scholarly journals Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Paweł Filipkowski ◽  
Olga Pietrow ◽  
Anna Panek ◽  
Józef Synowiecki
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Deinococcus Radiodurans ◽  
Trehalose Synthase ◽  
Reading Frame ◽  
Metal Affinity ◽  
C Terminus

A trehalose synthase gene from Deinococcus radiodurans (DSMZ 20539) containing 1659 bp reading frame encoding 552 amino acids was amplified using PCR. The gene was finally ligated into pET30Ek/LIC vector and expressed after isopropyl β-d-thiogalactopyranoside induction in Escherichia coli (DE3) Rosetta pLysS. The recombinant trehalose synthase (DraTreS) containing a His(6)-tag at the C-terminus was purified by metal affinity chromatography and characterized. The expressed enzyme is a homodimer with molecular mass of 126.9 kDa and exhibits the highest activity of 11.35 U/mg at pH 7.6 and at 30°C. DraTreS activity was almost unchanged after 2 h preincubation at 45°C and pH 7.6, and retained about 56% of maximal value after 8 h incubation at 50°C. The DraTreS was strongly inhibited by Cu(2+), Hg(2+), Zn(2+), Al(3+) and 10 mM Tris. The K(m) value of maltose conversion was 290.7 mM.

scholarly journals The C-Terminal Flexible Domain of the Heme Chaperone CcmE Is Important but Not Essential for Its Function

2003 ◽  
Vol 185 (13) ◽  
pp. 3821-3827 ◽  
Author(s):  
Elisabeth Enggist ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heme Binding ◽  
Membrane Anchor ◽  
Terminal Domain ◽  
Conserved Domain ◽  
C Terminus ◽  
Helical Turn ◽  
Low Activity

ABSTRACT CcmE is a heme chaperone active in the cytochrome c maturation pathway of Escherichia coli. It first binds heme covalently to strictly conserved histidine H130 and subsequently delivers it to apo-cytochrome c. The recently solved structure of soluble CcmE revealed a compact core consisting of a β-barrel and a flexible C-terminal domain with a short α-helical turn. In order to elucidate the function of this poorly conserved domain, CcmE was truncated stepwise from the C terminus. Removal of all 29 amino acids up to crucial histidine 130 did not abolish heme binding completely. For detectable transfer of heme to type c cytochromes, only one additional residue, D131, was required, and for efficient cytochrome c maturation, the seven-residue sequence 131DENYTPP137 was required. When soluble forms of CcmE were expressed in the periplasm, the C-terminal domain had to be slightly longer to allow detection of holo-CcmE. Soluble full-length CcmE had low activity in cytochrome c maturation, indicating the importance of the N-terminal membrane anchor for the in vivo function of CcmE.

scholarly journals UDP-sugar Pyrophosphorylase with Broad Substrate Specificity Toward Various Monosaccharide 1-Phosphates from Pea Sprouts

2004 ◽  
Vol 279 (44) ◽  
pp. 45728-45736 ◽  
Author(s):  
Toshihisa Kotake ◽  
Daisuke Yamaguchi ◽  
Hiroshi Ohzono ◽  
Sachiko Hojo ◽  
Satoshi Kaneko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Substrate Specificity ◽  
Molecular Mass ◽  
Glucuronic Acid ◽  
De Novo ◽  
Higher Plants ◽  
Maximum Activity ◽  
Broad Substrate Specificity ◽  
Salvage Pathways

UDP-sugars, activated forms of monosaccharides, are synthesized throughde novoand salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDP-sugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativumL.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDP-Gal, UDP-glucuronic acid, UDP-l-arabinose, and UDP-xylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5–7.5, and at 45 °C in the presence of 2 mmMg2+. The apparentKmvalues for Glc 1-phosphate andl-arabinose 1-phosphate were 0.34 and 0.96 mm, respectively.PsUSPcDNA was cloned by reverse transcriptase-PCR.PsUSPappears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues fromArabidopsisand rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed inEscherichia colicatalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDP-sugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.

scholarly journals Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

2000 ◽  
Vol 182 (22) ◽  
pp. 6440-6450 ◽  
Author(s):  
Jouko Sillanpää ◽  
Beatriz Martínez ◽  
Jenni Antikainen ◽  
Takahiro Toba ◽  
Nisse Kalkkinen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Type I Collagen ◽  
Signal Sequence ◽  
Type I ◽  
Binding Region ◽  
Sequence Comparisons ◽  
Collagen Binding ◽  
Reading Frame ◽  
Lactobacillus Crispatus ◽  
C Terminus

The cbsA gene of Lactobacillus crispatusstrain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. ThecbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three otherLactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsAwhere deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

scholarly journals Evidence for a Holin-Like Protein Gene Fully Embedded Out of Frame in the Endolysin Gene of Staphylococcus aureusBacteriophage 187

1999 ◽  
Vol 181 (15) ◽  
pp. 4452-4460 ◽  
Author(s):  
Martin J. Loessner ◽  
Susanne Gaeng ◽  
Siegfried Scherer
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
De Novo ◽  
Atp Synthesis ◽  
Large Cell ◽  
Lytic Enzyme ◽  
Reading Frame ◽  
C Terminus

ABSTRACT We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique Staphylococcus aureus phage 187. The endolysin gene ply187 encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined ply187 domains. This enzymatically active N terminus showed convincing amino acid sequence homology to anN-acetylmuramoyl-l-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5′ region of ply187 and was shown to encode a small, hydrophobic protein of holin-like function. The hol187 gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in Escherichia coli resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected S. aureus cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage λ Sam mutation in a nonsuppressing E. coli HB101 background. Plaque formation by λgt11::hol187 indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of ply187 and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected Staphylococcus cells.

scholarly journals Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

2009 ◽  
Vol 191 (15) ◽  
pp. 4996-5009 ◽  
Author(s):  
Dirk Tischler ◽  
Dirk Eulberg ◽  
Silvia Lakner ◽  
Stefan R. Kaschabek ◽  
Willem J. H. van Berkel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Styrene Oxide ◽  
Functional Expression ◽  
Rhodococcus Opacus ◽  
Reading Frame ◽  
Arthrobacter Aurescens ◽  
C Terminus ◽  
Two Component ◽  
Rhodococcus Opacus 1Cp

ABSTRACT Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

scholarly journals Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

2007 ◽  
Vol 73 (17) ◽  
pp. 5598-5606 ◽  
Author(s):  
Jeong Min Seo ◽  
Geun Eog Ji ◽  
Sang Hee Cho ◽  
Myung Soo Park ◽  
Hyong Joo Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Bifidobacterium Longum ◽  
Deae Cellulose ◽  
Cell Extract ◽  
Coding Region ◽  
Tryptic Fragment ◽  
C Terminus ◽  
A Cell

ABSTRACT A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.

scholarly journals Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 1005
Author(s):  
Yi-Ting Fang ◽  
Si-Yu Li ◽  
Nien-Jen Hu ◽  
Jie Yang ◽  
Jyung-Hurng Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Md Simulation ◽  
Cleavage Reaction ◽  
Chitin Binding Domain ◽  
First Order ◽  
C Terminus ◽  
Efficient Performance ◽  
Order Rate Equation

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.

Characterization of three terpenoid glycosyltransferase genes in ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck)

Genome ◽  
2010 ◽  
Vol 53 (10) ◽  
pp. 816-823 ◽  
Author(s):  
Jing Fan ◽  
Chunxian Chen ◽  
Qibin Yu ◽  
Zheng-Guo Li ◽  
Frederick G. Gmitter
Keyword(s):  
Amino Acids ◽  
Citrus Sinensis ◽  
Southern Blotting ◽  
Sweet Orange ◽  
Secondary Product ◽  
Reading Frame ◽  
C Terminus ◽  
Group D

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in ‘Valencia’ sweet orange ( Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730–874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.

scholarly journals Chemotaxis of Escherichia coli to Pyrimidines: a New Role for the Signal Transducer Tap

2007 ◽  
Vol 190 (3) ◽  
pp. 972-979 ◽  
Author(s):  
Xianxian Liu ◽  
Rebecca E. Parales
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Growth Conditions ◽  
Wild Type ◽  
E Coli ◽  
C Terminus ◽  
Signaling Domain ◽  
Chemotaxis Proteins ◽  
Periplasmic Domain

ABSTRACT Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coli RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.

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