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  E.coli news vol. 4  2003.11.19
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

  >>>aldo-keto reductase<<<

(JW2970)

FEMS Microbiol Lett. 2003 Jan 21;218(1):93-9. 
   
A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity.
Grant AW, Steel G, Waugh H, Ellis EM.
Department of Bioscience, University of Strathclyde, Royal College, 204 George Street, Glasgow G1 1XW, UK.

A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified. This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel beta-subunits in the AKR6 family. The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1. Sequences encoding putative homologues of this enzyme exist in many other bacteria. The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal. The K(m) for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM. Overexpression of the recombinant enzyme in E. coli leads to increased resistance to methylglyoxal. It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.

PMID: 12583903

*comment: YghZ is assayed by NADPH concentration (340 nm) in this paper, so product for the substrate (methylglyoxal) is unidentified. Methylglyoxal is converted from glycerone phosphate and is converted to pyruvate in vivo. YghZ may be another bypass pathway. One suggestion is methylglyoxal may be converted to lactaldehyde, this enzyme is missing in E.coli. Lactaldehyde is converted to lactate by aldA or aldB.

  >>>ascorbate transporter system<<<

(JW4149-JW4153)

J Bacteriol. 2003 Apr;185(7):2243-50. 
   
The ascorbate transporter of Escherichia coli.
Zhang Z, Aboulwafa M, Smith MH, Saier MH Jr.
Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0116, USA.

The sgaTBA genes of Escherichia coli encode a putative 12-transmembrane alpha-helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively. We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of L-ascorbate in vivo and its phosphoenolpyruvate-dependent phosphorylation in vitro. The transporter exhibits an apparent K(m) for L-ascorbate of 9 micro M and is highly specific. The sgaTBA genes are regulated at the transcriptional level by the yjfQ gene product, as well as by Crp and Fnr. The yjfR gene product is essential for L-ascorbate utilization and probably encodes a cytoplasmic L-ascorbate 6-phosphate lactonase. We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to L-ascorbate via the phospho

ryl transfer pathway: [pathway: see text].

PMID: 12644495

  >>>new carboxylesterase<<<

(JW3375)

J Biol Chem. 2003 Jul 11;278(28):26039-45.
   
Integrating structure, bioinformatics, and enzymology to discover function: BioH, a new carboxylesterase from Escherichia coli.
Sanishvili R, Yakunin AF, Laskowski RA, Skarina T, Evdokimova E, Doherty-Kirby A, Lajoie GA, Thornton JM, Arrowsmith CH, Savchenko A, Joachimiak A, Edwards AM.
Biosciences Division, Argonne National Laboratory, Argonne, Illinois, 60439, USA.

Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 A resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference f

or short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure.

PMID: 12732651

  >>>periplasmic copper-binding protein<<<

J Bacteriol. 2003 Jul;185(13):3804-12. 
   
Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli.
Franke S, Grass G, Rensing C, Nies DH.
Molekulare Mikrobiologie, Institut fur Mikrobiologie, Martin-Luther-Universitat Halle-Wittenberg, 06099 Halle, Germany.

The cus determinant of Escherichia coli encodes the CusCFBA proteins that mediate resistance to copper and silver by cation efflux. CusA and CusB were essential for copper resistance, and CusC and CusF were required for full resistance. Replacements of methionine residues 573, 623, and 672 with isoleucine in CusA resulted in loss of copper resistance, demonstrating their functional importance. Substitutions for several other methionine residues of this protein did not have any effect. The small 10-kDa protein CusF (previously YlcC) was shown to be a periplasmic protein. CusF bound one copper per polypeptide. The pink CusF copper protein complex exhibited an absorption maximum at around 510 nm. Methionine residues of CusF were involved in copper binding as shown by site-directed mutagenesis. CusF interacted with CusB and CusC polypeptides in a yeast two-hybrid assay. In contrast to other well-studied CBA-type heavy metal efflux systems, Cus was shown to be a tetrapa

rtite resistance system that involves the novel periplasmic copper-binding protein CusF. These data provide additional evidence for the hypothesis that Cu(I) is directly transported from the periplasm across the outer membrane by the Cus complex.

PMID: 12813074

  >>>CoA binding protein, may be CoA transferase<<<

(JW2371)

J Biol Chem. 2003 Sep 5;278(36):34582-6.
   
The crystal structure of the Escherichia coli YfdW gene product reveals a new fold of two interlaced rings identifying a wide family of CoA transferases.
Gruez A, Roig-Zamboni V, Valencia C, Campanacci V, Cambillau C.
Architecture et Fonction des Macromolecules Biologiques, UMR 6098, CNRS and Universites Aix-Marseille I and II, 31 chemin J. Aiguier, F-13402 Marseille, Cedex 20, France.

Because of its toxicity, oxalate accumulation from amino acid catabolism leads to acute disorders in mammals. Gut microflora are therefore pivotal in maintaining a safe intestinal oxalate balance through oxalate degradation. Oxalate catabolism was first identified in Oxalobacter formigenes, a specialized, strictly anaerobic bacterium. Oxalate degradation was found to be performed successively by two enzymes, a formyl-CoA transferase (frc) and an oxalate decarboxylase (oxc). These two genes are present in several bacterial genomes including that of Escherichia coli. The frc ortholog in E. coli is yfdW, with which it shares 61% sequence identity. We have expressed the YfdW open reading frame product and solved its crystal structure in the apo-form and in complex with acetyl-CoA and with a mixture of acetyl-CoA and oxalate. YfdW exhibits a novel and spectacular fold in which two monomers assemble as interlaced rings, defining the CoA binding site at their interface. F

rom the structure of the complex with acetyl-CoA and oxalate, we propose a putative formyl/oxalate transfer mechanism involving the conserved catalytic residue Asp169. The similarity of yfdW with bacterial orthologs (approximately 60% identity) and paralogs (approximately 20-30% identity) suggests that this new fold and parts of the CoA transfer mechanism are likely to be the hallmarks of a wide family of CoA transferases.

PMID: 12844490

  >>>novel toxin-antitoxin gene pairs<<<

(JW1987/JW0234/JW2627)

J Bacteriol. 2003 Nov;185(22):6600-8. 
   
A novel family of Escherichia coli toxin-antitoxin gene pairs.
Brown JM, Shaw KJ.
Johnson & Johnson Pharmaceutical Research and Development, LLC, La Jolla, California 92121, USA.

Bacterial toxin-antitoxin protein pairs (TA pairs) encode a toxin protein, which poisons cells by binding and inhibiting an essential enzyme, and an antitoxin protein, which binds the toxin and restores viability. We took an approach that did not rely on sequence homology to search for unidentified TA pairs in the genome of Escherichia coli K-12. Of 32 candidate genes tested, ectopic expression of 6 caused growth inhibition. In this report, we focus on the initial characterization of yeeV, ykfI, and ypjF, a novel family of toxin proteins. Coexpression of the gene upstream of each toxin restored the growth rate to that of the uninduced strain. Unexpectedly, we could not detect in vivo protein-protein interactions between the new toxin and antitoxin pairs. Instead, the antitoxins appeared to function by causing a large reduction in the level of cellular toxin protein.

PMID: 14594833

  >>>CsgD new function<<<

Microbiology. 2003 Feb;149(Pt 2):525-35. 
   
CsgD, a regulator of curli and cellulose synthesis, also regulates serine hydroxymethyltransferase synthesis in Escherichia coli K-12.
Chirwa NT, Herrington MB.
Biology Department, Concordia University, 1455 Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.

The homologous CsgD and AgfD proteins are members of the FixJ/UhpA/LuxR family and are proposed to regulate curli (thin aggregative fibres) and cellulose production by Escherichia coli and Salmonella enterica serovar Typhimurium, respectively. A plasmid containing part of the csgD gene was isolated during a screen for multicopy suppressors of glycine auxotrophy caused by deleting the folA gene in E. coli. The sequence of the plasmid suggests it encodes a chimaeric protein. Plasmids containing the intact csgD or agfD gene also caused suppression. Cells transformed with the recombinant plasmids contained higher serine hydroxymethyltransferase (SHMT) activity than controls. The increase could also be monitored by assaying beta-galactosidase activity from a reporter strain with part of the SHMT gene, glyA, fused to lacZ. The increase in SHMT activity was sufficient to correct the glycine auxotrophy of strains lacking folA. The recombinant plasmids also enabled K-12 str

ains that are not curli-proficient to make curli. Curlin, the major component of curli, contains more glycine than normal E. coli proteins. It is proposed that CsgD upregulates glyA to facilitate synthesis of curli. It is suggested that recombinant plasmids produce enough CsgD or chimaeric protein to titrate out a ligand that switches CsgD into its inactive form. As a result, sufficient active CsgD is present to activate genes in its regulon. It is concluded that CsgD increases expression of the glyA gene either directly or indirectly.

PMID: 12624214 [PubMed - indexed for MEDLINE]

***additional finding***

  >>>Fructoselysine kinase, Fructoselysine-6 phosphate deglycase<<<

(JW3337/JW3334)

J Biol Chem. 2002 Nov 8;277(45):42523-9. Epub 2002 Jul 29.
   
Identification of a pathway for the utilization of the Amadori product fructoselysine in Escherichia coli.
Wiame E, Delpierre G, Collard F, Van Schaftingen E.
Laboratory of Physiological Chemistry, University of Louvain and the Christian de Duve Institute of Cellular Pathology, B-1200 Brussels, Belgium.

Escherichia coli was found to grow on fructoselysine as an energetic substrate at a rate of about one-third of that observed with glucose. Extracts of cells grown on fructoselysine catalyzed in the presence of ATP the phosphorylation of fructoselysine and a delayed formation of glucose 6-phosphate from this substrate. Data base searches allowed us to identify an operon containing a putative kinase (YhfQ) belonging to the PfkB/ ribokinase family, a putative deglycase (YhfN), homologous to the isomerase domain of glucosamine-6-phosphate synthase, and a putative cationic amino acid transporter (YhfM). The proteins encoded by YhfQ and YhfN were overexpressed in E. coli, purified, and shown to catalyze the ATP-dependent phosphorylation of fructoselysine to a product identified as fructoselysine 6-phosphate by 31P NMR (YhfQ), and the reversible conversion of fructoselysine 6-phosphate and water to lysine and glucose 6-phosphate (YhfN). The K(m) of the kinase for fructose

lysine amounted to 18 microm, and the K(m) of the deglycase for fructoselysine 6-phosphate, to 0.4 mm. A value of 0.15 m was found for the equilibrium constant of the deglycase reaction. The kinase and the deglycase were both induced when E. coli was grown on fructoselysine and then reached activities sufficient to account for the rate of fructoselysine utilization.

PMID: 12147680

  >>>microcin related transport protein<<<

(JW3213/JW4194)

J Bacteriol. 2002 Jun;184(12):3224-31. 
   
The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation.
Allali N, Afif H, Couturier M, Van Melderen L.
Laboratoire de Genetique des Procaryotes, Institut de Biologie et de Medecine Moleculaires, Universite Libre de Bruxelles, 6041 Gosselies, Belgium.

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we fou

nd that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.

PMID: 12029038