scholarly journals Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

2020 ◽  
Author(s):  
Ekaterina A. Shmonova ◽  
Olga V. Voloshina ◽  
Maksim V. Ovsienko ◽  
Sergey V. Smirnov ◽  
Vera G. Doroshenko
Keyword(s):  
Pseudomonas Putida ◽  
Corynebacterium Glutamicum ◽  
Protocatechuic Acid ◽  
Microbial Production ◽  
E Coli ◽  
Sequence Identity ◽  
Terminal Domain ◽  
Monomeric Structure ◽  
Dehydratase Activity

AbstractThe dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum encoded by the qsuB gene is related to the previously described QuiC1 protein (39.9% identity) from Pseudomonas putida. QuiC1 and QsuB are both two-domain bacterial DSDs. The N-terminal domain provides dehydratase activity, while the C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate dioxygenase. Here, the QsuB protein and its DSD domain (N-QsuB) were expressed in the T7 system, purified and characterized. QsuB was present mainly in octameric form (60%), while N-QsuB had a predominantly monomeric structure (80%) in solution. Both proteins possessed DSD activity with one of the following cofactors (listed in order of decreasing activity): Co2+, Mg2+, Mn2+ or Ca2+. The Km and kcat values for QsuB were two and three times higher, respectively (Km ~ 1 mM, kcat ~ 61 s−1) than those for N-QsuB. Notably, 3,4-DHBA inhibited both enzymes via an uncompetitive mechanism. QsuB and N-QsuB were tested for 3,4-DHBA production from glucose in E. coli. MG1655ΔaroE Plac–qsuB produced at least two times more 3,4-DHBA than MG1655ΔaroE Plac–n-qsuB in the presence of isopropyl β-D-1-thiogalactopyranoside.

scholarly journals Characterization of the Corynebacterium glutamicum dehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid

PLoS ONE ◽  
2020 ◽  
Vol 15 (8) ◽  
pp. e0231560
Author(s):  
Ekaterina A. Shmonova ◽  
Olga V. Voloshina ◽  
Maksim V. Ovsienko ◽  
Sergey V. Smirnov ◽  
Dmitry E. Nolde ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Protocatechuic Acid ◽  
Microbial Production

scholarly journals NMR structure of the C-terminal domain of TonB protein from Pseudomonas aeruginosa

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5412 ◽  
Author(s):  
Jesper S. Oeemig ◽  
O.H. Samuli Ollila ◽  
Hideo Iwaï
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structural Changes ◽  
Nmr Structure ◽  
Dynamics Simulation ◽  
Solution Nmr ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Terminal Domain ◽  
Tonb Protein ◽  
Monomeric Structure

The TonB protein plays an essential role in the energy transduction system to drive active transport across the outer membrane (OM) using the proton-motive force of the cytoplasmic membrane of Gram-negative bacteria. The C-terminal domain (CTD) of TonB protein is known to interact with the conserved TonB box motif of TonB-dependent OM transporters, which likely induces structural changes in the OM transporters. Several distinct conformations of differently dissected CTDs of Escherichia coli TonB have been previously reported. Here we determined the solution NMR structure of a 96-residue fragment of Pseudomonas aeruginosa TonB (PaTonB-96). The structure shows a monomeric structure with the flexible C-terminal region (residues 338–342), different from the NMR structure of E. coli TonB (EcTonB-137). The extended and flexible C-terminal residues are confirmed by 15N relaxation analysis and molecular dynamics simulation. We created models for the PaTonB-96/TonB box interaction and propose that the internal fluctuations of PaTonB-96 makes it more accessible for the interactions with the TonB box and possibly plays a role in disrupting the plug domain of the TonB-dependent OM transporters.

scholarly journals Characterization of Two New Aminopeptidases in Escherichia coli

2005 ◽  
Vol 187 (11) ◽  
pp. 3671-3677 ◽  
Author(s):  
Yu Zheng ◽  
Richard J. Roberts ◽  
Simon Kasif ◽  
Chudi Guan
Keyword(s):  
Escherichia Coli ◽  
Stop Codon ◽  
Bacterial Species ◽  
Start Codon ◽  
Aminopeptidase Activity ◽  
Peptide Substrates ◽  
E Coli ◽  
Terminal Domain ◽  
Methionyl Aminopeptidase

ABSTRACT Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.

scholarly journals Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli

2004 ◽  
Vol 70 (12) ◽  
pp. 6968-6976 ◽  
Author(s):  
Taek Ho Yang ◽  
Jae Gu Pan ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Cell Viability ◽  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Fusion Proteins ◽  
Functional Expression ◽  
E Coli ◽  
Terminal Domain ◽  
Anchoring Motif ◽  
Periplasmic Enzyme

ABSTRACT The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme β-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.

Characterization of a phospholipase Dα cDNA from tomato fruit

2000 ◽  
Vol 28 (6) ◽  
pp. 819-821
Author(s):  
B. D. Whitaker ◽  
D. L. Smith ◽  
K. C. Green
Keyword(s):  
Castor Bean ◽  
Tomato Fruit ◽  
Molecular Genetic ◽  
Reading Frame ◽  
E Coli ◽  
Sequence Identity ◽  
Tomato Fruit Ripening ◽  
Sds Page

Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Total PL declines and phosphatidic acid increases in pericarp tissue during tomato fruit ripening, suggesting that increased PLD activity alters membrane structure. To assess the role of PLD in tomato ripening, we have begun a molecular genetic approach. Using a castor bean PLDα cDNA as a probe, a PLDα cDNA (LEPLD2) was isolated from our tomato fruit library. It has an open reading frame of 2421 nucleotides, encoding a polypeptide of 807 amino acids with a molecular mass of 92 kDa. The deduced LEPLD2 PLDα shares > 75% sequence identity with PLDαs from castor bean, tobacco and tomato. LEPLD2 transcript, detected by RNA gel-blot analysis, was very low in roots, low in stems, moderate in leaves, high in flowers, and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded active enzyme, and a FLAG-PLDα fusion protein produced by transformed E. coli migrated close to the calculated 94 kDa on SDS/PAGE.

scholarly journals Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering

2019 ◽  
Vol 20 (6) ◽  
pp. 1269 ◽  
Author(s):  
Sakda Yainoy ◽  
Thanawat Phuadraksa ◽  
Sineewanlaya Wichit ◽  
Maprang Sompoppokakul ◽  
Napat Songtawee ◽  
...  
Keyword(s):  
Rational Design ◽  
Specific Activity ◽  
Homology Modelling ◽  
Biochemical Properties ◽  
Wild Type ◽  
E Coli ◽  
Sequence Identity ◽  
High Expression Level ◽  
The Ideal

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at −20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.

scholarly journals The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

2020 ◽  
Vol 295 (18) ◽  
pp. 6225-6235 ◽  
Author(s):  
Alexander C. Anderson ◽  
Alysha J. N. Burnett ◽  
Lana Hiscock ◽  
Kenneth E. Maly ◽  
Joel T. Weadge
Keyword(s):  
Escherichia Coli ◽  
Catalytic Mechanism ◽  
Cellulose Synthase ◽  
Gram Negative Bacteria ◽  
Membrane Phospholipids ◽  
Colistin Resistance ◽  
E Coli ◽  
Terminal Domain

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

scholarly journals Cloning and Characterization of the Gene EncodingPasteurella haemolytica FnrP, a Regulator of theEscherichia coli Silent Hemolysin SheA

1999 ◽  
Vol 181 (12) ◽  
pp. 3845-3848 ◽  
Author(s):  
Gaylen A. Uhlich ◽  
Peter J. McNamara ◽  
John J. Iandolo ◽  
Derek A. Mosier
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Transcriptional Regulator ◽  
Pasteurella Haemolytica ◽  
Anaerobic Growth ◽  
Null Mutant ◽  
E Coli ◽  
Sequence Identity ◽  
Reductase Gene

ABSTRACT A Pasteurella haemolytica A1 gene was identified from a recombinant library clone that expressed hemolysis in hostEscherichia coli cells. The gene, designatedfnrP, had sequence identity to E. coli fnr, a global transcriptional regulator of genes required for conversion to anaerobic growth. FnrP complemented anaerobic deficiencies of afnr-null mutant strain of E. coli and increased expression of the Fnr-dependent, anaerobic terminal reductase gene,frdA. FnrP was purified, identified by immunoblotting, and shown to be nonhemolytic. When FnrP was expressed in E. coli ΔsheA, a null mutant of the cryptic hemolysin SheA, the transformants were nonhemolytic, indicating that FnrP activates this silent hemolysin.

scholarly journals Genetic Characterization of the Resorcinol Catabolic Pathway in Corynebacterium glutamicum

2006 ◽  
Vol 72 (11) ◽  
pp. 7238-7245 ◽  
Author(s):  
Yan Huang ◽  
Ke-Xin Zhao ◽  
Xi-Hui Shen ◽  
Muhammad Tausif Chaudhry ◽  
Cheng-Ying Jiang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Genetic Characterization ◽  
Gene Clusters ◽  
Sole Source ◽  
Cloning And Expression ◽  
Catabolic Pathway ◽  
E Coli ◽  
Genome Wide ◽  
Genome Wide Data

ABSTRACT Corynebacterium glutamicum grew on resorcinol as a sole source of carbon and energy. By genome-wide data mining, two gene clusters, designated NCgl1110-NCgl1113 and NCgl2950-NCgl2953, were proposed to encode putative proteins involved in resorcinol catabolism. Deletion of the NCgl2950-NCgl2953 gene cluster did not result in any observable phenotype changes. Disruption and complementation of each gene at NCgl1110-NCgl1113, NCgl2951, and NCgl2952 indicated that these genes were involved in resorcinol degradation. Expression of NCgl1112, NCgl1113, and NCgl2951 in Escherichia coli revealed that NCgl1113 and NCgl2951 both coded for hydroxyquinol 1,2-dioxygenases and NCgl1112 coded for maleylacetate reductases. NCgl1111 encoded a putative monooxygenase, but this putative hydroxylase was very different from previously functionally identified hydroxylases. Cloning and expression of NCgl1111 in E. coli revealed that NCgl1111 encoded a resorcinol hydroxylase that needs NADPH as a cofactor. E. coli cells containing Ncgl1111 and Ncgl1113 sequentially converted resorcinol into maleylacetate. NCgl1110 and NCgl2950 both encoded putative TetR family repressors, but only NCgl1110 was transcribed and functional. NCgl2953 encoded a putative transporter, but disruption of this gene did not affect resorcinol degradation by C. glutamicum. The function of NCgl2953 remains unclear.

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