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  E.coli news vol. 5  2003.12.20
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

  >>>JW5332/yefM, JW5331/yoeB, toxin-antitoxin system<<<

J Biol Chem. 2003 Dec 14 [Epub ahead of print].

The YefM antitoxin defines a family of natively unfolded proteins: Implications as a novel antibacterial target.
Cherny I, Gazit E.
Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978.

While natively unfolded proteins are being increasingly observed, their physiological role is not well understood. Here, we demonstrate that the Escherichia coli YefM protein is a natively unfolded antitoxin, lacking secondary structure even at low temperature or in the presence of stabilizing agent. This conformation of the protein is suggested to have a key role in its physiological regulatory activity. Due to the unfolded state of the protein, a linear determinant rather than a conformational one is presumably being recognized by its toxin partner, YoeB. A peptide array technology allowed the identification and validation of such a determinant. This recognition element may provide a novel antibacterial target. Indeed, a pair-constrained bioinformatic analysis facilitated the definite determination of novel YefM-YoeB toxin-antitoxin systems in a large number of bacteria including major pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobac

terium tuberculosis. Taken together, the YefM protein defines a new family of natively unfolded proteins. The existent of a large and conserved group of proteins with a clear physiologically- relevant unfolded state serves as a paradigm to understand the structural basis of this state.

PMID: 14672926

  >>>JW0491/yddB, selenophosphate-dependent tRNA 2-selenouridine synthase<<<

J Biol Chem. 2003 Oct 31 [Epub ahead of print]. 

Functional diversity of the Rhodanese homology domain: The Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase.
Wolfe MD, Ahmed F, Lacourciere GM, Lauhon CT, Stadtman TC, Larson TJ.
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061.

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-X-X-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant, but that 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction, but rather encodes a 2-selenouridine synth

ase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6)-tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys97 (but not Cys96) in the rhodanese sequence motif Cys96Cys97-X-X-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase, and identifies a new role for the rhodanese homology domain in enzymes.

PMID: 14594807

  >>>JW4028/yjcG, acetate permease<<<

J Bacteriol. 2003 Nov;185(21):6448-55. 

The gene yjcG, cotranscribed with the gene acs, encodes an acetate permease in Escherichia coli.
Gimenez R, Nunez MF, Badia J, Aguilar J, Baldoma L.
Department of Biochemistry, School of Pharmacy, University of Barcelona, E-08028 Barcelona, Spain.

We isolated an Escherichia coli mutant strain that suppresses the glycolate-negative phenotype of a strain deficient in both GlcA and LldP transporters of this compound. This suppressing phenotype was assigned to yjcG, a gene whose function was previously unknown, which was found to encode a membrane protein able to transport glycolate. On the basis of sequence similarity, the yjcG gene product was classified as a member of the sodium:solute symporter family. Northern experiments revealed that yjcG is cotranscribed with its neighbor, acs, encoding acetyl coenzyme A synthetase, which is involved in the scavenging acetate. The fortuitous presence of an IS2 element in acs, which impaired yjcG expression by polarity in our parental strain, allowed us to conclude that the alternative glycolate carrier became active after precise excision of IS2 in the suppressed strain. The finding that yjcG encodes a putative membrane carrier for glycolate and the cotranscription of yj

cG with acs suggested that the primary function of the yjcG gene product (proposed gene name, actP) could be acetate transport and allowed us to define an operon involved in acetate metabolism. The time course of [1,2-(14)C]acetate uptake and the results of a concentration kinetics analysis performed with cells expressing ActP or cells deficient in ActP supported the the hypothesis that this carrier is an acetate transporter and suggested that there may be another transport system for this monocarboxylate.

PMID: 14563880

  >>>JW1669/sufE, JW1670/sufS, cysteine desulfurase complex<<<

FEBS Lett. 2003 Dec 4;555(2):263-7. 

Mechanistic studies of the SufS-SufE cysteine desulfurase: evidence for sulfur transfer from SufS to SufE.
Ollagnier-de-Choudens S, Lascoux D, Loiseau L, Barras F, Forest E, Fontecave M.
Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DBMS-CB, CEA/CNRS/Universite Joseph Fourier, UMR 5047, 17 Avenue des Martyrs, 38054 Grenoble Cedex 09, France.

SufS is a cysteine desulfurase of the suf operon shown to be involved in iron-sulfur cluster biosynthesis under iron limitation and oxidative stress conditions. The enzyme catalyzes the conversion of L-cysteine to L-alanine and sulfide through the intermediate formation of a protein-bound cysteine persulfide in the active site. SufE, another component of the suf operon, has been previously shown to bind tightly to SufS and to drastically stimulate its cysteine desulfurase activity. Working with Escherichia coli proteins, we here demonstrate that a conserved cysteine residue in SufE at position 51 is essential for the SufS/SufE cysteine desulfurase activity. Mass spectrometry has been used to demonstrate (i). the ability of SufE to bind sulfur atoms on its cysteine 51 and (ii). the direct transfer of the sulfur atom from the cysteine persulfide of SufS to SufE. A reaction mechanism is proposed for this novel two-component cysteine desulfurase.

PMID: 14644425

  >>>JW1317/tpx, Sulfenic acid formation<<<

J Biol Chem. 2003 Mar 14;278(11):9203-11. Epub 2003 Jan 03.

Catalytic mechanism of thiol peroxidase from Escherichia coli. Sulfenic acid formation and overoxidation of essential CYS61.
Baker LM, Poole LB.
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.

Escherichia coli thiol peroxidase (Tpx, p20, scavengase) is part of an oxidative stress defense system that uses reducing equivalents from thioredoxin (Trx1) and thioredoxin reductase to reduce alkyl hydroperoxides. Tpx contains three Cys residues, Cys(95), Cys(82), and Cys(61), and the latter residue aligns with the N-terminal active site Cys of other peroxidases in the peroxiredoxin family. To identify the catalytically important Cys, we have cloned and purified Tpx and four mutants (C61S, C82S, C95S, and C82S,C95S). In rapid reaction kinetic experiments measuring steady-state turnover, C61S is inactive, C95S retains partial activity, and the C82S mutation only slightly affects reaction rates. Furthermore, a sulfenic acid intermediate at Cys(61) generated by cumene hydroperoxide (CHP) treatment was detected in UV-visible spectra of 4-nitrobenzo-2-oxa-1,3-diazole-labeled C82S,C95S, confirming the identity of Cys(61) as the peroxidatic center. In stopped-flow kinet

ic studies, Tpx and Trx1 form a Michaelis complex during turnover with a catalytic efficiency of 3.0 x 10(6) m(-1) s(-1), and the low K(m) (9.0 microm) of Tpx for CHP demonstrates substrate specificity toward alkyl hydroperoxides over H(2)O(2) (K(m) > 1.7 mm). Rapid inactivation of Tpx due to Cys(61) overoxidation is observed during turnover with CHP and a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid, but not H(2)O(2). Unlike most other 2-Cys peroxiredoxins, which operate by an intersubunit disulfide mechanism, Tpx contains a redox-active intrasubunit disulfide bond yet is homodimeric in solution.

PMID: 12514184

  >>>JW3909/metJ<<<

Arch Microbiol. 2003 Aug;180(2):88-100.

A transporter of Escherichia coli specific for L- and D-methionine is the prototype for a new family within the ABC superfamily.
Zhang Z, Feige JN, Chang AB, Anderson IJ, Brodianski VM, Vitreschak AG, Gelfand MS, Saier MH Jr.
Division of Biological Sciences, University of California at San Diego, CA 92093-0116, La Jolla, USA.

An ABC-type transporter in Escherichia coli that transports both L- and D-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that ma

ny MUT family members transport organic sulfur compounds.

PMID: 12819857

  >>>JW5089/lipB, lipoyl (octanoyl)-acyl carrier protein:protein transferase<<<

J Bacteriol. 2003 Mar;185(5):1582-9. 

The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase.
Jordan SW, Cronan JE Jr.
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.

In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thi

oesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.

PMID: 12591875

  >>>JW1736/astA, JW1734/astB<<<

FEBS Lett. 2003 Dec 18;555(3):505-10. 

Prediction of the structure and function of AstA and AstB, the first two enzymes of the arginine succinyltransferase pathway of arginine catabolism.
Shirai H, Mizuguchi K.
Department of Biochemistry, University of Cambridge, Old Addenbrooks Site, 80 Tennis Court Road, CB2 1GA, Cambridge, UK

Arginine succinyltransferase and succinylarginine dihydrolase catalyze the first two steps of arginine catabolism by the arginine succinyltransferase pathway. This route is the only major arginine catabolic pathway in Escherichia coli including its pathogenic strains O157 and CFT073. We have used fold recognition tools and identified novel homologies between each of these two enzymes and proteins of known three-dimensional structure: arginine succinyltransferase belongs to the acyl-CoA N-acyltransferase superfamily and succinylarginine dihydrolase belongs to the amidinotransferase superfamily. These findings shed light on the structures, catalytic mechanisms and evolution of diverse enzymes involved in arginine catabolism.

PMID: 14675764