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Escherichia coli K-12 substr. MG1655 Enzyme: prolyl-tRNA synthetase



Gene: proS Accession Numbers: EG10770 (EcoCyc), b0194, ECK0194

Synonyms: drp, drpA

Regulation Summary Diagram: ?

Subunit composition of prolyl-tRNA synthetase = [ProS]2
         prolyl-tRNA synthetase = ProS

Summary:
Prolyl-tRNA synthetase (ProRS) is a member of the family of aminoacyl-tRNA synthetases, which interpret the genetic code by covalently linking amino acids to their specific tRNA molecules. The reaction is driven by ATP hydrolysis. ProRS belongs to the Class II aminoacyl tRNA synthetases, which share three regions of homology [Eriani90, Cusack91].

ProRS of E. coli B is a dimer in solution [Lee69].

Specificity determinants within tRNAPro that are important for recognition by ProRS have been identified [Yap95, Liu94c, Liu95a, Liu95b, McClain94, Yap95a, Burke00, Hasegawa00]. Specificity determinants and residues within ProRS that are important for catalytic activity have been investigated [Stehlin97, Burke00].

Many aminoacyl tRNA synthetases have been shown to have editing functions. ProRS misactivates alanine, but has both a pre-transfer editing function, hydrolyzing the non-cognate amino acid before transfer to tRNAPro, and a post-transfer editing function that deacetylates mischarged Ala-tRNAPro [Beuning00]. The C433 [Beuning00] and K279 [Wong02] residues are critical for the post-transfer editing function. ProRS contains a large insertion domain between motifs 2 and 3 of the class II aminoacyl tRNA synthetases; this domain is independently folded and plays a role in the editing functions [Wong02, Wong03]. A chimeric yeast prolyl-tRNA synthetase engineered to contain this domain gains a post-transfer editing function [SternJohn07]. Pre-transfer editing is not stimulated by the addition of tRNAPro [Beuning00] and does not require a separate editing domain [Hati06].

ProRS exhibits a natural level of mischarging with cysteine, resulting in Cys-tRNAPro, which is not edited by the enzyme. The kcat/Km for proline is 369-fold higher than for cysteine [Ahel02].

Synthesis of ProRS is derepressed by starvation for proline [Archibold72]. A temperature-sensitive allele of proS has been isolated [Bohman80].

Review: [Ibba00]

Citations: [Kumar12]

Gene Citations: [Zhou90]

Locations: cytosol

Map Position: [217,057 <- 218,775] (4.68 centisomes)
Length: 1719 bp / 572 aa

Molecular Weight of Polypeptide: 63.693 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0000657 , CGSC:360 , DIP:DIP-10573N , EchoBASE:EB0763 , EcoGene:EG10770 , EcoliWiki:b0194 , Mint:MINT-1261583 , ModBase:P16659 , OU-Microarray:b0194 , PortEco:proS , PR:PRO_000023601 , Pride:P16659 , Protein Model Portal:P16659 , RefSeq:NP_414736 , RegulonDB:EG10770 , SMR:P16659 , String:511145.b0194 , UniProt:P16659

Relationship Links: InterPro:IN-FAMILY:IPR002314 , InterPro:IN-FAMILY:IPR002316 , InterPro:IN-FAMILY:IPR004154 , InterPro:IN-FAMILY:IPR004500 , InterPro:IN-FAMILY:IPR006195 , InterPro:IN-FAMILY:IPR007214 , InterPro:IN-FAMILY:IPR023717 , Panther:IN-FAMILY:PTHR11451:SF3 , Pfam:IN-FAMILY:PF00587 , Pfam:IN-FAMILY:PF03129 , Pfam:IN-FAMILY:PF04073 , Prints:IN-FAMILY:PR01046 , Prosite:IN-FAMILY:PS50862

In Paralogous Gene Group: 338 (2 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006433 - prolyl-tRNA aminoacylation Inferred from experiment Inferred by computational analysis [GOA01a, Bohman80]
GO:0006450 - regulation of translational fidelity Inferred by computational analysis Inferred from experiment [Wong03, GOA01a]
GO:0006412 - translation Inferred by computational analysis [UniProtGOA11a]
GO:0006418 - tRNA aminoacylation for protein translation Inferred by computational analysis [GOA01a]
Molecular Function: GO:0004827 - proline-tRNA ligase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Stehlin97]
GO:0043906 - Ala-tRNA(Pro) hydrolase activity Inferred from experiment [Wong03]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0002161 - aminoacyl-tRNA editing activity Inferred by computational analysis [GOA01a]
GO:0004812 - aminoacyl-tRNA ligase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA01a]

MultiFun Terms: information transfer protein related amino acid -activation

Essentiality data for proS knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 1]

Credits:
Last-Curated ? 17-Aug-2007 by Keseler I , SRI International


Enzymatic reaction of: prolyl-tRNA synthetase

Synonyms: ProRS

EC Number: 6.1.1.15/6.1.1.-

tRNApro + L-proline + ATP + H+ <=> L-prolyl-tRNApro + AMP + 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: tRNA charging

Summary:
The enzyme was initially purified from E. coli B [Lee69, Papas70, Papas71].

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
tRNApro
14140.0
0.24
[Hati06, BRENDA14]
ATP
600.0
[NORTON64, BRENDA14]
ATP
30.0
[Papas71]
ATP
81.0
[Stehlin97, BRENDA14]
L-proline
120.0
[NORTON64, BRENDA14]
L-proline
800.0
[Busiello80, BRENDA14]
L-proline
300.0
[Stehlin97, BRENDA14]
L-proline
290.0
14.0
[Ahel02, BRENDA14]
L-proline
250.0
70.0
[Hati06, BRENDA14]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 26 -> 27
[Eriani90, UniProt10a]
Alternate sequence: ML → IV; UniProt: (in Ref. 1);
Sequence-Conflict 127 -> 128
[Zhou90, UniProt10a]
Alternate sequence: QL → HV; UniProt: (in Ref. 2; AAA23710);
Sequence-Conflict 205
[Eriani90, UniProt10a]
[Zhou90, UniProt10a]
Alternate sequence: S → Q; UniProt: (in Ref. 1);
Alternate sequence: SASHEFQVLAQS → RPLTNSRCWRR; UniProt: (in Ref. 2; AAA23710);
Mutagenesis-Variant 257
[Wong02, UniProt11a]
Alternate sequence: T → A; UniProt: Reduces the posttransfer editing activity 5-fold, with little change in both the aminoacylation and the pretransfer editing activities.
Mutagenesis-Variant 279
[Wong02, UniProt11a]
Alternate sequence: K → A; UniProt: Severely affects the posttransfer editing activity, with little change in both the aminoacylation and the pretransfer editing activities.
Mutagenesis-Variant 350
[Wong02, UniProt11a]
Alternate sequence: D → A; UniProt: Abolishes the pretransfer editing activity and reduces the aminoacylation activity 20-fold and the posttransfer editing activity 5-fold.
Mutagenesis-Variant 369
[Wong02, UniProt11a]
Alternate sequence: H → A; UniProt: Reduces both the aminoacylation and the pretransfer editing activities 2.5- fold, and the posttransfer editing activity 5-fold. Loss of specificity in deacylation.
Mutagenesis-Variant 378
[Wong02, UniProt11a]
Alternate sequence: D → A; UniProt: Little change in both the aminoacylation and the editing activities.
Mutagenesis-Variant 386
[Wong02, UniProt11a]
Alternate sequence: D → A; UniProt: Reduces the posttransfer editing activity approximately 2-fold, with little change in both the aminoacylation and the pretransfer editing activities.
Mutagenesis-Variant 394
[Wong02, UniProt11a]
Alternate sequence: D → A; UniProt: Reduces the posttransfer editing activity approximately 2-fold, with little change in both the aminoacylation and the pretransfer editing activities.
Mutagenesis-Variant 443
[Beuning00, UniProt11a]
Alternate sequence: C → G; UniProt: 150-fold decrease in posttransfer editing activity against alanine, without change neither in pretransfer editing nor in aminoacylation.
Sequence-Conflict 516 -> 517
[Zhou90, UniProt10a]
Alternate sequence: PG → RA; UniProt: (in Ref. 2);
Sequence-Conflict 518 -> 572
[Zhou90, UniProt10a]
Alternate sequence: VMFADMELIGIPHTIVLGDRNLDNDDIEYKYRRNGEKQLIKTGDIVEYLVKQIKG → missing; UniProt: (in Ref. 2);


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b0194 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10770; confirmed by SwissProt match.


References

Ahel02: Ahel I, Stathopoulos C, Ambrogelly A, Sauerwald A, Toogood H, Hartsch T, Soll D (2002). "Cysteine activation is an inherent in vitro property of prolyl-tRNA synthetases." J Biol Chem 277(38);34743-8. PMID: 12130657

Archibold72: Archibold ER, Williams LS (1972). "Regulation of synthesis of methionyl-, prolyl-, and threonyl-transfer ribonucleic acid synthetases of Escherichia coli." J Bacteriol 109(3);1020-6. PMID: 4551738

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

Beuning00: Beuning PJ, Musier-Forsyth K (2000). "Hydrolytic editing by a class II aminoacyl-tRNA synthetase." Proc Natl Acad Sci U S A 97(16);8916-20. PMID: 10922054

Bohman80: Bohman K, Isaksson LA (1980). "A temperature-sensitive mutant in prolinyl-tRNA ligase of Escherichia coli K-12." Mol Gen Genet 177(4);603-5. PMID: 6991867

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Burke00: Burke B, Yang F, Chen F, Stehlin C, Chan B, Musier-Forsyth K (2000). "Evolutionary coadaptation of the motif 2--acceptor stem interaction in the class II prolyl-tRNA synthetase system." Biochemistry 39(50);15540-7. PMID: 11112540

Busiello80: Busiello V, Di Girolamo M, Cini C, de Marco C (1980). "Beta-selenaproline as competitive inhibitor of proline activation." Biochim Biophys Acta 606(2);347-52. PMID: 6243985

Cusack91: Cusack S, Hartlein M, Leberman R (1991). "Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases." Nucleic Acids Res 19(13);3489-98. PMID: 1852601

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Eriani90: Eriani G, Delarue M, Poch O, Gangloff J, Moras D (1990). "Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs." Nature 347(6289);203-6. PMID: 2203971

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."

Hasegawa00: Hasegawa T, Yokogawa T (2000). "Escherichia coli proline tRNA: structure and recognition sites for prolyl-tRNA synthetase." Nucleic Acids Symp Ser (44);7-8. PMID: 12903242

Hati06: Hati S, Ziervogel B, Sternjohn J, Wong FC, Nagan MC, Rosen AE, Siliciano PG, Chihade JW, Musier-Forsyth K (2006). "Pre-transfer editing by class II prolyl-tRNA synthetase: role of aminoacylation active site in "selective release" of noncognate amino acids." J Biol Chem 281(38);27862-72. PMID: 16864571

Ibba00: Ibba M, Soll D (2000). "Aminoacyl-tRNA synthesis." Annu Rev Biochem 69;617-50. PMID: 10966471

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Kumar12: Kumar S, Das M, Hadad CM, Musier-Forsyth K (2012). "Substrate and enzyme functional groups contribute to translational quality control by bacterial prolyl-tRNA synthetase." J Phys Chem B 116(23);6991-9. PMID: 22458656

Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726

Lee69: Lee ML, Muench KH (1969). "Prolyl transfer ribonucleic acid synthetase of Escherichia coli. I. Purification and evidence for subunits." J Biol Chem 244(2);223-30. PMID: 4886431

Liu94c: Liu H, Musier-Forsyth K (1994). "Escherichia coli proline tRNA synthetase is sensitive to changes in the core region of tRNA(Pro)." Biochemistry 33(42);12708-14. PMID: 7522561

Liu95a: Liu H, Yap LP, Stehlin C, Musier-Forsyth K (1995). "Molecular recognition of tRNA(Pro) by Escherichia coli proline-tRNA synthetase." Nucleic Acids Symp Ser (33);176-8. PMID: 8643363

Liu95b: Liu H, Peterson R, Kessler J, Musier-Forsyth K (1995). "Molecular recognition of tRNA(Pro) by Escherichia coli proline tRNA synthetase in vitro." Nucleic Acids Res 23(1);165-9. PMID: 7870582

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

McClain94: McClain WH, Schneider J, Gabriel K (1994). "Distinctive acceptor-end structure and other determinants of Escherichia coli tRNAPro identity." Nucleic Acids Res 22(3);522-9. PMID: 8127693

NORTON64: NORTON SJ (1964). "PURIFICATION AND PROPERTIES OF THE PROLYL RNA SYNTHETASE OF ESCHERICHIA COLI." Arch Biochem Biophys 106;147-52. PMID: 14217147

Papas70: Papas TS, Mehler AH (1970). "Analysis of the amino acid binding to the proline transfer ribonucleic acid synthetase of Escherichia coli." J Biol Chem 245(7);1588-95. PMID: 4314590

Papas71: Papas TS, Mehler AH (1971). "Kinetic studies of the prolyl transfer ribonucleic acid synthetase of Escherichia coli. Order of addition of substrates and release of products." J Biol Chem 246(19);5924-8. PMID: 4330060

Stehlin97: Stehlin C, Heacock DH, Liu H, Musier-Forsyth K (1997). "Chemical modification and site-directed mutagenesis of the single cysteine in motif 3 of class II Escherichia coli prolyl-tRNA synthetase." Biochemistry 36(10);2932-8. PMID: 9062123

SternJohn07: SternJohn J, Hati S, Siliciano PG, Musier-Forsyth K (2007). "Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain." Proc Natl Acad Sci U S A 104(7);2127-32. PMID: 17283340

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

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

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

Wong02: Wong FC, Beuning PJ, Nagan M, Shiba K, Musier-Forsyth K (2002). "Functional role of the prokaryotic proline-tRNA synthetase insertion domain in amino acid editing." Biochemistry 41(22);7108-15. PMID: 12033945

Wong03: Wong FC, Beuning PJ, Silvers C, Musier-Forsyth K (2003). "An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing." J Biol Chem 278(52);52857-64. PMID: 14530268

Yap95: Yap LP, Musier-Forsyth K (1995). "Transfer RNA aminoacylation: identification of a critical ribose 2'-hydroxyl-base interaction." RNA 1(4);418-24. PMID: 7493319

Yap95a: Yap LP, Stehlin C, Musier-Forsyth K (1995). "Use of semi-synthetic transfer RNAs to probe molecular recognition by Escherichia coli proline-tRNA synthetase." Chem Biol 2(10);661-6. PMID: 9383472

Zhou90: Zhou Z, Syvanen M (1990). "Identification and sequence of the drpA gene from Escherichia coli." J Bacteriol 172(1);281-6. PMID: 1688424


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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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