P00383 (DYR21_ECOLX) Reviewed, UniProtKB/Swiss-Prot

Names and origin

Protein names	Recommended name: Dihydrofolate reductase type 2 EC=1.5.1.3 Alternative name(s): Dihydrofolate reductase type II
Encoded on	Plasmid R67
Organism	Escherichia coli
Taxonomic identifier	562 [NCBI]
Taxonomic lineage	cellular organisms › Bacteria › Proteobacteria › Gammaproteobacteria › Enterobacteriales › Enterobacteriaceae › Escherichia

Protein attributes

Sequence length	78 AA.
Sequence status	Complete.
Protein existence	Evidence at protein level

General annotation (Comments)

Function	Key enzyme in folate metabolism. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis.
Catalytic activity	5,6,7,8-tetrahydrofolate + NADP+ = 7,8-dihydrofolate + NADPH.
Pathway	Cofactor biosynthesis; tetrahydrofolate biosynthesis; 5,6,7,8-tetrahydrofolate from 7,8-dihydrofolate: step 1/1.
Subunit structure	Homotetramer. Ref.3 Ref.4 Ref.5
Domain	The active site is situated at the inner surface of a pore formed by the four subunits. Ref.4 Ref.5
Miscellaneous	Type II plasmid-specified enzyme is practically insensitive to trimethoprim and methotrexate.
Biophysicochemical properties	Kinetic parameters: KM=5.8 µM for dihydrofolate Ref.3 KM=3.0 µM for NADPH

Ontologies

Keywords
Biological process	Antibiotic resistance Methotrexate resistance One-carbon metabolism Trimethoprim resistance
Ligand	NADP
Molecular function	Oxidoreductase
Technical term	3D-structure Direct protein sequencing Plasmid
Gene Ontology (GO)
Biological_process	one-carbon metabolic process Inferred from electronic annotation. Source: UniProtKB-KW response to antibiotic Inferred from electronic annotation. Source: UniProtKB-KW response to drug Inferred from electronic annotation. Source: InterPro response to methotrexate Inferred from electronic annotation. Source: UniProtKB-KW tetrahydrofolate biosynthetic process Inferred from electronic annotation. Source: UniProtKB-UniPathway
Molecular_function	dihydrofolate reductase activity Inferred from electronic annotation. Source: EC
Complete GO annotation...

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Chain

1 – 78

78

Dihydrofolate reductase type 2

PRO_0000186435

Regions

Nucleotide binding

66 – 69

4

NADP

Sites

Binding site

32

1

NADP

Binding site

68

1

Substrate; via amide nitrogen

Experimental info

Mutagenesis

65

1

S → A: No effect. Ref.3

Mutagenesis

67

1

Q → C: Decreases affinity for NADPH and dihydrofolate about 9-fold. Ref.3

Mutagenesis

67

1

Q → H: Increases affinity for dihydrofolate 36-fold. Increases affinity for NADPH 110-fold. Ref.3

Mutagenesis

68

1

I → L or M: Decreases affinity for dihydrofolate about 5-fold. Decreases affinity for NADPH about 7-fold. Ref.3

Mutagenesis

69

1

Y → F: Decreases affinity for dihydrofolate about 9-fold. Decreases affinity for NADPH about 22-fold. Ref.3

Mutagenesis

69

1

Y → H: Decreases affinity for dihydrofolate about 9-fold. Decreases affinity for NADPH about 60-fold. Ref.3

Secondary structure

1

.

.

.

.

.

.

.

.

.

.

.

78

Helix Strand Turn

Details...

Beta strand

29 – 36

8

Beta strand

39 – 46

8

Beta strand

52 – 62

11

Beta strand

66 – 70

5

Helix

71 – 73

3

Beta strand

74 – 76

3

Sequences

	Sequence		Length	Mass (Da)
	P00383 [UniParc]. Last modified July 21, 1986. Version 1. Checksum: 0BDB0B9146529417	FASTA	78	8,446
	10 20 30 40 50 60 MERSSNEVSN PVAGNFVFPS NATFGMGDRV RKKSGAAWQG QIVGWYCTNL TPEGYAVESE 70 AHPGSVQIYP VAALERIN

References

[1]	"Nucleotide sequence of the dihydrofolate-reductase gene borne by the plasmid R67 and conferring methotrexate resistance." Brisson N., Hohn T. Gene 28:271-275(1984) [PubMed] [Europe PMC] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA].
[2]	"The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67." Stone D., Smith S.L. J. Biol. Chem. 254:10857-10861(1979) [PubMed] [Europe PMC] [Abstract] Cited for: PROTEIN SEQUENCE.
[3]	"Role of S65, Q67, I68, and Y69 residues in homotetrameric R67 dihydrofolate reductase." Strader M.B., Smiley R.D., Stinnett L.G., VerBerkmoes N.C., Howell E.E. Biochemistry 40:11344-11352(2001) [PubMed] [Europe PMC] [Abstract] Cited for: BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, MASS SPECTROMETRY, MUTAGENESIS OF SER-65; GLN-67; ILE-68 AND TYR-69.
[4]	"A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site." Narayana N., Matthews D.A., Howell E.E., Nguyen-Huu X. Nat. Struct. Biol. 2:1018-1025(1995) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.7 ANGSTROMS) OF 17-78 IN COMPLEX WITH FOLATE, DOMAIN, SUBUNIT.
[5]	"High-resolution structure of a plasmid-encoded dihydrofolate reductase: pentagonal network of water molecules in the D2-symmetric active site." Narayana N. Acta Crystallogr. D 62:695-706(2006) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.1 ANGSTROMS) OF 17-78 OF APOPROTEIN, DOMAIN, SUBUNIT.
[6]	"Crystal structure of a type II dihydrofolate reductase catalytic ternary complex." Krahn J.M., Jackson M.R., DeRose E.F., Howell E.E., London R.E. Biochemistry 46:14878-14888(2007) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (0.96 ANGSTROMS) OF 17-78 IN COMPLEX WITH DIHYDROFOLATE AND NADP.
[7]	"Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode." Divya N., Grifith E., Narayana N. Protein Sci. 16:1063-1068(2007) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.15 ANGSTROMS) OF 17-78 OF MUTANT HIS-67 IN COMPLEX WITH NADP.
+	Additional computationally mapped references.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ

K02118 Genomic DNA. Translation: AAA26083.1.

PIR

RDECD6. A91512.

3D structure databases

PDBe
RCSB PDB
PDBj

Entry	Method	Resolution (Å)	Chain	Positions	PDBsum
1VIE	X-ray	1.70	A	17-78	[»]
1VIF	X-ray	1.80	A	17-78	[»]
2GQV	X-ray	1.10	A	17-78	[»]
2P4T	X-ray	1.15	A	17-78	[»]
2RH2	X-ray	0.96	A	17-78	[»]
2RK1	X-ray	1.26	A	17-78	[»]
2RK2	X-ray	1.90	A	17-78	[»]
3SFM	X-ray	1.40	A	17-78	[»]

ProteinModelPortal

P00383.

SMR

P00383. Positions 19-78.

ModBase

Search...

Protocols and materials databases

StructuralBiologyKnowledgebase

Search...

Enzyme and pathway databases

SABIO-RK

P00383.

UniPathway

UPA00077; UER00158.

Family and domain databases

InterPro

IPR009159. Dhfr_type_II.
IPR008990. Elect_transpt_acc-like_dom.
[Graphical view]

Pfam

PF06442. DHFR_2. 1 hit.
[Graphical view]

PIRSF

PIRSF000199. Dhfr_type_II. 1 hit.

SUPFAM

SSF50090. E_transp_acc. 1 hit.

ProtoNet

Search...

Other

EvolutionaryTrace

P00383.

Entry information

Entry name

DYR21_ECOLX

Accession

Primary (citable) accession number: P00383

Entry history

Integrated into UniProtKB/Swiss-Prot:	July 21, 1986
Last sequence update:	July 21, 1986
Last modified:	April 3, 2013
This is version 81 of the entry and version 1 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation program

Prokaryotic Protein Annotation Program