (ReCR) demonstrated strict (S)-stereoselectivity and catalyzed the irreversible reduction of N-Boc-3-piperidone (NBPO) to (S)-N-Boc-3-hydroxypiperidine [(S)-NBHP], a key chiral intermediate in the synthesis of ibrutinib. The NAD(H)-specific enzyme was active within broad ranges of pH and temperature and had remarkable activity in the presence of higher concentration of organic solvents. The amino acid residue at position 54 was critical for the activity and the substitution of Tyr54 to Phe significantly enhanced the catalytic efficiency of ReCR. The kcat/Km values of ReCR Y54F for NBPO, (R/S)-2-octanol, and 2-propanol were 49.17 s−1 mM−1, 56.56 s−1 mM−1, and 20.69 s−1 mM−1, respectively. In addition, the (S)-NBHP yield was as high as 95.92% when whole cells of E. coli overexpressing ReCR variant Y54F catalyzed the asymmetric reduction of 1.5 M NBPO for 12 h in the aqueous/(R/S)-2-octanol biphasic system, demonstrating the great potential of ReCR variant Y54F for practical applications.

scholarly journals Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Thomas Hilberath ◽  
Alessandra Raffaele ◽  
Leonie M. Windeln ◽  
Vlada B. Urlacher
Keyword(s):  
Cytochromes P450 ◽  
Rhodococcus Erythropolis ◽  
Resting Cells ◽  
P450 Monooxygenase ◽  
Monooxygenase Activity ◽  
Whole Cells ◽  
E Coli ◽  
Redox Partner ◽  
Starting Point ◽  
P450 Activity

AbstractCytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.

Capabilities of Four Microorganisms for Bioremediation of Lead Contaminated Soil

2020 ◽  
Vol 838 ◽  
pp. 125-134
Author(s):  
Edobor Kingsley Osagie
Keyword(s):  
Contaminated Soils ◽  
Lead Concentration ◽  
Soil Samples ◽  
Decision Makers ◽  
Pseudomonas Sp ◽  
Bacillus Sp ◽  
E Coli ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

This work studied the potentials of indigenous Micrococcus sp., Bacillus sp., Pseudomonas sp., and Escherichia coli (E. coli) for bioremediation of lead contaminated soils collected from Amita forest in Ebonyi State of Nigeria.The organisms isolated from the soils were conditioned with the predetermined optimum factors in inoculated soil samples. The samples were tested for residual lead concentration at times 8, 16, 24, 32, 40, 48, and 56 days with Atomic Absorption Spectrophotometer.The performances of the organisms were in the decreasing order of Micrococcus sp., Bacillus sp., Pseudomonas sp., and E. coli. Micrococcus sp. and Bacillus sp. performed earlier at time 16 days as against Pseudomonas sp., and E. coli at 24 days. The maximum efficiencies were discovered at time 56 days as 76.68%, 72.24%, 70.11% and 55.47% for Micrococcus sp., Bacillus sp., Pseudomonas sp., and E. coli respectively with respective residual concentrations of 31.55 mg/kg, 37.55 mg/kg, 40.44 mg/kg and 60.24 mg/kg at the respective efficiencies.The rates of removals were in the decreasing order of -0.0524d-1 for Pseudomonas sp., -0.0714 d-1 for Bacillus sp., -0.0743d-1 Micrococcus sp., and 0.113 d-1 E. coli. The fitted models showed diffusion as the rate-limiting step for removals by Pseudomonas sp., Bacillus sp., and Micrococcus sp.; while chemisorption was the rate-limiting step for removal by E. coli. This information will be helpful to researchers and decision makers for the remediation of lead contaminated soils.

Efficient expression in E. coli of an enantioselective nitrile hydratase from Rhodococcus erythropolis

2007 ◽  
Vol 30 (4) ◽  
pp. 755-762 ◽  
Author(s):  
Liya Song ◽  
Hong-Jie Yuan ◽  
Lee Coffey ◽  
John Doran ◽  
Mei-Xiang Wang ◽  
...  
Keyword(s):  
Nitrile Hydratase ◽  
Rhodococcus Erythropolis ◽  
E Coli ◽  
Efficient Expression

scholarly journals A hybrid cyt c maturation system enhances the bioelectrical performance of engineered Escherichia coli by improving the rate-limiting step

2020 ◽  
Author(s):  
Lin Su ◽  
Tatsuya Fukushima ◽  
Caroline M. Ajo-Franklin
Keyword(s):  
Escherichia Coli ◽  
Electron Flux ◽  
Electrical Response ◽  
Shewanella Oneidensis ◽  
Electrical Performance ◽  
E Coli ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cyt C ◽  
Rate Limiting

ABSTRACTBioelectronic devices can use electron flux to enable communication between biotic components and abiotic electrodes. We have modified Escherichia coli to electrically interact with electrodes by expressing the cytochrome c from Shewanella oneidensis MR-1. However, we observe inefficient electrical performance, which we hypothesize is due to the limited compatibility of the E. coli cytochrome c maturation (Ccm) systems with MR-1 cytochrome c. Here we test whether the bioelectronic performance of E. coli can be improved by constructing hybrid Ccm systems containing protein domains from both E. coli and S. oneidensis MR-1. The hybrid CcmH increased cytochrome c expression by increasing the abundance of CymA 60%, while only slightly changing the abundance of the other cytochromes c. Electrochemical measurements showed that the overall current from the hybrid ccm strain increased 121% relative to the wildtype ccm strain, with an electron flux per cell of 12.3 ± 0.3 fA·cell-1. Additionally, the hybrid ccm strain doubled its electrical response with the addition of exogenous flavin, and quantitative analysis of this demonstrates CymA is the rate-limiting step in this electron conduit. These results demonstrate that this hybrid Ccm system can enhance the bioelectrical performance of the cyt c expressing E. coli, allowing the construction of more efficient bioelectronic devices.

A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III

1994 ◽  
Vol 14 (1) ◽  
pp. 822-830
Author(s):  
G Rameau ◽  
K Puglia ◽  
A Crowe ◽  
I Sethy ◽  
I Willis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Structural Motif ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Polymerase Iii ◽  
Rate Limiting

In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.

scholarly journals The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase

1994 ◽  
Vol 300 (3) ◽  
pp. 765-770 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
E Coli ◽  
Rate Limiting Step ◽  
Rate Limiting

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

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