scholarly journals Enzymic synthesis of N-acetyl-l-phenylalanine in Escherichia coli K12

1971 ◽  
Vol 124 (5) ◽  
pp. 905-913 ◽  
Author(s):  
R. V. Krishna ◽  
P. R. Krishnaswamy ◽  
D. Rajagopal Rao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
E Coli ◽  
Enzymic Synthesis ◽  
Escherichia Coli K12

1. Cell-free extracts of Escherichia coli K12 catalyse the synthesis of N-acetyl-l-phenylalanine from acetyl-CoA and l-phenylalanine. 2. The acetyl-CoA–l-phenylalanine α-N-acetyltransferase was purified 160-fold from cell-free extracts. 3. The enzyme has a pH optimum of 8 and catalyses the acetylation of l-phenylalanine. Other l-amino acids such as histidine and alanine are acetylated at slower rates. 4. A transacylase was also purified from E. coli extracts and its substrate specificity studied. 5. The properties of both these enzymes were compared with those of other known amino acid acetyltransferases and transacylases.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Amino acid deprivation and its effect on mating ability inEscherichia coliK12

1966 ◽  
Vol 8 (1) ◽  
pp. 115-118 ◽  
Author(s):  
K. W. Fisher
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Deprivation ◽  
Mating Ability ◽  
Chromosome Transfer ◽  
E Coli ◽  
Donor Cells ◽  
Relationship Of ◽  
The Relationship

The conclusion by Suit, Matney, Doudney & Billen (1964) that Hfr donor cells ofEscherichia coliK12, starved of required amino acids can mate, has been re-examined. It appears that their conclusion is not valid and that apparent fertility of amino-acid starved cells is due to cross-feeding by the F−cells. The relationship of this result to the alternative mechanisms for chromosome transfer inE. coliis discussed.

scholarly journals Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli

1995 ◽  
Vol 309 (2) ◽  
pp. 411-417 ◽  
Author(s):  
K V Lu ◽  
M F Rohde ◽  
A R Thomason ◽  
W C Kenney ◽  
H S Lu
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Growth Factor ◽  
Translation Termination ◽  
Termination Codon ◽  
Platelet Derived Growth Factor ◽  
E Coli ◽  
Translation Termination Codon ◽  
Terminal Amino

The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.

Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, fromKlebsiellasp. 601

2008 ◽  
Vol 54 (9) ◽  
pp. 725-733 ◽  
Author(s):  
Yang Li ◽  
Jiao Yin ◽  
Guosheng Qu ◽  
Luchao Lv ◽  
Yadong Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Kinetic Studies ◽  
Multicopper Oxidase ◽  
Coli Strain ◽  
Gene Encoding ◽  
E Coli ◽  
C Terminus ◽  
Optimal Ph

A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2′-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat/Kmvalues of 0.49 mmol·L–1, 1.08 × 103s–1, and 2.23 × 103s–1·mmol–1·L, respectively, for DMP, 5.63 mmol·L–1, 6.64 × 103s–1, and 1.18 × 103s–1·mmol–1·L for ABTS, and 0.023 mmol·L–1, 11 s–1, and 4.68 × 102s–1·mmol–1·L for SGZ.

scholarly journals A Four-Step Enzymatic Cascade for Efficient Production of L-Phenylglycine from Biobased L-Phenylalanine

2022 ◽  
Author(s):  
Yuling Zhu ◽  
Jifeng Yuan
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Industrial Applications ◽  
Efficient Production ◽  
E Coli ◽  
Amycolatopsis Orientalis ◽  
Enzyme Cascade ◽  
Pharmaceutical Industries ◽  
Rate Limiting

Enantiopure amino acids are of particular interest in the agrochemical and pharmaceutical industries. Here, we reported a multi-enzyme cascade for efficient production of L-phenylglycine (L-Phg) from biobased L-phenylalanine (L-Phe). We first attempted to engineer Escherichia coli for expressing L-amino acid deaminase (LAAD) from Proteus mirabilis, hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis, (S)-mandelate dehydrogenase (SMDH) from Pseudomonas putida, the endogenous aminotransferase (AT) encoded by ilvE and L-glutamate dehydrogenase (GluDH) from E. coli. However, 10 mM L-Phe only afforded the synthesis of 7.21 mM L-Phg. The accumulation of benzoylformic acid suggested that the transamination step might be rate-limiting. We next used leucine dehydrogenase (LeuDH) from Bacillus cereus to bypass the use of L-glutamate as amine donor, and 40 mM L-Phe gave 39.97 mM (6.04 g/L) L-Phg, reaching 99.9% conversion. In summary, this work demonstrated a concise four-step enzymatic cascade for the L-Phg synthesis from biobased L-Phe, with a potential for future industrial applications.

scholarly journals Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

2020 ◽  
Author(s):  
Hong Liang ◽  
Xiaoqiang Ma ◽  
Wenbo Ning ◽  
Yurou Liu ◽  
Anthony J. Sinskey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Structural Similarity ◽  
List Type ◽  
Acetyl Coa ◽  
E Coli ◽  
Utilization Pathway ◽  
Aspartate Family

AbstractEngineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 hours. We also engineered E. coli strain to produced 24 mg/L of prenol from 10 g/L of ethanol in 48 hours, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.HighlightsEngineered Escherichia coli strains to grow on ethanol as sole carbon sourceDemonstrated that ethanol was converted into acetyl-CoA (AcCoA) through two pathways (acetaldehyde-acetate-AcCoA and acetaldehyde-AcCoA)Converted ethanol into two acetyl-CoA derived products with low structural similarity (polyhydroxybutyrate and prenol)Discovered that supplementation of the aspartate family amino acids can substantially improve cell growth on ethanol

scholarly journals Different Rifampin Sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA Polymerases Are Not Explained by the Difference in the β-Subunit Rifampin Regions I and II

2005 ◽  
Vol 49 (4) ◽  
pp. 1587-1590 ◽  
Author(s):  
N. Zenkin ◽  
A. Kulbachinskiy ◽  
I. Bass ◽  
V. Nikiforov
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Β Subunit ◽  
E Coli ◽  
Increased Sensitivity ◽  
The Difference

ABSTRACT Mycobacterium tuberculosis RNA polymerase is 1,000-fold more sensitive to rifampin than Escherichia coli RNA polymerase. Chimeric E. coli RNA polymerase in which the β-subunit segment encompassing rifampin regions I and II (amino acids [aa] 463 through 590) was replaced with the corresponding region from M. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. Thus, the difference in amino acid sequence between the rifampin regions I and II of the two species does not account for the difference in rifampin sensitivity of the two polymerases.

scholarly journals Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

2010 ◽  
Vol 55 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Akiko Shimizu-Ibuka ◽  
Mika Oishi ◽  
Shoko Yamada ◽  
Yoshikazu Ishii ◽  
Kiyoshi Mura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Binding Site ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Hydrolysis Of

ABSTRACTToho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced inEscherichia colistrains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum

1980 ◽  
Vol 58 (9) ◽  
pp. 696-706 ◽  
Author(s):  
David Morse ◽  
Harry W. Duckworth
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Citrate Synthase ◽  
Amino Acid Sequences ◽  
Aerobic Bacterium ◽  
Acetyl Coa ◽  
Gram Negative ◽  
E Coli ◽  
Sulfhydryl Reagent ◽  
Complete Inactivation

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, K1 = 2 μM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 μM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968)) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4′-dithiodipyridine (4,4′-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a rise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4′-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.

scholarly journals The rfaE Gene from Escherichia coli Encodes a Bifunctional Protein Involved in Biosynthesis of the Lipopolysaccharide Core Precursor ADP-l-glycero-d-manno-Heptose

2000 ◽  
Vol 182 (2) ◽  
pp. 488-497 ◽  
Author(s):  
Miguel A. Valvano ◽  
Cristina L. Marolda ◽  
Mauricio Bittner ◽  
Mike Glaskin-Clay ◽  
Tania L. Simon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Inner Core ◽  
Structural Features ◽  
Predicted Amino Acid Sequence ◽  
E Coli ◽  
K 12 ◽  
Domain I

ABSTRACT The intermediate steps in the biosynthesis of the ADP-l-glycero-d-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement therfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation ofSalmonella enterica SL1102. DNA sequencing of therfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation inR. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains ofrfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis ofd-glycero-d-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-d-glycero-d-manno-heptose.

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