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1.
Aims: To investigate the effect of an absence of aminopeptidase PepS on the growth of Streptococcus thermophilus on different media and at different temperatures.
Methods and Results: Using gene interruption, a negative mutant of the Strep. thermophilus CNRZ385 strain was constructed for the aminopeptidase PepS (strain Δ pepS ). Checks were first of all made using biochemical assays that the Δ pepS strain lacks the peptide hydrolase activity of aminopeptidase PepS. It was demonstrated that the absence of the aminopeptidase PepS exerted a negative effect on growth whatever the culture medium (M17, chemically defined medium, milk). The role of aminopeptidase PepS in growth was enhanced at a high temperature (45°C vs 37°C). The Δ pepS strain was more resistant to lysozyme than the wild-type strain .
Conclusions: We were able to demonstrate that aminopeptidase PepS probably plays a pleiotropic role through its involvement in growth via nitrogen nutrition, as well as via other cellular functions/metabolisms (such as peptidoglycane metabolism).
Significance and Impact of the Study: This study constitutes the first report on the role of a member of the M29 MEROPS family of metallopeptidases ( http://merops.sanger.ac.uk/ ). 相似文献
Methods and Results: Using gene interruption, a negative mutant of the Strep. thermophilus CNRZ385 strain was constructed for the aminopeptidase PepS (strain Δ pepS ). Checks were first of all made using biochemical assays that the Δ pepS strain lacks the peptide hydrolase activity of aminopeptidase PepS. It was demonstrated that the absence of the aminopeptidase PepS exerted a negative effect on growth whatever the culture medium (M17, chemically defined medium, milk). The role of aminopeptidase PepS in growth was enhanced at a high temperature (45°C vs 37°C). The Δ pepS strain was more resistant to lysozyme than the wild-type strain .
Conclusions: We were able to demonstrate that aminopeptidase PepS probably plays a pleiotropic role through its involvement in growth via nitrogen nutrition, as well as via other cellular functions/metabolisms (such as peptidoglycane metabolism).
Significance and Impact of the Study: This study constitutes the first report on the role of a member of the M29 MEROPS family of metallopeptidases ( http://merops.sanger.ac.uk/ ). 相似文献
2.
The X-ray crystallographic structure of a thioredoxin from Thermus thermophilus was solved to 1.8 A resolution by molecular replacement. The crystals' space group was C2 with cell dimensions of a = 40.91, b = 95.44, c = 56.68 A, beta =91.41 degrees, with two molecules in the asymmetric unit. Unlike the reported thioredoxin structures, the biological unit of T. thermophilus thioredoxin is a dimer both in solution and in the crystal. The fold conforms to the "thioredoxin fold" that is common over a class of nine protein families including thioredoxin; however, the folded portion of this protein is much more compact than other thioredoxins previously solved by X-ray crystallography being reduced by one alpha-helix and one beta-strand. As with other thioredoxins, the active site is highly conserved even though the variation in sequence can be quite large. The T. thermophilus thioredoxin has some variability at the active site, especially compared with previously solved structures from bacterial sources. 相似文献
3.
James A. Fee Tatsuro Yoshida Kristene K. Surerus Michael W. Mather 《Journal of bioenergetics and biomembranes》1993,25(2):103-114
The subject of this short review is the cytochromec oxidase (caa
3) from the thermophilic bacteriumThermus thermophilus. First, some of the extensive physical and enzymological results obtained with this enzyme are reviewed, and two experiments are described, involving isotope substitutions in combination with Mössbauer and ENDOR spectroscopies, which have provided novel insight into the active sites of the enzyme. Second, we summarize recent molecular genetic work showing thatThermus cytochromecaa
3 is abona fide member of the superfamily of heme-copper oxidases. Finally, we present a rough three-dimensional model and speculate about certain features of the metal-binding sites. 相似文献
4.
The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme. 相似文献
5.
An intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100. The enzyme had a molecular weight of 89,000. It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35 degrees C with Km = 1.80 mmol/l; above 55 degrees C the enzyme activity declined rapidly. The aminopeptidase was capable of degrading substrates by hydrolysis of the N-terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity. The enzyme was strongly inactivated by EDTA. Serine and sulphydryl group reagents had no effect on enzyme activity. 相似文献
6.
E. TSAKALIDOU AND G. KALANTZOPOULOS. 1992. An intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100. The enzyme had a molecular weight of 89 000. It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35°C with K m = 1.80 mmol/l; above 55°C the enzyme activity declined rapidly. The aminopeptidase was capable of degrading substrates by hydrolysis of the N -terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity. The enzyme was strongly inactivated by EDTA. Serine and sulphydryl group reagents had no effect on enzyme activity. 相似文献
7.
Mingardon F Bagert JD Maisonnier C Trudeau DL Arnold FH 《Applied and environmental microbiology》2011,77(4):1436-1442
Cellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three homologs: Clostridium cellulolyticum Cel9G, Thermobifida fusca Cel9A, and C. thermocellum Cel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without a T. fusca fibronectin type 3 homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, but C. thermocellum Cel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation. 相似文献
8.
H Ragg 《Nucleic acids research》1986,14(2):1073-1088
A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II. 相似文献
9.
Anastasiou R Papadelli M Georgalaki MD Kalantzopoulos G Tsakalidou E 《Journal of applied microbiology》2002,93(1):52-59
AIMS: To clone and sequence the pepX gene from Streptococcus thermophilus. METHODS AND RESULTS: Three pairs of primers were used in polymerase chain reactions using as template the total DNA from Strep. thermophilus ACA-DC 4 in order to amplify, clone and sequence the pepX gene. Sequence analysis revealed an open reading frame of 2268 nucleotides encoding a protein of 755 amino acids. The calculated molecular mass of 85 632 Da agreed well with the apparent molecular mass of 80 000 Da previously determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration for the monomeric form of the purified enzyme. CONCLUSIONS: The pepX gene from Strep. thermophilus ACA-DC 4 was cloned and sequenced. The PepX protein showed significant sequence similarity with PepX enzymes from other lactic acid bacteria and contained a motif which was almost identical with the active site motif of the serine-dependent PepX family. SIGNIFICANCE AND IMPACT OF THE STUDY: There are economic and technological incentives for accelerating and controlling the process of cheese ripening. To achieve this, starters may be modified by introducing appropriate genes from other food-grade bacteria. New or additional peptidase activities may alter or improve the proteolytic properties of lactic acid bacteria. 相似文献
10.
A human T cell-specific molecule is a member of a new gene family 总被引:40,自引:0,他引:40
T J Schall J Jongstra B J Dyer J Jorgensen C Clayberger M M Davis A M Krensky 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(3):1018-1025
We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules. 相似文献
11.
A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding. 相似文献
12.
A new member of the bacterial ribonuclease inhibitor family from Saccharopolyspora erythraea 总被引:1,自引:0,他引:1
We have identified Sti, the gene of a ribonuclease inhibitor from Saccharopolyspora erythraea, by using a T7 phage display system. A specific phage has been isolated from a genome library by a biopanning procedure, using RNase Sa3, a ribonuclease from Streptomyces aureofaciens, as bait. Sti, a protein of 121 amino acid residues, with molecular mass 13059 Da, is a homolog of barstar and other microbial ribonuclease inhibitors. To overexpress its gene in Escherichia coli, we optimized the secondary structure of its mRNA by introducing a series of silent mutations. Soluble protein was isolated and purified to homogeneity. Inhibition constants of complex of Sti and RNase Sa3 or barnase were determined at pH 7 as 5 x 10(-12) or 7 x 10(-7), respectively. 相似文献
13.
14.
15.
A new inhibitor of metalloproteinases from chicken: ChIMP-3. A third member of the TIMP family. 总被引:7,自引:0,他引:7
N Pavloff P W Staskus N S Kishnani S P Hawkes 《The Journal of biological chemistry》1992,267(24):17321-17326
We report cDNA cloning and primary structure of a new metalloproteinase inhibitor (ChIMP-3) produced by chicken embryo fibroblasts. ChIMP-3, formerly called the 21-kDa protein, is one of five ChIMPs (Chicken Inhibitor of MetalloProteinases). In this paper, we report that of the three most abundant ChIMPs, ChIMP-3 and ChIMP-a are extracellular matrix components, whereas ChIMP-2 is found in the media conditioned by the cells. Treatment of ChIMP-3 and ChIMP-a with N-glycosidase-F indicates that ChIMP-a is N-glycosylated whereas ChIMP-3 is not. The deduced amino acid sequence of ChIMP-3 predicts a protein whose properties are consistent with experimental measurements. Analysis of sequence alignments with the two previously described members of the TIMP (tissue inhibitor of metalloproteinases) family, TIMP-1 and TIMP-2, from various species indicates that ChIMP-3 is a related but distinct protein. This conclusion is supported by lack of significant binding with anti-TIMP-1 and anti-TIMP-2 antibodies. Based on these data, its unusual solubility properties, and its exclusive location in the matrix, we propose that ChIMP-3 is a new member of this family of metalloproteinase inhibitors, a TIMP-3. 相似文献
16.
Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus 总被引:6,自引:0,他引:6
The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli. 相似文献
17.
Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaABd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The
final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme
was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal
activity at 50°C and pH 7.5. LaaABd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was
largely restored by the addition of Co2+ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co2+, suggesting that LaaABd is a Co2+-dependent enzyme. LaaABd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaABd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence
of seven open-reading frames (ORFs), one of which (laaA
Bd
) encodes the amidase. LaaABd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits
significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA
Bd
gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaABd in cell-free extracts of the E. coli transformant was 25.9 units mg−1 with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720. 相似文献
18.
Rosselli LK Oliveira CL Azzoni AR Tada SF Catani CF Saraiva AM Soares JS Medrano FJ Torriani IL Souza AP 《Archives of biochemistry and biophysics》2006,453(2):143-150
The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants. 相似文献
19.