P00957 (SYA_ECOLI) Reviewed, UniProtKB/Swiss-Prot

Names and origin

Protein names

Recommended name:
Alanine--tRNA ligase
EC=6.1.1.7

Alternative name(s):
Alanyl-tRNA synthetase
Short name=AlaRS

Gene names

Name:	alaS
Synonyms:	lovB
Ordered Locus Names:	b2697, JW2667

Organism

Escherichia coli (strain K12)

Taxonomic identifier

83333 [NCBI]

Taxonomic lineage

Bacteria › Proteobacteria › Gammaproteobacteria › Enterobacteriales › Enterobacteriaceae › Escherichia

Protein attributes

Sequence length	876 AA.
Sequence status	Complete.
Sequence processing	The displayed sequence is further processed into a mature form.
Protein existence	Evidence at protein level

General annotation (Comments)

Function	Catalyzes the attachment of alanine to tRNA(Ala) in a two-step reaction: alanine is first activated by ATP to form Ala-AMP and then transferred to the acceptor end of tRNA(Ala). AlaRS also incorrectly activates the sterically smaller than alanine amino acid glycine as well as the sterically larger amino acid serine. These incorrectly charged amino acids occur because the of inherent physicochemical limitations on discrimination between closely related amino acids (glycine and serine) in the charging step. Ref.12 Ref.13 Ref.16 Edits incorrectly charged Ser-tRNA(Ala) and Gly-tRNA(Ala) but not incorrectly charged Ser-tRNA(Thr). Ref.12 Ref.13 Ref.16
Catalytic activity	ATP + L-alanine + tRNA(Ala) = AMP + diphosphate + L-alanyl-tRNA(Ala). HAMAP MF_00036_B
Cofactor	Binds 1 zinc ion per subunit; it is not clear where this binding occurs.
Subunit structure	Homotetramer. Ref.6
Subcellular location	Cytoplasm HAMAP MF_00036_B.
Domain	Consists of three domains; the N-terminal catalytic domain, the editing domain and the C-terminal C-Ala domain. The editing domain removes incorrectly charged amino acids, while the C-Ala domain, along with tRNA(Ala), serves as a bridge to cooperatively bring together the editing and aminoacylation centers thus stimulating deacylation of misacylated tRNAs. Ref.14 Ref.16 The editing domain removes incorrectly charged amino acids, i.e. Ser-tRNA(Ala) or Gly-tRNA(Ala) become uncharged tRNA(Ala) and the amino acid. It is specific for the acceptor stem of tRNA(Ala). Ref.14 Ref.16 The C-terminal C-Ala domain, along with tRNA(Ala), serves as a bridge to cooperatively bring together the editing and aminoacylation centers thus stimulating deacylation of misacylated tRNAs. This C-Ala domain can be replaced in vitro by the corresponding domain of Aquifex aeolicus or man. Recognition of tRNA(Ala) by the 2 domains is independent, that is one enzyme recognizes the same tRNA(Ala) in 2 different manners. Ref.14 Ref.16
Sequence similarities	Belongs to the class-II aminoacyl-tRNA synthetase family.

Ontologies

Keywords
Biological process	Protein biosynthesis
Cellular component	Cytoplasm
Ligand	ATP-binding Metal-binding Nucleotide-binding RNA-binding Zinc tRNA-binding
Molecular function	Aminoacyl-tRNA synthetase Ligase
PTM	Acetylation
Technical term	3D-structure Complete proteome Direct protein sequencing Reference proteome
Gene Ontology (GO)
Biological process	alanyl-tRNA aminoacylation Inferred from direct assay Ref.6. Source: EcoCyc negative regulation of transcription, DNA-dependent Inferred from direct assay. Source: EcoCyc
Cellular component	cytoplasm Inferred from electronic annotation. Source: UniProtKB-SubCell membrane Inferred from direct assay. Source: UniProtKB
Molecular function	ATP binding Inferred from electronic annotation. Source: UniProtKB-KW alanine-tRNA ligase activity Inferred from direct assay Ref.6. Source: EcoCyc aminoacyl-tRNA editing activity Inferred from direct assay Ref.14. Source: UniProtKB tRNA binding Inferred from electronic annotation. Source: UniProtKB-KW zinc ion binding Inferred from direct assay Ref.11. Source: EcoCyc
Complete GO annotation...

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Initiator methionine

1

1

Removed Ref.6

Chain

2 – 876

875

Alanine--tRNA ligase HAMAP MF_00036_B

PRO_0000075113

Regions

Region

2 – 461

460

Catalytic HAMAP MF_00036_B

Region

553 – 705

153

Editing HAMAP MF_00036_B

Region

699 – 808

110

Important for oligomerization HAMAP MF_00036_B

Region

766 – 875

110

C-Ala domain HAMAP MF_00036_B

Sites

Metal binding

564

1

Zinc By similarity

Metal binding

568

1

Zinc By similarity

Metal binding

666

1

Zinc By similarity

Metal binding

670

1

Zinc By similarity

Amino acid modifications

Modified residue

74

1

N6-acetyllysine Ref.15

Experimental info

Mutagenesis

179

1

C → S: Inactivates the enzyme. Ref.11

Mutagenesis

182

1

C → S: No inactivation. Ref.11

Mutagenesis

184

1

E → Q: No inactivation. Ref.11

Mutagenesis

188

1

D → N: No inactivation. Ref.11

Mutagenesis

189

1

H → Q: Inactivates the enzyme. Ref.11

Mutagenesis

191

1

D → N: No inactivation. Ref.11

Mutagenesis

192

1

H → Q: Inactivates the enzyme. Ref.11

Mutagenesis

236

1

D → A or N: Decreases affinity for alanine without improving discrimination against serine. Ref.17

Mutagenesis

238

1

G → A: Greatly decreases affinity for alanine with only small changes in affinity for serine and glycine, in a 2-442 residue construct. Ref.17

Mutagenesis

564

1

H → A: No effect on tRNA editing. Ref.12

Mutagenesis

568

1

H → A: No effect on tRNA editing. Ref.12

Mutagenesis

584

1

Q → H: Loss of mischarged tRNA editing activity; when associated with A-666. Ref.12 Ref.13

Mutagenesis

666

1

C → A: Loss of mischarged tRNA editing activity; when associated with H-584 the effect is more pronounced. Ref.12 Ref.13

Mutagenesis

670

1

H → A: No effect on tRNA editing. Ref.12

Mutagenesis

693

1

R → K: Reduces specificity of editing activity for tRNA(Ala), allows editing of tRNA(Thr). Ref.14

Sequence conflict

29 – 33

5

LVPHN → RYPIT Ref.4

Sequence conflict

29 – 33

5

LVPHN → RYPIT in AAA03208. Ref.5

Sequence conflict

158

1

G → N Ref.4

Sequence conflict

158

1

G → N in AAA03208. Ref.5

Sequence conflict

168

1

D → G in AAA03208. Ref.5

Sequence conflict

172

1

Q → R in AAA03208. Ref.5

Sequence conflict

175

1

D → G in AAA03208. Ref.5

Sequence conflict

180

1

G → D in AAA03208. Ref.5

Sequence conflict

584

1

Q → H in AAA03208. Ref.5

Sequence conflict

619

1

Q → L in AAA03208. Ref.5

Secondary structure

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876

Helix Strand Turn

Details...

Helix

5 – 18

14

Helix

45 – 47

3

Helix

48 – 51

4

Beta strand

60 – 63

4

Beta strand

66 – 69

4

Helix

78 – 80

3

Beta strand

89 – 94

6

Beta strand

96 – 98

3

Helix

104 – 116

13

Turn

118 – 121

4

Helix

125 – 127

3

Beta strand

128 – 133

6

Helix

137 – 145

9

Helix

151 – 153

3

Beta strand

154 – 157

4

Beta strand

181 – 188

8

Beta strand

208 – 221

14

Beta strand

227 – 239

13

Helix

242 – 248

7

Helix

254 – 256

3

Helix

258 – 270

13

Helix

279 – 296

18

Helix

309 – 319

11

Helix

331 – 334

4

Helix

338 – 341

4

Helix

348 – 351

4

Helix

353 – 368

16

Helix

376 – 383

8

Helix

394 – 401

8

Helix

406 – 414

9

Turn

415 – 417

3

Helix

426 – 439

14

Sequences

	Sequence		Length	Mass (Da)
	P00957 [UniParc]. Last modified November 1, 1997. Version 2. Checksum: 73F69C69FCF8C08C	FASTA	876	96,032
	10 20 30 40 50 60 MSKSTAEIRQ AFLDFFHSKG HQVVASSSLV PHNDPTLLFT NAGMNQFKDV FLGLDKRNYS 70 80 90 100 110 120 RATTSQRCVR AGGKHNDLEN VGYTARHHTF FEMLGNFSFG DYFKHDAIQF AWELLTSEKW 130 140 150 160 170 180 FALPKERLWV TVYESDDEAY EIWEKEVGIP RERIIRIGDN KGAPYASDNF WQMGDTGPCG 190 200 210 220 230 240 PCTEIFYDHG DHIWGGPPGS PEEDGDRYIE IWNIVFMQFN RQADGTMEPL PKPSVDTGMG 250 260 270 280 290 300 LERIAAVLQH VNSNYDIDLF RTLIQAVAKV TGATDLSNKS LRVIADHIRS CAFLIADGVM 310 320 330 340 350 360 PSNENRGYVL RRIIRRAVRH GNMLGAKETF FYKLVGPLID VMGSAGEDLK RQQAQVEQVL 370 380 390 400 410 420 KTEEEQFART LERGLALLDE ELAKLSGDTL DGETAFRLYD TYGFPVDLTA DVCRERNIKV 430 440 450 460 470 480 DEAGFEAAME EQRRRAREAS GFGADYNAMI RVDSASEFKG YDHLELNGKV TALFVDGKAV 490 500 510 520 530 540 DAINAGQEAV VVLDQTPFYA ESGGQVGDKG ELKGANFSFA VEDTQKYGQA IGHIGKLAAG 550 560 570 580 590 600 SLKVGDAVQA DVDEARRARI RLNHSATHLM HAALRQVLGT HVSQKGSLVN DKVLRFDFSH 610 620 630 640 650 660 NEAMKPEEIR AVEDLVNTQI RRNLPIETNI MDLEAAKAKG AMALFGEKYD ERVRVLSMGD 670 680 690 700 710 720 FSTELCGGTH ASRTGDIGLF RIISESGTAA GVRRIEAVTG EGAIATVHAD SDRLSEVAHL 730 740 750 760 770 780 LKGDSNNLAD KVRSVLERTR QLEKELQQLK EQAAAQESAN LSSKAIDVNG VKLLVSELSG 790 800 810 820 830 840 VEPKMLRTMV DDLKNQLGST IIVLATVVEG KVSLIAGVSK DVTDRVKAGE LIGMVAQQVG 850 860 870 GKGGGRPDMA QAGGTDAAAL PAALASVKGW VSAKLQ

References

	« Hide 'large scale' referencesShow 'large scale' references »
[1]	"Construction of a contiguous 874-kb sequence of the Escherichia coli-K12 genome corresponding to 50.0-68.8 min on the linkage map and analysis of its sequence features." Yamamoto Y., Aiba H., Baba T., Hayashi K., Inada T., Isono K., Itoh T., Kimura S., Kitagawa M., Makino K., Miki T., Mitsuhashi N., Mizobuchi K., Mori H., Nakade S., Nakamura Y., Nashimoto H., Oshima T. , Oyama S., Saito N., Sampei G., Satoh Y., Sivasundaram S., Tagami H., Takahashi H., Takeda J., Takemoto K., Uehara K., Wada C., Yamagata S., Horiuchi T. DNA Res. 4:91-113(1997) [PubMed: 9205837] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. Strain: K12 / W3110 / ATCC 27325 / DSM 5911.
[2]	"The complete genome sequence of Escherichia coli K-12." Blattner F.R., Plunkett G. III, Bloch C.A., Perna N.T., Burland V., Riley M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew G.F., Gregor J., Davis N.W., Kirkpatrick H.A., Goeden M.A., Rose D.J., Mau B., Shao Y. Science 277:1453-1474(1997) [PubMed: 9278503] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. Strain: K12 / MG1655 / ATCC 47076.
[3]	"Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110." Hayashi K., Morooka N., Yamamoto Y., Fujita K., Isono K., Choi S., Ohtsubo E., Baba T., Wanner B.L., Mori H., Horiuchi T. Mol. Syst. Biol. 2:E1-E5(2006) [PubMed: 16738553] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. Strain: K12 / W3110 / ATCC 27325 / DSM 5911.
[4]	"Mass spectra of partial protein hydrolysates as a multiple phase check for long polypeptides deduced from DNA sequences: NH2-terminal segment of alanine tRNA synthetase." Herlihy W.C., Royal N.J., Biemann K., Putney S.D., Schimmel P.R. Proc. Natl. Acad. Sci. U.S.A. 77:6531-6535(1980) [PubMed: 7005898] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-166, PARTIAL PROTEIN SEQUENCE.
[5]	"Primary structure of a large aminoacyl-tRNA synthetase." Putney S.D., Royal N.J., de Vegvar H.N., Herlihy W.C., Biemann K., Schimmel P. Science 213:1497-1501(1981) [PubMed: 7025207] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 2-876.
[6]	"Purification and properties of alanine tRNA synthetase from Escherichia coli A tetramer of identical subunits." Putney S.D., Sauer R.T., Schimmel P.R. J. Biol. Chem. 256:198-204(1981) [PubMed: 7005211] [Abstract] Cited for: PROTEIN SEQUENCE OF 2-12, SUBUNIT.
[7]	"Acceptor helix interactions in a class II tRNA synthetase: photoaffinity cross-linking of an RNA miniduplex substrate." Musier-Forsyth K., Schimmel P. Biochemistry 33:773-779(1994) [PubMed: 8292605] [Abstract] Cited for: PROTEIN SEQUENCE OF 163-173.
[8]	"Evidence for involvement of Escherichia coli genes pmbA, csrA and a previously unrecognized gene tldD, in the control of DNA gyrase by letD (ccdB) of sex factor F." Murayama N., Shimizu H., Takiguchi S., Baba Y., Amino H., Horiuchi T., Sekimizu K., Miki T. J. Mol. Biol. 256:483-502(1996) [PubMed: 8604133] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 799-876. Strain: KP4714.
[9]	"Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties." Romeo T., Gong M., Liu M.-Y., Brun-Zinkernagel A.-M. J. Bacteriol. 175:4744-4755(1993) [PubMed: 8393005] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 866-876. Strain: K12.
[10]	"Cloning, partial sequencing, and in vitro transcription of the gene for alanine tRNA synthetase." Putney S.D., Melendez D.L., Schimmel P.R. J. Biol. Chem. 256:205-211(1981) [PubMed: 6256345] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-13.
[11]	"Evidence for a 'cysteine-histidine box' metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase." Miller W.T., Hill K.A.W., Schimmel P. Biochemistry 30:6970-6976(1991) [PubMed: 1712632] [Abstract] Cited for: ZINC-BINDING, MUTAGENESIS OF CYS-179; CYS-182; GLU-184; ASP-188; HIS-189; ASP-191 AND HIS-192.
[12]	"Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability." Beebe K., Ribas De Pouplana L., Schimmel P. EMBO J. 22:668-675(2003) [PubMed: 12554667] [Abstract] Cited for: FUNCTION IN TRNA EDITING, MUTAGENESIS OF HIS-564; HIS-568; GLN-584; CYS-666 AND HIS-670.
[13]	"Natural homolog of tRNA synthetase editing domain rescues conditional lethality caused by mistranslation." Chong Y.E., Yang X.L., Schimmel P. J. Biol. Chem. 283:30073-30078(2008) [PubMed: 18723508] [Abstract] Cited for: FUNCTION IN TRNA EDITING, MUTAGENESIS OF GLN-584 AND CYS-666. Strain: K12.
[14]	"Distinct domains of tRNA synthetase recognize the same base pair." Beebe K., Mock M., Merriman E., Schimmel P. Nature 451:90-93(2008) [PubMed: 18172502] [Abstract] Cited for: DOMAIN EDITING, MUTAGENESIS OF ARG-693.
[15]	"Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli." Zhang J., Sprung R., Pei J., Tan X., Kim S., Zhu H., Liu C.F., Grishin N.V., Zhao Y. Mol. Cell. Proteomics 8:215-225(2009) [PubMed: 18723842] [Abstract] Cited for: ACETYLATION [LARGE SCALE ANALYSIS] AT LYS-74, MASS SPECTROMETRY. Strain: K12 / JW1106 and K12 / MG1655 / ATCC 47076.
[16]	"The C-Ala domain brings together editing and aminoacylation functions on one tRNA." Guo M., Chong Y.E., Beebe K., Shapiro R., Yang X.-L., Schimmel P. Science 325:744-747(2009) [PubMed: 19661429] [Abstract] Cited for: FUNCTION IN TRNA-BINDING VIA C-ALA DOMAIN, DOMAIN EXCHANGE EXPERIMENTS.
[17]	"Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma." Guo M., Chong Y.E., Shapiro R., Beebe K., Yang X.L., Schimmel P. Nature 462:808-812(2009) [PubMed: 20010690] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.90 ANGSTROMS) OF 2-442 WITH AND WITHOUT CHARGED CORRECT AND INCORRECT AMINO ACID, MUTAGENESIS OF ASP-236 AND GLY-238.
+	Additional computationally mapped references.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ

U00096 Genomic DNA. Translation: AAC75739.1.
AP009048 Genomic DNA. Translation: BAA16559.1.
J01581 Unassigned DNA. Translation: AAA03208.1.
L07596 Unassigned DNA. Translation: AAA71918.1. Sequence problems.
D44453 Genomic DNA. Translation: BAA21554.1.
Z28405 Genomic DNA. Translation: CAA82247.1.

PIR

SYECAT. E65049.

RefSeq

NP_417177.1. NC_000913.2.

3D structure databases

PDBe
RCSB PDB
PDBj

Entry	Method	Resolution (Å)	Chain	Positions	PDBsum
3HXU	X-ray	2.10	A	2-442	[»]
3HXV	X-ray	1.93	A	2-442	[»]
3HXW	X-ray	1.93	A	2-442	[»]
3HXX	X-ray	2.11	A	2-442	[»]
3HXY	X-ray	2.27	A	2-442	[»]
3HXZ	X-ray	1.99	A/B/C/D	2-442	[»]
3HY0	X-ray	1.90	A/B	2-442	[»]
3HY1	X-ray	2.79	A/B	2-442	[»]

ProteinModelPortal

P00957.

SMR

P00957. Positions 2-875.

ModBase

Search...

Protein-protein interaction databases

DIP

DIP-9080N.

IntAct

P00957. 21 interactions.

MINT

MINT-1266342.

2D gel databases

SWISS-2DPAGE

P00957.

ECO2DBASE

F093.0. 6TH EDITION.

Proteomic databases

PRIDE

P00957.

Protocols and materials databases

StructuralBiologyKnowledgebase

Search...

Genome annotation databases

EnsemblBacteria

EBESCT00000002492; EBESCP00000002492; EBESCG00000002032.
EBESCT00000014987; EBESCP00000014278; EBESCG00000014047.

GeneID

947175.

GenomeReviews

Gene locus JW2667 in contig AP009048_GR.
Gene locus b2697 in contig U00096_GR.

KEGG

ecj:JW2667.
eco:b2697.

PATRIC

32120790. VBIEscCol129921_2787.

Organism-specific databases

EchoBASE

EB0033.

EcoGene

EG10034. alaS.

Phylogenomic databases

eggNOG

COG0013.

GeneTree

EBGT00050000010345.

HOGENOM

HBG354397.

OMA

EIDIFRT.

PhylomeDB

P00957.

ProtClustDB

PRK00252.

Enzyme and pathway databases

BioCyc

EcoCyc:ALAS-MONOMER.
MetaCyc:ALAS-MONOMER.

Gene expression databases

Genevestigator

P00957.

Family and domain databases

HAMAP

MF_00036_B. Ala_tRNA_synth_B.
[Tree]

InterPro

IPR002318. Ala-tRNA-synth_IIc.
IPR018162. Ala-tRNA-synth_IIc_anticod-bd.
IPR018165. Ala-tRNA-synth_IIc_core.
IPR018164. Ala-tRNA-synth_IIc_N.
IPR023033. Ala_tRNA_synth_euk/bac.
IPR003156. Pesterase_DHHA1.
IPR018163. Thr/Ala-tRNA-synth_IIc_edit.
IPR012947. tRNA_SAD.
[Graphical view]

KO

K01872.

Pfam

PF02272. DHHA1. 1 hit.
PF01411. tRNA-synt_2c. 1 hit.
PF07973. tRNA_SAD. 1 hit.
[Graphical view]

PRINTS

PR00980. TRNASYNTHALA.

SMART

SM00863. tRNA_SAD. 1 hit.
[Graphical view]

SUPFAM

SSF101353. Ala-tRNA-synth_IIc_anticod-bd. 1 hit.
SSF55186. Thr/Ala-tRNA-synth_IIc_edit. 1 hit.

TIGRFAMs

TIGR00344. AlaS. 1 hit.

PROSITE

PS50860. AA_TRNA_LIGASE_II_ALA. 1 hit.
[Graphical view]

ProtoNet

Search...

Entry information

Entry name

SYA_ECOLI

Accession

Primary (citable) accession number: P00957
Secondary accession number(s): P78279

Entry history

Integrated into UniProtKB/Swiss-Prot:	July 21, 1986
Last sequence update:	November 1, 1997
Last modified:	January 25, 2012
This is version 124 of the entry and version 2 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation program

Prokaryotic Protein Annotation Program