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  E.coli news vol. 1  2003.7.3
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

  >>>Putrescine:2-oxoglutarate aminotransferase<<<

BMC Microbiol. 2003 Jan 31 [Epub ahead of print]. 
   
Molecular cloning and characterization of Escherichia coli K12 ygjG gene.
Samsonova NN, Smirnov SV, Altman IB, Ptitsyn LR.
Ajinomoto-Genetika Research Institute, 1st Dorozhny pr, 1, Moscow, 113545, Russia.

BACKGROUND: Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source. Transaminase and dehydrogenase enzymes seem to be implicated in the degradative pathway of putrescine, in which this compound is converted into gamma-aminobutyrate. But genes coding for these enzymes have not been identified so far. RESULTS: The 1.8-kbp DNA fragment containing E. coli K12 ygjG gene with aer-ygjG intergenic region was examined. It was found that the fragment contains sigma54-depended open reading frame (ORF) of 1,380 nucleotides encoding a 459-amino acid polypeptide of approximately 49.6 kDa. The cytidine (C) residue localized 10 bp downstream of the sigma54 promoter sequence was identified as the first mRNA base. The UUG translation initiation codon is situated 36 nucleotides downstream of the mRNA start. The YgjG was expressed as a his6-tag fused protein and purified to homogeneity. The prote

in catalyzed putrescine:2-oxoglutaric acid (2-OG) aminotransferase reaction (PATase, EC 2.6.1.29). The Km values for putrescine and 2-OG were found to be 9.2 mM and 19.0 mM, respectively. The recombinant enzyme also was able to transaminate cadaverine and, in lower extent, spermidine, and gave maximum activity at pH 9.0. CONCLUSION: Expression of E. coli K12 ygjG coding region revealed sigma54-depended ORF which encodes a 459-amino acid protein with putrescine:2-OG aminotransferase activity. The enzyme also was able to transaminate cadaverine and, in lower extent, spermidine.

PMID: 12617754

  >>>New Shikimate Dehydrogenase<<<

J Biol Chem. 2003 May 23;278(21):19463-72. Epub 2003 Mar 12.
   
Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB: A COMMON STRUCTURAL FRAMEWORK FOR DIFFERENT ACTIVITIES.
Michel G, Roszak AW, Sauve V, Maclean J, Matte A, Coggins JR, Cygler M, Lapthorn AJ.
Biotechnology Research Institute, NRC Macromolecular Structure Group, Montreal, Quebec H4P 2R2, Canada.

Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 A resolution, respectively. Both enzymes display a similar architecture with two alpha/beta domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence ana

lysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms.

PMID: 12637497

  >>>novel anaerobic beta-oxidation pathway<<<

Mol Microbiol. 2003 Feb;47(3):793-805. 
   
A new Escherichia coli metabolic competency: growth on fatty acids by a novel anaerobic beta-oxidation pathway.
Campbell JW, Morgan-Kiss RM, E Cronan J Jr.
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Escherichia coli uses fatty acids as a sole carbon and energy source during aerobic growth by means of the enzymes encoded by the fad regulon. We report that this bacterium can also grow on fatty acids under anaerobic conditions provided that a terminal respiratory electron acceptor such as nitrate is available. This anaerobic utilization pathway is distinct from the well-studied aerobic pathway in that (i). it proceeds normally in mutant strains lacking various enzymes of the aerobic pathway; (ii). it functions with fatty acids (octanoate and decanoate) that cannot be used by wild-type E. coli strains under aerobic conditions; and (iii). super-repressor mutants of the fadR regulatory locus that block aerobic growth on fatty acids fail to block the anaerobic pathway. We have identified homologues of the FadA, FadB and FadD proteins required for aerobic fatty acid utilization called YfcY, YfcX and YdiD, respectively, which are involved in anaerobic growth on fatty a

cids. A strong FadR binding site was detected upstream of the yfcY gene consistent with microarray analyses, indicating that yfcYX expression is negatively regulated by FadR under aerobic growth conditions. In contrast, transcriptional regulation of ydiD appears to be independent of FadR, and anaerobic growth on fatty acids is not under FadR control. These three genes are conserved in the available genome sequences of pathogenic E. coli, Shigella and Salmonella strains.

PMID: 12535077

  >>>D-Arabinose 5-phosphate isomerase<<<

J Biol Chem. 2003 Jun 12 [Epub ahead of print].

Escherichia coli YrbH is a D-Arabinose 5-phosphate isomeraseo.
Meredith TC, Woodard RW.
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065.

A gene encoding for arabinose 5-phosphate isomerase (API), which catalyzes the interconversion of D-ribulose 5-phosphate (Ru5P) and D-arabinose 5-phosphate (A5P), has been identified from the genome of Escherichia coli K-12. API is the first enzyme in th e biosynthesis of 3-deoxy-D-manno-octulosonate (KDO), a sugar moiety located in the lipopolysaccharide layer of most Gram-negative bacteria. The API gene yrbH is located next to the recently identified specific KDO 8-P phosphatase gene, yrbI. The 328 a m ino acid open reading frame (orf) yrbH was cloned, overexpressed, and characterized. The purified recombinant enzyme is a tetramer and is sensitive to inhibition by zinc cations. API has optimal activity at pH 8.4 and catalytic residues with estimated p Ka values of 6.5 and 10.3. The enzyme is specific for A5P and Ru5P, with apparent Km values of 0.61.06 mM for A5P and of 0.35.08 mM for Ru5P. The apparent kcat in the A5P to Ru5P direction is 157 4 sec-1 and in the

Ru5P to A5P direction it is 255 16 sec-1. The value of Keq ([Ru5P]/[A5P]) is 0.5.06. Homology searches of the E. coli genome suggest yrbH may be one of multiple genes which encode proteins with API activity.i3

PMID: 12805358

***others***

  >>>acyl coenzyme A dehydrogenase<<<

J Bacteriol. 2002 Jul;184(13):3759-64. 
   
The enigmatic Escherichia coli fadE gene is yafH.
Campbell JW, Cronan JE Jr.
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

The identity of the gene encoding acyl coenzyme A dehydrogenase is a major remaining mystery of the Escherichia coli fatty acid degradation (fad) regulon. Our prior genome array analyses showed that transcription of the yafH gene is controlled by the FadR regulatory protein. We now report direct experimental proof that yafH and fadE are the same gene.

PMID: 12057976

  >>>sucrose metabolism<<<

J Bacteriol. 2002 Oct;184(19):5307-16. 
   
Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132.
Jahreis K, Bentler L, Bockmann J, Hans S, Meyer A, Siepelmeyer J, Lengeler JW.
Arbeitsgruppe Genetik, Fachbereich Biologie/Chemie, Universitat Osnabruck, D-49069 Osnabruck, Germany.

Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.

PMID: 12218016