|Gene:||aat||Accession Numbers: EG11112 (EcoCyc), b0885, ECK0876|
L/F-transferase (Aat) attaches leucine and phenylalanine to exposed amino-terminal arginines and lysines on proteins. This is a key step in the ClpAP-dependent N-end rule degradation pathway.
L/F-transferase catalyzes the transfer of leucine and phenylalanine from charged tRNA to amino-terminal lysines and arginines on substrate proteins [Momose66, Leibowitz69, Kaji65, Leibowitz70, Kuno03]. When assayed with small peptides, Aat only modifies peptides bearing amino-terminal L-arginine and L-lysine, although the simple dipeptide D-lysyl-D-valine is not a substrate [Soffer73]. Testing with purified Aat and actual protein substrates shows that Aat will only transfer to amino-terminal, rather than internal, arginines in proteins [Leibowitz71, Leibowitz71a]. Though addition of both leucine and phenylalanine occurs in vitro, an in vivo experiment with arginine-β-galactosidase yielded only leucine addition [Shrader93]. In one case, purified L/F-transferase has been shown to transfer methionine onto the amino-terminus of substrate peptides [Scarpulla76]. L/F-transferase has also been shown to transfer to an unidentified 12-kilodalton protein component of the 30S ribosome [Leibowitz71a].
L/F-transferase is required for the degradation of some substrates for the N-end rule pathway, by which a protein undergoes rapid degradation by the ClpAP protease if its amino-terminal residue is arginine, lysine, leucine, phenylalanine, tyrosine or tryptophan. Arginine and lysine are considered secondary destabilizing residues, as the addition of leucine or phenylalanine by L/F-transferase is required to allow degradation [Tobias91].
The chaperone protein GroEL copurifies with L/F-transferase. Recombinant L/F-transferase with no RNA present is as active as purified wild-type protein which may contain some RNA [Abramochkin95].
A number of structural studies have been carried out on L/F-transferase. Crystal structures of isolated Aat to 2.4 Å and 1.6 Å resolution and of Aat bound to the aminoacyl-tRNA analog puromycin to 2.8 Å resolution show that Aat comprises two compact domains. Both domains are involved in puromycin, and presumably tRNA, binding via a hydrophobic pocket that is required for Aat activity [Suto06, Dong07]. Crystal structures of Aat complexed with a phenylalanyl-tRNA analog with or without an associated peptide substrate show that the phenylalanine side chain binds in the hydrophobic pocket as suggested by the puromycin analysis. In addition, this work shows that except for the amino-terminal residue, recognition of peptide substrates by L/F-transferase is sequence independent [Watanabe07]. Truncation of the Aat amino-terminus by 33 or 78 residues results in a protein that is capable of measureable transferase activity and that has wild type substrate specificity [Ichetovkin97].
Mutants lacking L/F-transferase have diminished activities of L-phenylalanyl-transfer ribonucleic acid synthetase and tryptophanase and accumulate high levels of enterochelin during iron limitation. Neither synthetase nor tryptophanase are substrates for L/F-transferase, however [Deutch77]. When grown on glycerol, L/F-transferase mutants are partial proline auxotrophs and show a fourfold increase in proline oxidase activity [Deutch75]. This increase in activity is due to an increase in the amount of proline oxidase, rather than in the enzymatic activity of individual proline oxidases [Scarpulla79].
|Map Position: [925,951 <- 926,655] (19.96 centisomes)||Length: 705 bp / 234 aa|
Molecular Weight of Polypeptide: 26.618 kD (from nucleotide sequence)
Unification Links: ASAP:ABE-0003009 , DIP:DIP-48235N , EchoBASE:EB1103 , EcoGene:EG11112 , EcoliWiki:b0885 , OU-Microarray:b0885 , PortEco:aat , PR:PRO_000022026 , Protein Model Portal:P0A8P1 , RefSeq:NP_415405 , RegulonDB:EG11112 , SMR:P0A8P1 , String:511145.b0885 , UniProt:P0A8P1
Relationship Links: InterPro:IN-FAMILY:IPR004616 , InterPro:IN-FAMILY:IPR016181 , PDB:Structure:2CXA , PDB:Structure:2DPS , PDB:Structure:2DPT , PDB:Structure:2Z3K , PDB:Structure:2Z3L , PDB:Structure:2Z3M , PDB:Structure:2Z3N , PDB:Structure:2Z3O , PDB:Structure:2Z3P , Pfam:IN-FAMILY:PF03588
|Biological Process:||GO:0030163 - protein catabolic process
GO:0071596 - ubiquitin-dependent protein catabolic process via the N-end rule pathway [Tobias91]
|Molecular Function:||GO:0008914 - leucyltransferase activity
[GOA01, GOA01a, Abramochkin95, Leibowitz70]
GO:0016740 - transferase activity [UniProtGOA11a]
GO:0016746 - transferase activity, transferring acyl groups [UniProtGOA11a]
|Cellular Component:||GO:0005737 - cytoplasm
[UniProtGOA11, UniProtGOA11a, Leibowitz70]
GO:0005829 - cytosol [DiazMejia09]
|MultiFun Terms:||information transfer → protein related → turnover, degradation|
|metabolism → degradation of macromolecules → proteins/peptides/glycopeptides|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB enriched||Yes||37||Aerobic||6.95||Yes [Gerdes03, Comment 1]|
|LB Lennox||Yes||37||Aerobic||7||Yes [Baba06, Comment 2]|
|M9 medium with 1% glycerol||Yes||37||Aerobic||7.2||0.35||Yes [Joyce06, Comment 3]|
|MOPS medium with 0.4% glucose||Yes||37||Aerobic||7.2||0.22||Yes [Baba06, Comment 2]|
Enzymatic reaction of: leucyl, phenylalanyl-tRNA-protein transferase
Synonyms: leucyl/phenylalanyl-tRNA-protein transferase, L/F-transferase
EC Number: 220.127.116.11
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
Reversibility of this reaction is unspecified.
1/26/1998 (pkarp) Merged genes G8002/aat and EG11112/aat
Abramochkin95: Abramochkin G, Shrader TE (1995). "The leucyl/phenylalanyl-tRNA-protein transferase. Overexpression and characterization of substrate recognition, domain structure, and secondary structure." J Biol Chem 270(35);20621-8. PMID: 7657641
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
Deutch77: Deutch CE, Scarpulla RC, Sonnenblick EB, Soffer RL (1977). "Pleiotropic phenotype of an Escherichia coli mutant lacking leucyl-, phenylalanyl-transfer ribonucleic acid-protein transferase." J Bacteriol 129(1);544-6. PMID: 137233
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
Dong07: Dong X, Kato-Murayama M, Muramatsu T, Mori H, Shirouzu M, Bessho Y, Yokoyama S (2007). "The crystal structure of leucyl/phenylalanyl-tRNA-protein transferase from Escherichia coli." Protein Sci 16(3);528-34. PMID: 17242373
Ebhardt09: Ebhardt HA, Xu Z, Fung AW, Fahlman RP (2009). "Quantification of the post-translational addition of amino acids to proteins by MALDI-TOF mass spectrometry." Anal Chem 81(5);1937-43. PMID: 19186990
Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938
Ichetovkin97: Ichetovkin IE, Abramochkin G, Shrader TE (1997). "Substrate recognition by the leucyl/phenylalanyl-tRNA-protein transferase. Conservation within the enzyme family and localization to the trypsin-resistant domain." J Biol Chem 272(52);33009-14. PMID: 9407082
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
Kaji65: Kaji A, Kaji H, Novelli GD (1965). "Soluble amino acid-incorporating system. II. Soluble nature of the system and the characterization of the radioactive product." J Biol Chem 240;1192-7. PMID: 14284725
Kuno03: Kuno A, Taki M, Kaneko S, Taira K, Hasegawa T (2003). "Leucyl/phenylalanyl (L/F)-tRNA-protein transferase-mediated N-terminal specific labelling of a protein in vitro." Nucleic Acids Res Suppl (3);259-60. PMID: 14510479
Leibowitz69: Leibowitz MJ, Soffer RL (1969). "A soluble enzyme from Escherichia coli which catalyzes the transfer of leucine and phenylalanine from tRNA to acceptor proteins." Biochem Biophys Res Commun 36(1);47-53. PMID: 4894363
Leibowitz70: Leibowitz MJ, Soffer RL (1970). "Enzymatic modification of proteins. 3. Purification and properties of a leucyl, phenylalanyl transfer ribonucleic acid protein transferase from Escherichia coli." J Biol Chem 245(8);2066-73. PMID: 4909560
Leibowitz71: Leibowitz MJ, Soffer RL (1971). "Enzymatic modification of proteins. VI. Site of acylation of bovine serum albumin in the leucine, phenylalanine-transfer reaction." J Biol Chem 246(14);4431-8. PMID: 4937128
Leibowitz71a: Leibowitz MJ, Soffer RL (1971). "Modification of a specific ribosomal protein catalyzed by leucyl, phenylalanyl-tRNA: protein transferase." Proc Natl Acad Sci U S A 68(8);1866-9. PMID: 4942916
Momose66: Momose K, Kaji A (1966). "Soluble amino acid-incorporating system. 3. Further studies on the product and its relation to the ribosomal system for incorporation." J Biol Chem 241(14);3294-307. PMID: 5330431
Scarpulla79: Scarpulla RC, Soffer RL (1979). "Regulation of proline dehydrogenase activity in Escherichia coli by leucyl-, phenylalanyl-tRNA:protein transferase." J Biol Chem 254(5);1724-5. PMID: 368074
Shrader93: Shrader TE, Tobias JW, Varshavsky A (1993). "The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat." J Bacteriol 175(14);4364-74. PMID: 8331068
Suto06: Suto K, Shimizu Y, Watanabe K, Ueda T, Fukai S, Nureki O, Tomita K (2006). "Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog." EMBO J 25(24);5942-50. PMID: 17110926
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