Tyr-503 of β-galactosidase (Escherichia coli) plays an important role in degalactosylation

1999 ◽  
Vol 77 (3) ◽  
pp. 229-236 ◽  
Author(s):  
Robert M Penner ◽  
Nathan J Roth ◽  
Beatrice Rob ◽  
Helga Lay ◽  
Reuben E Huber
Keyword(s):  
Escherichia Coli ◽  
Acid Catalysis ◽  
Covalent Bond ◽  
Reaction Rates ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Burst Kinetics ◽  
Inhibition Studies ◽  
Ks Values ◽  
Lines Of Evidence

Substitutions for Tyr-503 of β-galactosidase caused large decreases of the activity. Both the galactosylation (k2) and degalactosylation (k3) rates were decreased. Substitutions by residues without transferable protons, caused k3 to decrease much more than k2 while substitutions with residues having transferable protons, caused approximately equal decreases of k2 and k3. Several lines of evidence showed this. The Km values of the substituted enzymes were much smaller than those for the wild type if the substituted amino acid residues did not have transferable protons; this was not the case when the substituted residues had transferable protons. Inhibition studies showed that the Km values were not small because of small Ks values but were small because of relatively small k3 values (compared with the k2 values). The conclusion that the k3 values are small relative to k2 upon substitution with residues without transferable protons is also based upon other studies: studies indicating that the reaction rates were similar with different substrates, studies in the presence of alcohol acceptors, studies showing that the rate of inactivation by 2,4-dinitrophenyl-2-deoxy-2-F-β-D-galactopyranoside decreased much less than the rate of reactivation; studies on burst kinetics, and pH studies. The data suggest that Tyr-503 may be important for the degalactosylation reaction because of its ability to transfer protons and thereby facilitate cleavage of the transient covalent bond between galactose and Glu-537. Key words: β-galactosidase, tyrosine, mechanism, acid catalysis.

scholarly journals Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

1999 ◽  
Vol 181 (14) ◽  
pp. 4318-4325 ◽  
Author(s):  
Masaru Ohara ◽  
Henry C. Wu ◽  
Krishnan Sankaran ◽  
Paul D. Rick
Keyword(s):  
Escherichia Coli ◽  
Direct Consequence ◽  
Insertion Mutant ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
E Coli ◽  
K 12 ◽  
Identification And Characterization ◽  
Lines Of Evidence

ABSTRACT We report here the identification of a new lipoprotein, NlpI, inEscherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp(polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.

scholarly journals Escherichia coli DNA Topoisomerase I and Suppression of Killing by Tn5 Transposase Overproduction: Topoisomerase I Modulates Tn5 Transposition

1998 ◽  
Vol 180 (22) ◽  
pp. 5866-5874 ◽  
Author(s):  
Hesna Yigit ◽  
William S. Reznikoff
Keyword(s):  
Escherichia Coli ◽  
Chromosome Segregation ◽  
Topoisomerase I ◽  
Dna Gyrase ◽  
Wild Type ◽  
Dna Topoisomerase ◽  
Dna Relaxation ◽  
Tn5 Transposase ◽  
Lines Of Evidence

ABSTRACT Tn5 transposase (Tnp) overproduction is lethal toEscherichia coli. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing, stkD10, is localized in topA (the gene for topoisomerase I). ThestkD10 mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in rpoH, stkA14 (H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704–1713, 1997), can be explained by an increase in topA expression; and third, overexpression of wild-type topA partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn5 transposition up to 30-fold in vivo.

scholarly journals The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

2000 ◽  
Vol 74 (9) ◽  
pp. 4229-4235 ◽  
Author(s):  
Nina Nilsson ◽  
Ann-Christin Malmborg ◽  
Carl A. K. Borrebaeck
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Functional Role ◽  
Infection Process ◽  
Proper Function ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Linker Region ◽  
Extensive Evaluation ◽  
Type Phage

ABSTRACT The filamentous bacteriophage infects Escherichia coliby interaction with the F pilus and the TolQRA complex. The virus-encoded protein initiating this process is the gene 3 protein (g3p). The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions. Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions. Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region. A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage. This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression.

scholarly journals Identification of valine/leucine/isoleucine and threonine/alanine/glycine proton-spin systems of Escherichia coli adenylate kinase by selective deuteration and selective protonation

1991 ◽  
Vol 273 (2) ◽  
pp. 311-316 ◽  
Author(s):  
I Bock-Möbius ◽  
M Brune ◽  
A Wittinghofer ◽  
H Zimmermann ◽  
R Leberman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Adenylate Kinase ◽  
Spin Systems ◽  
Proton Spin ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Auxotrophic Strain ◽  
E Coli ◽  
Selective Deuteration ◽  
The One

Adenylate kinase from two types of Escherichia coli strains, a wild-type and a leucine-auxotrophic strain, was purified. On the one hand, growing the leucine-auxotrophic bacteria on a medium containing deuterated leucine yielded E. coli adenylate kinase with all leucine residues deuterated. On the other hand, by growing the wild-type bacteria on deuterated medium with phenylalanine, threonine and isoleucine present as protonated specimens, 80% randomly deuterated enzyme with protonated phenylalanine, threonine and isoleucine residues could be prepared. Use of these proteins enabled identification of the spin systems of these amino acid residues in the n.m.r. spectra of the protein.

scholarly journals Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5491-5499 ◽  
Author(s):  
Sun Nyunt Wai ◽  
Marie Westermark ◽  
Jan Oscarsson ◽  
Jana Jass ◽  
Elke Maier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Single Channel ◽  
Dominant Negative ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Mutant Proteins ◽  
Negative Feature

ABSTRACT We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

scholarly journals In Vivo Reconstitution of the FhuA Transport Protein of Escherichia coli K-12

2003 ◽  
Vol 185 (18) ◽  
pp. 5508-5518 ◽  
Author(s):  
Michael Braun ◽  
Franziska Endriss ◽  
Helmut Killmann ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Globular Domain ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Colicin M ◽  
Genetic Deletion ◽  
K 12 ◽  
Microcin J25

ABSTRACT The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and φ80 and for colicin M and microcin J25. The crystal structure reveals a β-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a β-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the β-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type β-barrel to form an active FhuA. Previous claims that the β-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the β-barrel, and they suggest that in wild-type FhuA the β-barrel is formed prior to the insertion of the cork.

scholarly journals Overproduction of l-Cysteine andl-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

1998 ◽  
Vol 64 (5) ◽  
pp. 1607-1611 ◽  
Author(s):  
Shigeru Nakamori ◽  
Shin-ichiro Kobayashi ◽  
Chitose Kobayashi ◽  
Hiroshi Takagi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Serine Acetyltransferase ◽  
Methionine Residue ◽  
Cysteine Desulfhydrase ◽  
K 12

ABSTRACT Organisms that overproduced l-cysteine andl-cystine from glucose were constructed by usingEscherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg ofl-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production ofl-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production ofl-cysteine plus l-cystine was markedly increased compared to production in JM39.

scholarly journals Cloning and thermostability of TaqI endonuclease isoschizomers from Thermus species SM32 and Thermus filiformis Tok6A1

1998 ◽  
Vol 333 (2) ◽  
pp. 425-431 ◽  
Author(s):  
Weiguo CAO ◽  
Jing LU ◽  
Simon G. WELCH ◽  
Ralph A. D. WILLIAMS ◽  
Francis BARANY
Keyword(s):  
Escherichia Coli ◽  
Sequence Data ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Nucleotide Sequence Data ◽  
Structural Context ◽  
Low Salt ◽  
Thermus Filiformis ◽  
Loop Regions

Two TaqI endonuclease (hereafter referred to as TaqI) isoschizomer genes, tsp32IR from Thermus species SM32 of Azores and tfiTok6A1I from T. filiformis Tok6A1 of New Zealand, were cloned in Escherichia coli. The overexpressed enzymes were partly purified and their thermostability was determined. In the medium-salt buffer, Tsp32IR, TfiTok6A1I and one previously cloned TaqI isoschizomer (TthHB8I) were more thermostable than TaqI. Tsp32IR remained partly active up to 90 °C in the low-salt buffer. Six amino acid residues that are identical in the three high thermostability isoschizomers (Tsp32IR, TfiTok6A1I and TthHB8I) but differ in TaqI might provide added rigidity for thermostabilization. These include four proline residues located in or near loop regions, and one alanine and one arginine located at helix regions in the predicted TaqI endonuclease secondary structure. The possible role of these residues in thermostabilization was evaluated by mutagenizing the TaqI enzyme. Mutants generated at these six positions were less thermostable than wild-type TaqI. The results suggest that the surrounding sequence or structural context might be as important as the mutation itself. The nucleotide sequence data reported in this paper for TfiTok6A1I and Tsp32IR appear in the GenBank Database under the accession numbers U86869 and U86870 respectively.

scholarly journals Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450

2021 ◽  
Author(s):  
Yohei Iizaka ◽  
Ryusei Arai ◽  
Akari Takahashi ◽  
Mikino Ito ◽  
Miho Sakai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Random Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
P450 Monooxygenase ◽  
Cell Assays ◽  
Allylic Hydroxylation ◽  
Reaction Profile

Abstract MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.

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