scholarly journals Identification of Conserved Amino Acid Residues of the Salmonella σS Chaperone Crl Involved in Crl-σS Interactions

2009 ◽  
Vol 192 (4) ◽  
pp. 1075-1087 ◽  
Author(s):  
Véronique Monteil ◽  
Annie Kolb ◽  
Jacques D'Alayer ◽  
Pierre Beguin ◽  
Françoise Norel
Keyword(s):  
General Rule ◽  
Site Directed Mutagenesis ◽  
Ferric Uptake Regulator ◽  
Amino Acid Residues ◽  
Bacterial Genomes ◽  
E Coli ◽  
Core Enzyme ◽  
Serovar Typhimurium ◽  
Σ Factor ◽  
The Relationship

ABSTRACT Proteins that bind σ factors typically attenuate the function of the σ factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor σS (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of σS-dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a Δcrl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-σS interaction and that residues 1 to 71 in σS are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli.

scholarly journals Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase

2000 ◽  
Vol 182 (9) ◽  
pp. 2536-2543 ◽  
Author(s):  
Rachel A. L. Lassy ◽  
Charles G. Miller
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Serine Hydrolase ◽  
Genome Sequences ◽  
New Family ◽  
The Family ◽  
Serovar Typhimurium ◽  
Structural Classes

ABSTRACT Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available.

scholarly journals Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase

2000 ◽  
Vol 182 (4) ◽  
pp. 869-873 ◽  
Author(s):  
Lise Tourneux ◽  
Nadia Bucurenci ◽  
Cosmin Saveanu ◽  
Pierre Alexandre Kaminski ◽  
Madeleine Bouzon ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Gene Encoding ◽  
E Coli ◽  
Salmonella Enterica Serovar Typhi ◽  
Acid Protein ◽  
Serovar Typhimurium ◽  
Amino Acid Protein

ABSTRACT We identified in the genome of Salmonella entericaserovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between theuhpA and ilvN genes, is absent inEscherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. colicorrespond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coliribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased theV max of deoxyribokinase by a factor of 2.5 and increased the K m for deoxyribose by a factor of 70, compared to the parent enzyme.

scholarly journals Expression of the Isoamylase Gene ofFlavobacterium odoratum KU in Escherichia coliand Identification of Essential Residues of the Enzyme by Site-Directed Mutagenesis

1999 ◽  
Vol 65 (9) ◽  
pp. 4163-4170 ◽  
Author(s):  
Jun-ichi Abe ◽  
Chiaki Ushijima ◽  
Susumu Hizukuri
Keyword(s):  
Culture Medium ◽  
Active Form ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
E Coli ◽  
Protein X ◽  
Mature Enzyme ◽  
Flavobacterium Odoratum ◽  
Branched Chains ◽  
Different Origins

ABSTRACT The isoamylase gene from Flavobacterium odoratum KU was cloned into and expressed in Escherichia coli JM109. The promoter of the gene was successful in E. coli, and the enzyme produced was excreted into the culture medium, depending on the amount of the enzyme expressed. The enzyme found in the culture medium showed almost the same M r, heat-inactivating constant, and N-terminal sequence as those of the enzyme accumulated in the periplasmic space. This result indicated that the enzyme accumulated in an active form at the periplasm was transported out of the cell. The primary sequence of the enzyme, which was deduced from its nucleotide sequence, showed that the mature enzyme consisted of 741 amino acid residues. By changing five possible residues to Ala independently, it was found that Asp-374, Glu-422, and Asp-497 were essential. The sequences around those residues were highly conserved in isoamylases of different origins and the glycogen operon protein X, GlgX. The comparison of the distance between these essential residues with those of various amylases suggested that the bacterial and plant isoamylase but not GlgX had a longer fourth loop than the other amylases. This longer fourth loop had a possible role in accommodating the long branched chains of native glycogens and starches.

scholarly journals Conserved Structural Regions Involved in the Catalytic Mechanism of Escherichia coli K-12 WaaO (RfaI)

1998 ◽  
Vol 180 (20) ◽  
pp. 5313-5318 ◽  
Author(s):  
Keigo Shibayama ◽  
Shinji Ohsuka ◽  
Toshihiko Tanaka ◽  
Yoshichika Arakawa ◽  
Michio Ohta
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Glycosidic Linkage ◽  
Hydrophobic Cluster Analysis ◽  
E Coli ◽  
Catalytic Sites ◽  
K 12

ABSTRACT Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.

scholarly journals Mutagenesis of Region 4 of Sigma 28 from Chlamydia trachomatis Defines Determinants for Protein-Protein and Protein-DNA Interactions

2008 ◽  
Vol 191 (2) ◽  
pp. 651-660 ◽  
Author(s):  
Ziyu Hua ◽  
Xiancai Rao ◽  
Xiaogeng Feng ◽  
Xudong Luo ◽  
Yanmei Liang ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Morphological Differentiation ◽  
Amino Acid Residues ◽  
E Coli ◽  
Promoter Recognition ◽  
Protein Dna Interactions ◽  
Serovar Typhimurium ◽  
Insight Into

ABSTRACT Transcription factor σ28 in Chlamydia trachomatis (σ28 Ct) plays a role in the regulation of genes that are important for late-stage morphological differentiation. In vitro mutational and genetic screening in Salmonella enterica serovar Typhimurium was performed in order to identify mutants with mutations in region 4 of σ28 Ct that were defective in σ28-specific transcription. Specially, the previously undefined but important interactions between σ28 Ct region 4 and the flap domain of the RNA polymerase β subunit (β-flap) or the −35 element of the chlamydial hctB promoter were examined. Our results indicate that amino acid residues E206, Y214, and E222 of σ28 Ct contribute to an interaction with the β-flap when σ28 Ct associates with the core RNA polymerase. These residues function in contacts with the β-flap similarly to their counterpart residues in Escherichia coli σ70. Conversely, residue Q236 of σ28 Ct directly binds the chlamydial hctB −35 element. The conserved counterpart residue in E. coli σ70 has not been reported to interact with the −35 element of the σ70 promoter. Observed functional disparity between σ28 Ct and σ70 region 4 is consistent with their divergent properties in promoter recognition. This work provides new insight into understanding the molecular basis of gene regulation controlled by σ28 Ct in C. trachomatis.

Site-Directed Mutagenesis of Proline-285 to Leucine in Cephalosporium acremonium Isopenicillin N - Synthase Affects Catalysis and Increases Soluble Expression at Higher Temperatures

2001 ◽  
Vol 56 (5-6) ◽  
pp. 413-415 ◽  
Author(s):  
Paxton Loke ◽  
Tiow-Suan Sim
Keyword(s):  
Biosynthetic Pathway ◽  
Soluble Expression ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cephalosporium Acremonium ◽  
E Coli ◽  
Measurable Activity ◽  
Important Enzyme ◽  
Isopenicillin N

The conversion of δ-(ʟ-α-aminoadipyl)-ʟ-cysteinyl-ᴅ-valine (ACV) to isopenicillin N is dependant on the catalytic action of isopenicillin N - synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. One of the amino acid residues suggested by the Aspergillus nidulans IPNS crystal structure for interaction with the valine isopropyl group of ACV is proline-283. Site-directed mutagenesis of the corresponding proline- 285 to leucine in Cephalosporium acremonium IPNS resulted in non-measurable activity but an increased soluble expression at higher temperatures in a heterologous E. coli host.

scholarly journals Engineering Promiscuous Alcohol Dehydrogenase Activity of a Reductive Aminase AspRedAm for Selective Reduction of Biobased Furans

2021 ◽  
Vol 9 ◽  
Author(s):  
Hao-Yu Jia ◽  
Zi-Yue Yang ◽  
Qi Chen ◽  
Min-Hua Zong ◽  
Ning Li
Keyword(s):  
Alcohol Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Selective Reduction ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
E Coli ◽  
Alcohol Dehydrogenase Activity ◽  
Starting Point

Catalytic promiscuity is a promising starting point for improving the existing enzymes and even creating novel enzymes. In this work, site-directed mutagenesis was performed to improve promiscuous alcohol dehydrogenase activity of reductive aminase from Aspergillus oryzae (AspRedAm). AspRedAm showed the cofactor preference toward NADPH in reductive aminations, while it favored NADH in the reduction reactions. Some key amino acid residues such as N93, I118, M119, and D169 were identified for mutagenesis by molecular docking. Variant N93A showed the optimal pH and temperature of 8 and 30°C, respectively, in the reduction of 5-hydroxymethylfurfural (HMF). The thermostability was enhanced upon mutation of N93 to alanine. The catalytic efficiency of variant N93A (kcat/Km, 23.6 mM−1 s−1) was approximately 2-fold higher compared to that of the wild-type (WT) enzyme (13.1 mM−1 s−1). The improved catalytic efficiency of this variant may be attributed to the reduced steric hindrance that stems from the smaller side chain of alanine in the substrate-binding pocket. Both the WT enzyme and variant N93A had broad substrate specificity. Escherichia coli (E. coli) cells harboring plain vector enabled selective reduction of biobased furans to target alcohols, with the conversions of 35–95% and the selectivities of >93%. The introduction of variant N93A to E. coli resulted in improved substrate conversions (>98%) and selectivities (>99%).

Application Of Electron Probe Analyses Of Minerals In Discussing The Relationship Between Ductile Deformation And Metamorphism

1990 ◽  
Vol 48 (2) ◽  
pp. 250-251
Author(s):  
Fan Guochuan ◽  
Sun Zhongshi
Keyword(s):  
Chemical Composition ◽  
Shear Deformation ◽  
Electron Probe ◽  
General Rule ◽  
Granulite Facies ◽  
Ductile Deformation ◽  
Higher Temperature ◽  
Ductile Shear ◽  
The Relationship ◽  
Ductile Shear Deformation

Under influence of ductile shear deformation, granulite facies mineral paragenesis underwent metamorphism and changes in chemical composition. The present paper discusses some changes in chemical composition of garnet in hypers thene_absent felsic gnesiss and of hypersthene in rock in early and late granulite facies undergone increasing ductile shear deformation .In garnet fetsic geniss, band structures were formed because of partial melting and resulted in zoning from massive⟶transitional⟶melanocrate zones in increasing deformed sequence. The electron-probe analyses for garnet in these zones are listed in table 1 . The Table shows that Mno, Cao contents in garnet decrease swiftly from slightly to intensely deformed zones.In slightly and moderately deformed zones, Mgo contents keep unchanged and Feo is slightly lower. In intensely deformed zone, Mgo contents increase, indicating a higher temperature. This is in accord with the general rule that Mgo contents in garnet increase with rising temperature.

The relationship between health effects in triathletes and microbiological quality of freshwater

1995 ◽  
Vol 31 (5-6) ◽  
pp. 19-26 ◽  
Author(s):  
G. J. Medema ◽  
I. A. van Asperen ◽  
J. M. Klokman-Houweling ◽  
A. Nooitgedagt ◽  
M. J. W. van de Laar ◽  
...  
Keyword(s):  
Water Quality ◽  
Geometric Mean ◽  
Microbiological Quality ◽  
Quality Parameters ◽  
Health Complaints ◽  
E Coli ◽  
Thermotolerant Coliforms ◽  
Microbiological Water Quality ◽  
The Relationship ◽  
Faecal Streptococci

This pilot study was carried out to determine the relationship between microbiological water quality parameters and the occurrence of health complaints among triathletes. Data were collected at an Olympic distance triathlon (n=314) and a run-bike-run (n=81; controls for exposure to fresh water). At the time of the triathlon, the concentrations of Escherichia coli , thermotolerant coliforms, faecal streptococci, entero- and reoviruses, F-specific RNA phages, Salmonella, Campylobacter, Aeromonas, Plesiomonas shigelloides, Pseudomonas aeruginosa and Staphylococcus aureus were examined over the swimming course. Information on the occurrence of health complaints during the competition and in the week thereafter was collected through a written questionnaire. The results show that triathletes and run-bike-runners are comparable with respect to factors other than water exposure (age, sex, training history, physical stress, lower intestinal health complaints during the competition) that may influence the occurrence of health complaints in the week after the competition. Triathletes and run-bike-runners reported gastro-intestinal (7.7% vs 2.5%), respiratory (5.5% vs 3.7%), skin/mucosal (2.6% vs 1.2%), general (3.5% vs 1.2%) and total symptoms (14.8% vs 7.4%) in the week after the event. The health risks for triathletes for all symptom groups are not significantly higher than for run-bike-runners. The geometric mean concentration of faecal indicator bacteria is relatively low: E. coli 170/100 ml; faecal streptococci 13/100 ml, enteroviruses were present at concentrations of 0.1/l. The group of triathletes was homogeneusly and relatively intensely exposed to water; they all swam in the same body of water at the same time and 75% reported to have swallowed freshwater. It was concluded that this study design is suitable to study the relationship between health complaints and microbiological water quality. In the summers of 1993 and 1994, a study will be carried out concerning several run-bike-runs and triathlons in freshwaters of different quality.

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