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Escherichia coli K-12 substr. MG1655 Enzyme: PII uridylyltransferase / uridylyl removing enzyme



Gene: glnD Accession Numbers: EG11411 (EcoCyc), b0167, ECK0165

Synonyms: glnD5, UTase/UR

Regulation Summary Diagram: ?

Regulation summary diagram for glnD

Summary:
Uridylyltransferase (UTase) encoded by glnD is a bifunctional protein that catalyzes the uridylylation as well as the de-uridylylation of the regulatory protein PII. Uridylylation and deuridylylation are distinct reactions; uridylylation involves the transfer of a UMP group (derived from UTP) to PII to form PII-UMP and pyrophosphate; deuridylylation of PII-UMP is a hydrolytic reaction that results in the release of UMP and PII [Garcia80, Garcia83]. GlnD may function to sense cellular glutamine concentration and relay this information via the uridylylation status of PII (see [vanHeeswijk13]

Under limiting nitrogen conditions PII-UMP interacts with glutamine synthetase (GS) adenylyltransferase which catalyzes removal of AMP from (and consequent activation of) glutamine synthetase. In the absence of UTase there is very little activation of glutamine synthetase in response to nitrogen deprivation [Bloom78, Bueno85, Ninfa86]. Glutamine inhibits the uridylylation reaction and stimulates the deuridylylation reaction; Mg2+ is the physiologically relevent metal ion cofactor [Jiang98]. UTase can use other nucleotides to modify PII in vitro but PII-UMP is most effective for activating NtrB phosphatase activity and GlnE GS deadenylylation activity [Jiang98].

GlnD can also catalyse the uridylylation and deuridylylation of the glnK encoded PII protein (PII 2) [vanHeeswijk96, Atkinson99]; the deuridylylation of GlnK-UMP by GlnD is very slow [Atkinson99].

GlnD contains 4 domains: an N-terminal nucleotidyltransferase (NT) domain associated with uridylylation activity; a central HD (conserved histidine (H) and aspartate (D)) domain essential for deuridylylation activity and two C-terminal ACT (aspartokinase, chorismate mutase, and TyrA ) domains that may be involved in sensing glutamine [Zhang10]. The uridylylation and deuridylylation reactions may [Jiang98] or may not [Zhang10] share the same active site. The central HD domain may act to pass the signal from the glutamine sensing ACT domains to the N-terminal NT domain; PII inhibits the uridylylation activity of GlnD by binding to the HD domain [Jiang12]. The UTase activity of GlnD is subject to substrate inhibition by PII [Jiang12a].

glnD is expressed constitutively at a low level [vanHeeswijk93, Kim98]. ArcA appears to activate glnD gene expression under anaerobiosis. A putative ArcA binding site is located 560 bp upstream of this gene [Salmon05].

Citations: [Adler75, Atkinson94, Kamberov95, Rhee85, Magasanik85, Tondervik06]

Locations: cytosol

Map Position: [185,978 <- 188,650] (4.01 centisomes, 14°)
Length: 2673 bp / 890 aa

Molecular Weight of Polypeptide: 102.39 kD (from nucleotide sequence), 95.0 kD (experimental) [Garcia83 ]

pI: 6.68

Unification Links: ASAP:ABE-0000568 , CGSC:704 , DIP:DIP-9779N , EchoBASE:EB1383 , EcoGene:EG11411 , EcoliWiki:b0167 , Mint:MINT-1252261 , ModBase:P27249 , OU-Microarray:b0167 , PortEco:glnD , PR:PRO_000022784 , Pride:P27249 , Protein Model Portal:P27249 , RefSeq:NP_414709 , RegulonDB:EG11411 , SMR:P27249 , String:511145.b0167 , UniProt:P27249

Relationship Links: InterPro:IN-FAMILY:IPR002912 , InterPro:IN-FAMILY:IPR002934 , InterPro:IN-FAMILY:IPR003607 , InterPro:IN-FAMILY:IPR006674 , InterPro:IN-FAMILY:IPR010043 , InterPro:IN-FAMILY:IPR013546 , Panther:IN-FAMILY:PTHR13734:SF1 , Pfam:IN-FAMILY:PF01842 , Pfam:IN-FAMILY:PF01909 , Pfam:IN-FAMILY:PF01966 , Pfam:IN-FAMILY:PF08335 , Prosite:IN-FAMILY:PS51671 , Smart:IN-FAMILY:SM00471

In Paralogous Gene Group: 57 (2 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for glnD

GO Terms:

Biological Process: GO:0006464 - cellular protein modification process Inferred from experiment [Garcia83]
GO:0006808 - regulation of nitrogen utilization Inferred from experiment Inferred by computational analysis [GOA06, Bueno85]
GO:0006807 - nitrogen compound metabolic process Inferred by computational analysis [GOA01a]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Rajagopala14, Burillo04]
GO:0008773 - [protein-PII] uridylyltransferase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, vanHeeswijk96, Jiang98, Bloom78, Garcia83]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a]
GO:0008081 - phosphoric diester hydrolase activity Inferred by computational analysis [GOA06]
GO:0016597 - amino acid binding Inferred by computational analysis [GOA01a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016779 - nucleotidyltransferase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred by curator

MultiFun Terms: information transfer protein related posttranslational modification
metabolism biosynthesis of building blocks amino acids glutamine
metabolism metabolism of other compounds nitrogen metabolism

Essentiality data for glnD knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 1]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 2]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 1]

Credits:
Revised 16-Feb-2015 by Mackie A , Macquarie University
Last-Curated ? 16-Feb-2015 by Mackie A , Macquarie University


Enzymatic reaction of: [protein-PII] uridylyltransferase (PII uridylyltransferase / uridylyl removing enzyme)

Synonyms: protein PII uridylyltransferase, PII uridylyltransferase

EC Number: 2.7.7.59

a PII protein + UTP <=> a uridylylated PII protein + diphosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for UTP: ATP [Jiang98 ] , CTP [Jiang98 ] , GTP [Jiang98 ] , dTTP [Jiang98 ] , dCTP [Jiang98 ]

In Pathways: Nitrogen Regulation Two-Component System

Cofactors or Prosthetic Groups: Mn2+ [Jiang98], Mg2+ [Jiang98]

Inhibitors (Unknown Mechanism): L-glutamine [Adler75, Atkinson94, Kamberov95, Jiang98]

Primary Physiological Regulators of Enzyme Activity: L-glutamine

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
GTP
940.0
[Jiang98]
a PII protein
3.0
2.28
0.76
[Jiang98]
CTP
380.0
[Jiang98]
ATP
1870.0
[Jiang98]
UTP
40.0
[Jiang98]


Enzymatic reaction of: PII-UMP deuridylylation (PII uridylyltransferase / uridylyl removing enzyme)

a uridylylated PII protein + H2O <=> a PII protein + UMP

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

Cofactors or Prosthetic Groups: Mg2+ [Jiang98], Mn2+ [Jiang98]

Activators (Unknown Mechanism): L-glutamine [Jiang98]

Primary Physiological Regulators of Enzyme Activity: L-glutamine

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
a uridylylated PII protein
0.82
10.08
12.3
[Jiang98]


Enzymatic reaction of: [protein-PII 2] uridylyltransferase (PII uridylyltransferase / uridylyl removing enzyme)

EC Number: 2.7.7.59

GlnK + UTP <=> uridylyl-[GlnK] + diphosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

In Pathways: Nitrogen Regulation Two-Component System


Sequence Features

Protein sequence of PII uridylyltransferase / uridylyl removing enzyme with features indicated

Feature Class Location Common Name Catalytic Activity Citations Comment
Protein-Segment 1 -> 349    
[UniProt14a]
UniProt: Uridylyltransferase; Sequence Annotation Type: region of interest.
Sequence-Conflict 47    
[Park92, UniProt15]
UniProt: (in Ref. 1; AAA23878).
Catalytic-Domain 74 -> 169 nucleotidyltransferase (NT) domain [protein-PII] uridylyltransferase
[Zhang10]
 
Mutagenesis-Variant 93    
[Zhang10, UniProt15]
G → L or V: Loss of UTase activity, while no significant effect on UR activity.
Mutagenesis-Variant 94    
[Zhang10, UniProt15]
UniProt: Loss of UTase activity, while no significant effect on UR activity.
Mutagenesis-Variant 107    
[Zhang10, UniProt15]
D → A, V or Y: Loss of UTase activity, while no significant effect on UR activity.
Sequence-Conflict 225    
[Fujita94, UniProt10a]
UniProt: (in Ref. 3);
Protein-Segment 350 -> 708    
[UniProt14a]
UniProt: Uridylyl-removing; Sequence Annotation Type: region of interest.
Sequence-Conflict 357 -> 358    
[Park92, UniProt15]
UniProt: (in Ref. 1; AAA23878).
Catalytic-Domain 468 -> 601 HD domain PII-UMP deuridylylation
[UniProt14a, Zhang10]
UniProt: HD. Essnetial for uridylyl removing activity
Mutagenesis-Variant 514 -> 515    
[Zhang10, UniProt15]
HD → AA or QN: Loss of UR activity, while no significant effect on UTase activity.
Sequence-Conflict 523    
[Park92, UniProt15]
UniProt: (in Ref. 1; AAA23878).
Sequence-Conflict 703    
[vanHeeswijk93, UniProt10a]
UniProt: (in Ref. 2; CAA79887);
Conserved-Region 708 -> 771 ACT 1  
[Zhang10]
analysis of mutants suggests this domain may play a role in the response to glutamine
Conserved-Region 709 -> 789    
[UniProt13]
UniProt: ACT 1.
Conserved-Region 815 -> 877 ACT 2  
[Zhang10]
analysis of mutants suggest this domain may play a role in the response to glutamine
Conserved-Region 816 -> 890    
[UniProt13]
UniProt: ACT 2.


Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Notes:

History:
10/20/97 Gene b0167 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11411; confirmed by SwissProt match.


References

Adler75: Adler SP, Purich D, Stadtman ER (1975). "Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme." J Biol Chem 1975;250(16);6264-72. PMID: 239942

Atkinson94: Atkinson MR, Kamberov ES, Weiss RL, Ninfa AJ (1994). "Reversible uridylylation of the Escherichia coli PII signal transduction protein regulates its ability to stimulate the dephosphorylation of the transcription factor nitrogen regulator I (NRI or NtrC)." J Biol Chem 1994;269(45);28288-93. PMID: 7961766

Atkinson99: Atkinson MR, Ninfa AJ (1999). "Characterization of the GlnK protein of Escherichia coli." Mol Microbiol 32(2);301-13. PMID: 10231487

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Bloom78: Bloom FR, Levin MS, Foor F, Tyler B (1978). "Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase." J Bacteriol 134(2);569-77. PMID: 26660

Bueno85: Bueno R, Pahel G, Magasanik B (1985). "Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli." J Bacteriol 1985;164(2);816-22. PMID: 2865248

Burillo04: Burillo S, Luque I, Fuentes I, Contreras A (2004). "Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis." J Bacteriol 186(11);3346-54. PMID: 15150219

Fujita94: Fujita N, Mori H, Yura T, Ishihama A (1994). "Systematic sequencing of the Escherichia coli genome: analysis of the 2.4-4.1 min (110,917-193,643 bp) region." Nucleic Acids Res 22(9);1637-9. PMID: 8202364

Garcia80: Garcia E, Federici M, Rhee SG, Berberich MA (1980). "Glutamine synthetase cascade: enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids." Arch Biochem Biophys 203(1);181-9. PMID: 6105849

Garcia83: Garcia E, Rhee SG (1983). "Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII uridylyltransferase and uridylyl-removing enzyme." J Biol Chem 1983;258(4);2246-53. PMID: 6130097

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Jiang12: Jiang P, Zhang Y, Atkinson MR, Ninfa AJ (2012). "The robustness of the Escherichia coli signal-transducing UTase/UR-PII covalent modification cycle to variation in the PII concentration requires very strong inhibition of the UTase activity of UTase/UR by glutamine." Biochemistry 51(45);9032-44. PMID: 23088522

Jiang12a: Jiang P, Ventura AC, Ninfa AJ (2012). "Characterization of the reconstituted UTase/UR-PII-NRII-NRI bicyclic signal transduction system that controls the transcription of nitrogen-regulated (Ntr) genes in Escherichia coli." Biochemistry 51(45);9045-57. PMID: 23088566

Jiang98: Jiang P, Peliska JA, Ninfa AJ (1998). "Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein." Biochemistry 37(37);12782-94. PMID: 9737855

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kamberov95: Kamberov ES, Atkinson MR, Ninfa AJ (1995). "The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP." J Biol Chem 1995;270(30);17797-807. PMID: 7629080

Kim98: Kim IH, Kwak SJ, Kang J, Park SC (1998). "Transcriptional control of the glnD gene is not dependent on nitrogen availability in Escherichia coli." Mol Cells 8(4);483-90. PMID: 9749538

Magasanik85: Magasanik B, Bueno R (1985). "The role of uridylyltransferase and PII in the regulation of the synthesis of glutamine synthetase in Escherichia coli." Curr Top Cell Regul 27;215-20. PMID: 2868841

Ninfa86: Ninfa AJ, Magasanik B (1986). "Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli." Proc Natl Acad Sci U S A 1986;83(16);5909-13. PMID: 2874557

Park92: Park S.-C., Kim I.H., Rhee S.G. (1992). "Molecular cloning and sequencing of gldD, the gene for uridylyl transferase and uridyl removing enzyme of E. coli." Data submission to EMBL/GenBank/DDBJ databases on 1992-07.

Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554

Rhee85: Rhee SG, Park SC, Koo JH (1985). "The role of adenylyltransferase and uridylyltransferase in the regulation of glutamine synthetase in Escherichia coli." Curr Top Cell Regul 27;221-32. PMID: 2868842

Salmon05: Salmon KA, Hung SP, Steffen NR, Krupp R, Baldi P, Hatfield GW, Gunsalus RP (2005). "Global gene expression profiling in Escherichia coli K12: effects of oxygen availability and ArcA." J Biol Chem 280(15);15084-96. PMID: 15699038

Tondervik06: Tondervik A, Torgersen HR, Botnmark HK, Strom AR (2006). "Transposon mutations in the 5' end of glnD, the gene for a nitrogen regulatory sensor, that suppress the osmosensitive phenotype caused by otsBA lesions in Escherichia coli." J Bacteriol 188(12);4218-26. PMID: 16740928

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

UniProt14a: UniProt Consortium (2014). "UniProt version 2014-01 released on 2014-01-01 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

vanHeeswijk13: van Heeswijk WC, Westerhoff HV, Boogerd FC (2013). "Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective." Microbiol Mol Biol Rev 77(4);628-95. PMID: 24296575

vanHeeswijk93: van Heeswijk WC, Rabenberg M, Westerhoff HV, Kahn D (1993). "The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli." Mol Microbiol 9(3);443-57. PMID: 8412694

vanHeeswijk96: van Heeswijk WC, Hoving S, Molenaar D, Stegeman B, Kahn D, Westerhoff HV (1996). "An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli." Mol Microbiol 1996;21(1);133-46. PMID: 8843440

Zhang10: Zhang Y, Pohlmann EL, Serate J, Conrad MC, Roberts GP (2010). "Mutagenesis and functional characterization of the four domains of GlnD, a bifunctional nitrogen sensor protein." J Bacteriol 192(11);2711-21. PMID: 20363937

Other References Related to Gene Regulation

Kumar11: Kumar R, Shimizu K (2011). "Transcriptional regulation of main metabolic pathways of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic continuous cultures." Microb Cell Fact 10;3. PMID: 21272324


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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