Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK)

Boyle, Stephen M.; Markham, George D.; Hafner, Edmund W.; Wright, Jonathan; Tabor, Herbert; Tabor, Celia White

doi:10.1016/0378-1119(84)90113-6

Cited by 78 publications

(59 citation statements)

References 30 publications

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“…Activity was measured as described (43). Recombinant S-adenosylmethionine synthetase from E. coli was overproduced from pK8 (44) in E. coli DH5␣ (pK8, a generous gift of Dr. G. Markham, Fox Chase Cancer Center). Expression and streptomycin sulfate and ammonium sulfate purification steps were as described (45).…”

Section: Methodsmentioning

confidence: 99%

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

Lanen

Kinzie

Matthieu

et al. 2003

Journal of Biological Chemistry

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The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA Tyr in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-14 C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg 2؉and Mn 2؉ in millimolar concentrations. The steady-state kinetic parameters were determined to be K m AdoMet ‫؍‬ 101.4 M, K m tRNA ‫؍‬ 1.5 M, and k cat ‫؍‬ 2.5 min ؊1 . A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K m RNA ‫؍‬ 37.7 M and k cat ‫؍‬ 14.7 min ؊1 ), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.

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Section: Methodsmentioning

confidence: 99%

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

Lanen

Kinzie

Matthieu

et al. 2003

Journal of Biological Chemistry

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“…The primer extension and the S1 nuclease assays were performed as described previously (16) (1,250 Ci/mmol, 12.5 mCi/ml, 10.4 mmol/ml), which was purchased from E. I. du Pont de Nemours & Co., Inc. (Wilmington, Del. ), was used in each reaction; the reaction mixture was incubated at 37°C for 10 min, followed by the addition of 2 ,ul of 0.5 mM solution of all four deoxynucleoside triphosphates and an additional 5 min of incubation at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Analysis and sequence of the speB gene encoding agmatine ureohydrolase, a putrescine biosynthetic enzyme in Escherichia coli

Szumanski¹,

Boyle²

1990

J Bacteriol

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The speB gene of Escherichia coli encodes the enzyme agmatine ureohydrolase (AUH). AUH catalyzes the hydrolysis of agmatine to urea and putrescine in one of the two polyamine biosynthetic pathways in E. coli. Sequencing of a 2.97-kilobase-pair fragment of the E. coli chromosome containing speB revealed the presence of three intact open reading frames (ORFs), ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as weUl as a truncated ORF, ORF4, which terminated 92 kilobase pairs upstream from ORF3. ORF3 contained the coding sequence of the speB gene, as confirmed by complementation analysis. Two ORF3 transcripts were detected: a shorter transcript that included only ORF3 and a longer transcript that included both ORF3 and ORF4. The short transcript was abundantly expressed when the ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter from which the shorter transcript was produced contained a TATACT sequence at position -12, but sequences upstream from the -12 position seemed to be irrelevant for promoter activity. The predicted amino acid sequence of AUH contained three regions of high homology to the arginases of yeasts, rats, and humans.In Escherichia coli putrescine is synthesized either by decarboxylation of ornithine or by decarboxylation of arginine to agmatine followed by hydrolysis of agmatine to putrescine and urea (14). The last two reactions are catalyzed by the enzymes arginine decarboxylase and agmatine ureohydrolase (AUH), respectively. The AUH protein has previously been purified from an E. coli isolate that was transformed with the plasmid pKA5; the enzymatic properties of AUH have been characterized previously (9). The subunit size of AUH, as deduced from its mobility on a sodium dodecyl sulfate-polyacrylamide gel, is 38 kilodaltons. The expression of AUH activity is antagonistically regulated by cyclic AMP and agmatine. In the presence of the cyclic AMP receptor protein, cyclic AMP represses the expression of the speB gene, while agmatine induces it. These two modulators appear to act independently from each other (10); the mechanism of this differential regulation is unknown. The speB gene coding for AUH is located at approximately 63.5 min on the E. coli chromosome. Although a large chromosomal fragment corresponding to this region, including the speB gene, is present in the pKA5 plasmid, the exact location of speB was not previously established. Here we report the nucleotide sequence of the speB structural gene and identify the promoter responsible for transcription of one of two mRNAs encoding AUH. We also report the mapping of the mRNA resulting from transcription that initiated at this promoter. MATERIALS AND METHODSBacterial hosts, media, and growth conditions. E. coli CB806(A1acZ galK phoA8 rpsL thi recA56) (11) was used for all experiments involving the promoter vector pCB267 and its derivatives. E. coli DHSa [F- The bacteria wer...

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“…Putrescine is synthesized in Escherichia coli cells from ornithine by ornithine decarboxylase (SpeC) or from arginine by the sequential actions of arginine decarboxylase (SpeA) and agmatinase (SpeB) (9). Putrescine is converted to spermidine, another polyamine, by the addition of an aminopropyl group derived from decarboxylated S-adenosylmethionine by spermidine synthase (SpeE) (10).…”

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confidence: 99%

A Novel Putrescine Importer Required for Type 1 Pili-driven Surface Motility Induced by Extracellular Putrescine in Escherichia coli K-12

Kurihara

Suzuki

Oshida

et al. 2011

Journal of Biological Chemistry

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Recently, many studies have reported that polyamines play a role in bacterial cell-to-cell signaling processes. The present study describes a novel putrescine importer required for induction of type 1 pili-driven surface motility. The surface motility of the Escherichia coli ⌬speAB ⌬speC ⌬potABCD strain, which cannot produce putrescine and cannot import spermidine from the medium, was induced by extracellular putrescine. Introduction of the gene deletions for known polyamine importers (⌬potE, ⌬potFGHI, and ⌬puuP) or a putative polyamine importer (⌬ydcSTUV) into the ⌬speAB ⌬speC ⌬potABCD strain did not affect putrescine-induced surface motility. The deletion of yeeF, an annotated putative putrescine importer, in the ⌬speAB ⌬speC ⌬potABCD ⌬ydcSTUV strain abolished surface motility in putrescine-supplemented medium. Complementation of yeeF by a plasmid vector restored surface motility. The surface motility observed in the present study was abolished by the deletion of fimA, suggesting that the surface motility is type 1 pili-driven. A transport assay using the yeeF ؉ or ⌬yeeF strains revealed that YeeF is a novel putrescine importer. The K m of YeeF (155 M) is 40 to 300 times higher than that of other importers reported previously. On the other hand, the V max of YeeF (9.3 nmol/min/mg) is comparable to that of PotABCD, PotFGHI, and PuuP. The low affinity of YeeF for putrescine may allow E. coli to sense the cell density depending on the concentration of extracellular putrescine.

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Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK)

Cited by 78 publications

References 30 publications

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

Analysis and sequence of the speB gene encoding agmatine ureohydrolase, a putrescine biosynthetic enzyme in Escherichia coli

A Novel Putrescine Importer Required for Type 1 Pili-driven Surface Motility Induced by Extracellular Putrescine in Escherichia coli K-12

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