A possible glycine radical in anaerobic ribonucleotide reductase from Escherichia coli: nucleotide sequence of the cloned nrdD gene.

Sun, Xueyin; Harder, Jens; Krook, Maria; Jörnvall, Hans; Sjöberg, Britt‐Marie; Reichard, Peter

doi:10.1073/pnas.90.2.577

Cited by 114 publications

(103 citation statements)

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“…treC is located immediately upstream of nrdD, which encodes the anaerobic ribonucleotide reductase at 96.5 min on the genetic map of E. coli (49). It is separated from nrdD by a putative transcriptional termination site (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Trehalose-6-phosphate hydrolase of Escherichia coli

1994

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The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli. At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant. However, they can also degrade trehalose as the sole source of carbon under both high-and low-osmolarity growth conditions. The modes of trehalose utilization are different under the two conditions and have to be well regulated (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB. The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC. We have cloned treC, contained in an operon with treB as the promoter-proximal gene. We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a V.. of at least 5.5iimol of trehalose-6-phosphate hydrolyzed per min per mg of protein. The enzyme also very effectively hydrolyzes p-nitrophenyl-a-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins. treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781. The amino acid sequence deduced from the DNA sequence shows homology (50%6 identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacilus spp. but not with those of other disaccharide phosphate hydrolases. This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells.The disaccharide trehalose serves as an osmoprotectant in many different organisms. Its two glucose molecules are linked 1-1 oa-glycosidically. Thus, the sugar is nonreducing and has the unique quality of maintaining the fluidity of membranes under conditions of dryness and desiccation (16,17). In Escherichia coli, trehalose is synthesized internally as an answer to osmotic stress (23,45,48). Among other, more prominent osmoprotectants such as glycine betaine or proline (11,36), trehalose can contribute up to 20% of the entire capacity for osmotic protection of the cell (21, 32). To synthesize the osmoprotectant trehalose at high osmolarity, UDP-glucose and glucose 6-phosphate are used to form trehalose-6-phosphate, regardless of the carbon source. Trehalose-6-phosphate is subsequently dephosphorylated to give free trehalose. Trehalose-6-phosphate synthase is encoded by otsA (osmotic trehalose synthesis), and trehalose-6-phosphate phosphatase is encoded by otsB. The two genes are localized at 42 min on the chromosome, otsB...

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Section: Discussionmentioning

confidence: 99%

“…The bacterium has developed two different 882658. Fax: 49 7531 883356. systems of trehalose metabolism, one for high osmolarity and one for low osmolarity.…”

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confidence: 99%

Trehalose-6-phosphate hydrolase of Escherichia coli

1994

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“…Escherichia coli possess three such enzymes, aerobic essential NrdA͞NrdB, aerobic nonessential NrdE͞NrdF and anaerobic NrdD͞NrdG (3)(4)(5)(6)(7)(8). In addition to host DNA degradation, certain bacteriophages supply nucleotides by a de novo pathway, in which phage-encoded RR play(s) a central role.…”

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confidence: 99%

Introns and intein coding sequence in the ribonucleotide reductase genes of Bacillus subtilis temperate bacteriophage SPβ

Lazarević

Soldo

Düsterhöft

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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The two putative ribonucleotide reductase subunits of the Bacillus subtilis bacteriophage SP␤ are encoded by the bnrdE and bnrdF genes that are highly similar to corresponding host paralogs, located on the opposite replication arm. In contrast to their bacterial counterparts, bnrdE and bnrdF each are interrupted by a group I intron, efficiently removed in vivo by mRNA processing. The bnrdF intron contains an ORF encoding a polypeptide similar to homing endonucleases responsible for intron mobility, whereas the bnrdE intron has no obvious trace of coding sequence. The downstream bnrdE exon harbors an intervening sequence not excised at the level of the primary transcript, which encodes an in-frame polypeptide displaying all the features of an intein. Presently, this is the only intein identified in bacteriophages. In addition, bnrdE provides an example of a group I intron and an intein coding sequence within the same gene.

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“…However, the radical (5'-deoxyadenosyl) formed by this cofactor is not directly responsible for the initial hydrogen abstraction reaction, and apparently a protein-radical participates also in this case, perhaps in the form of a cysteinyl species [23]. In anaerobically grown Escherichia coli a third type of the enzyme has recently been isolated; this form apparently uses a glycyl radical analogous to the one found in pyruvate for-mate lyase [29].…”

Section: Ribonucleotide Reductasementioning

confidence: 99%

Protein‐radical enzymes

Pedersen

Finazzi‐Agrò

1993

FEBS Letters

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Protein-radical enzymes use a free radical located on an intrinsic amino acid residue as a cofactor. The amino acid involved can be a tyrosine (ribonucleotide reductase, photosystem II, prostaglandin H synthase), a modified tyrosine (amine oxidase, galactose oxidase), a tryptophan (cytochrome c peroxidase), a modified tryptophan (methylamine dehydrogenase) or a glycine (ribonucleotide reductase, pyruvate formate lyase). The mechanistic role of these radicals appears to be that of a one-electron gate, allowing the separation of single reducing equivalents in time and space.

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A possible glycine radical in anaerobic ribonucleotide reductase from Escherichia coli: nucleotide sequence of the cloned nrdD gene.

Cited by 114 publications

References 23 publications

Trehalose-6-phosphate hydrolase of Escherichia coli

Trehalose-6-phosphate hydrolase of Escherichia coli

Introns and intein coding sequence in the ribonucleotide reductase genes of Bacillus subtilis temperate bacteriophage SPβ

Protein‐radical enzymes

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