Identification of Important Amino Acid Residues That Modulate Binding of Escherichia coli GroEL to Its Various Cochaperones

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 507-517
Author(s):  
Gracjana Klein ◽  
Costa Georgopoulos
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
En Bloc ◽  
E Coli ◽  
Important Amino Acid ◽  
Suppressor Mutations ◽  
Sensitive Allele ◽  
Intragenic Suppressor

Abstract Genetic experiments have shown that the GroEL/GroES chaperone machine of Escherichia coli is absolutely essential, not only for bacterial growth but also for the propagation of many bacteriophages including λ. The virulent bacteriophages T4 and RB49 are independent of the host GroES function, because they encode their own cochaperone proteins, Gp31 and CocO, respectively. E. coli groEL44 mutant bacteria do not form colonies above 42° nor do they propagate bacteriophages λ, T4, or RB49. We found that the vast majority (40/46) of spontaneous groEL44 temperature-resistant colonies at 43° were due to the presence of an intragenic suppressor mutation. These suppressors define 21 different amino acid substitutions in GroEL, each affecting one of 13 different amino acid residues. All of these amino acid residues are located at or near the hinge, which regulates the large en bloc movements of the GroEL apical domain. All of these intragenic suppressors support bacteriophages λ, T4, and RB49 growth to various extents in the presence of the groEL44 allele. Since it is known that the GroEL44 mutant protein does not interact effectively with Gp31, the suppressor mutations should enhance cochaperone binding. Analogous intragenic suppressor studies were conducted with the groEL673 temperature-sensitive allele.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase

2001 ◽  
Vol 360 (3) ◽  
pp. 617-623 ◽  
Author(s):  
Qian HAN ◽  
Jianmin FANG ◽  
Jianyong LI
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartate Aminotransferase ◽  
Expression System ◽  
Xanthurenic Acid ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Kynurenine Aminotransferase ◽  
E Coli ◽  
Terminal Amino

The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT). KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E. coli cells. Separation of the E. coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme. N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E. coli AspAT. Recombinant AspAT (R-AspAT), homologously expressed in an E. coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (Km = 3mM; Vmax = 7.9μmol·min−1·mg−1) and 3-hydroxy-dl-kynurenine (Km = 3.7mM; Vmax = 1.25μmol·min−1·mg−1) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50–60°C and at a pH of approx. 7.0. Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (Km = 8mM; Vmax = 20.6μmol·min−1·mg−1). The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E. coli protein is AspAT.

scholarly journals Molecular Analysis of the Multiple GroEL Proteins of Chlamydiae

2003 ◽  
Vol 185 (6) ◽  
pp. 1958-1966 ◽  
Author(s):  
Karuna P. Karunakaran ◽  
Yasuyuki Noguchi ◽  
Timothy D. Read ◽  
Artem Cherkasov ◽  
Jeffrey Kwee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Developmental Cycle ◽  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
E Coli ◽  
Chaperonin Groel ◽  
Mutant Complementation

ABSTRACT Genome sequencing revealed that all six chlamydiae genomes contain three groEL-like genes (groEL1, groEL2, and groEL3). Phylogenetic analysis of groEL1, groEL2, and groEL3 indicates that these genes are likely to have been present in chlamydiae since the beginning of the lineage. Comparison of deduced amino acid sequences of the three groEL genes with those of other organisms showed high homology only for groEL1, although comparison of critical amino acid residues that are required for polypeptide binding of the Escherichia coli chaperonin GroEL revealed substantial conservation in all three chlamydial GroELs. This was further supported by three-dimensional structural predictions. All three genes are expressed constitutively throughout the developmental cycle of Chlamydia trachomatis, although groEL1 is expressed at much higher levels than are groEL2 and groEL3. Transcription of groEL1, but not groEL2 and groEL3, was elevated when HeLa cells infected with C. trachomatis were subjected to heat shock. Western blot analysis with polyclonal antibodies raised against recombinant GroEL1, GroEL2, and GroEL3 demonstrated the presence of the three proteins in C. trachomatis elementary bodies, with GroEL1 being present in the largest amount. Only C. trachomatis groEL1 and groES together complemented a temperature-sensitive E. coli groEL mutant. Complementation did not occur with groEL2 or groEL3 alone or together with groES. The role for each of the three GroELs in the chlamydial developmental cycle and in disease pathogenesis requires further study.

scholarly journals Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli.

1992 ◽  
Vol 118 (1) ◽  
pp. 33-42 ◽  
Author(s):  
A Fujishige ◽  
K R Smith ◽  
J L Silen ◽  
D A Agard
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Outer Membrane ◽  
Temperature Sensitive ◽  
Extracellular Medium ◽  
E Coli ◽  
Extracellular Secretion ◽  
In Trans ◽  
In Cis ◽  
Pro Region

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.

scholarly journals The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa

1988 ◽  
Vol 251 (2) ◽  
pp. 313-322 ◽  
Author(s):  
P J White ◽  
G Millar ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Neurospora Crassa ◽  
Gel Filtration ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Chorismate Synthase ◽  
E Coli ◽  
A Subunit

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.

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 Effect of Drug Transporter Genes on Cysteine Export and Overproduction in Escherichia coli

2006 ◽  
Vol 72 (7) ◽  
pp. 4735-4742 ◽  
Author(s):  
Satoshi Yamada ◽  
Naoki Awano ◽  
Kyoko Inubushi ◽  
Eri Maeda ◽  
Shigeru Nakamori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Tetracycline Resistance ◽  
Industrial Applications ◽  
Genetically Engineered ◽  
Drug Transporter ◽  
E Coli ◽  
Important Amino Acid ◽  
Gene Assay

ABSTRACT l-Cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of l-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in l-cysteine degradation, increased l-cysteine productivity in E. coli. The use of an l-cysteine efflux system could be promising for breeding l-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as l-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on l-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by l-cysteine. We also found that overexpression of these eight genes reduces intracellular l-cysteine levels after cultivation in the presence of l-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes l-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more l-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in l-cysteine export and overproduction in genetically engineered E. coli cells.

scholarly journals Evolution of a Pathway to Novel Long-Chain Carotenoids

2004 ◽  
Vol 186 (5) ◽  
pp. 1531-1536 ◽  
Author(s):  
Daisuke Umeno ◽  
Frances H. Arnold
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Residues ◽  
Laboratory Evolution ◽  
Important Amino Acid ◽  
Naturally Occurring ◽  
Long Chain

ABSTRACT Using methods of laboratory evolution to force the C30 carotenoid synthase CrtM to function as a C40 synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C45 and C50 carotenoid backbones (mono- and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.

scholarly journals Isolation and characterization of lipoylated and unlipoylated domains of the E2p subunit of the pyruvate dehydrogenase complex of Escherichia coli

1990 ◽  
Vol 271 (1) ◽  
pp. 139-145 ◽  
Author(s):  
S T Ali ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Amino Acid Residues ◽  
E Coli ◽  
Isolation And Characterization ◽  
Lysine Residues ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx. 80 amino acid residues. These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits. A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product. A method has been devised for purifying the three types of independently folded domain from crude extracts of E. coli, based on their pH-(and heat-)stabilities. The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity. This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution.

scholarly journals Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase

1988 ◽  
Vol 249 (2) ◽  
pp. 319-326 ◽  
Author(s):  
I A Anton ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Shikimate Dehydrogenase ◽  
Gene Encoding ◽  
E Coli ◽  
Multifunctional Enzymes ◽  
Aroe Gene ◽  
Terminal Amino

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.

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