P62623 (ISPH_ECOLI) Reviewed, UniProtKB/Swiss-Prot

Names and origin

Protein names

Recommended name:
4-hydroxy-3-methylbut-2-enyl diphosphate reductase
EC=1.17.1.2

Gene names

Name:	ispH
Synonyms:	lytB, yaaE
Ordered Locus Names:	b0029, JW0027

Organism

Escherichia coli (strain K12) [Reference proteome] [HAMAP]

Taxonomic identifier

83333 [NCBI]

Taxonomic lineage

cellular organisms › Bacteria › Proteobacteria › Gammaproteobacteria › Enterobacteriales › Enterobacteriaceae › Escherichia › Escherichia coli

Protein attributes

Sequence length	316 AA.
Sequence status	Complete.
Protein existence	Evidence at protein level

General annotation (Comments)

Function	Converts 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Is also involved in penicillin tolerance and control of the stringent response. Seems to directly or indirectly interact with RelA to maintain it in an inactive form during normal growth. Ref.13 Ref.14 Ref.15
Catalytic activity	Isopentenyl diphosphate + NAD(P)+ + H2O = (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H. Ref.9 Ref.11 Dimethylallyl diphosphate + NAD(P)+ + H2O = (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate + NAD(P)H. Ref.9 Ref.11
Cofactor	Binds 1 3Fe-4S cluster per subunit. Ref.13 Ref.14 Ref.15
Enzyme regulation	Addition of Ca2+, Mg2+, Mn2+, Ni2+, Co2+ or Fe2+ decreases the activity. Addition of Zn2+ results in complete loss of activity. Ref.12
Pathway	Isoprenoid biosynthesis; dimethylallyl diphosphate biosynthesis; dimethylallyl diphosphate from (2E)-4-hydroxy-3-methylbutenyl diphosphate: step 1/1. Ref.7 Ref.8 Isoprenoid biosynthesis; isopentenyl diphosphate biosynthesis via DXP pathway; isopentenyl diphosphate from 1-deoxy-D-xylulose 5-phosphate: step 6/6. Ref.7 Ref.8
Subunit structure	Homodimer. Ref.10 Ref.13 Ref.14 Ref.15
Miscellaneous	Ref.10 and Ref.15 authors have reported the presence of a 4Fe-4S iron-sulfur cluster. It could be due to experimental conditions. HAMAP-Rule MF_00191
Sequence similarities	Belongs to the IspH family.
Biophysicochemical properties	Kinetic parameters: KM=30 µM for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate Ref.12 pH dependence: Optimum pH is 7.0.

Ontologies

Keywords
Biological process	Isoprene biosynthesis
Ligand	3Fe-4S Iron Iron-sulfur Metal-binding NADP
Molecular function	Oxidoreductase
Technical term	3D-structure Complete proteome Reference proteome
Gene Ontology (GO)
Biological_process	dimethylallyl diphosphate biosynthetic process Inferred from electronic annotation. Source: UniProtKB-UniPathway isopentenyl diphosphate biosynthetic process, mevalonate-independent pathway Inferred from electronic annotation. Source: HAMAP response to antibiotic Inferred from direct assay Ref.11. Source: EcoCyc terpenoid biosynthetic process Inferred from electronic annotation. Source: HAMAP ubiquinone biosynthetic process Inferred from direct assay Ref.11. Source: EcoCyc
Molecular_function	3 iron, 4 sulfur cluster binding Inferred from electronic annotation. Source: HAMAP 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase activity Inferred from electronic annotation. Source: HAMAP hydroxymethylbutenyl pyrophosphate reductase activity Inferred from direct assay Ref.11. Source: EcoCyc metal ion binding Inferred from electronic annotation. Source: UniProtKB-KW
Complete GO annotation...

Binary interactions

With	Entry	#Exp.	IntAct	Notes
dnaK	P0A6Y8	1	EBI-554022,EBI-542092

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Chain

1 – 316

316

4-hydroxy-3-methylbut-2-enyl diphosphate reductase HAMAP-Rule MF_00191

PRO_0000128812

Regions

Region

225 – 227

3

Substrate binding HAMAP-Rule MF_00191

Sites

Metal binding

12

1

Iron-sulfur (3Fe-4S)

Metal binding

96

1

Iron-sulfur (3Fe-4S)

Metal binding

197

1

Iron-sulfur (3Fe-4S)

Binding site

41

1

Substrate

Binding site

74

1

Substrate

Binding site

124

1

Substrate

Binding site

167

1

Substrate

Binding site

269

1

Substrate

Experimental info

Mutagenesis

12

1

C → S: Loss of activity. Ref.12 Ref.14

Mutagenesis

41

1

H → N: No effect on the activity. Ref.13

Mutagenesis

74

1

H → N: Reduces activity 2-fold. Ref.13

Mutagenesis

96

1

C → S: Loss of activity. Ref.12 Ref.14

Mutagenesis

99

1

V → A: No effect on the activity. Ref.13

Mutagenesis

124

1

H → N: Reduces activity 100-fold. Ref.13

Mutagenesis

126

1

E → D or Q: Reduces activity 100-fold. Ref.13 Ref.15

Mutagenesis

167

1

T → C: Reduces activity 3-fold. Ref.13 Ref.15

Mutagenesis

167

1

T → S: No effect on the activity. Ref.13 Ref.15

Mutagenesis

197

1

C → S: Loss of activity. Ref.12 Ref.14

Mutagenesis

225

1

S → C: Reduces activity 100-fold. Ref.13

Mutagenesis

227

1

N → Q: Reduces activity 20-fold. Ref.13

Secondary structure

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316

Helix Strand Turn

Details...

Beta strand

2 – 5

4

Helix

13 – 29

17

Beta strand

33 – 37

5

Beta strand

39 – 41

3

Helix

43 – 51

9

Beta strand

54 – 59

6

Helix

60 – 62

3

Beta strand

68 – 71

4

Helix

78 – 86

9

Beta strand

90 – 93

4

Helix

97 – 112

16

Beta strand

115 – 120

6

Helix

125 – 131

7

Beta strand

137 – 139

3

Beta strand

141 – 144

4

Helix

147 – 152

6

Beta strand

160 – 165

6

Beta strand

167 – 169

3

Helix

171 – 184

14

Beta strand

191 – 193

3

Helix

198 – 213

16

Beta strand

215 – 220

6

Helix

226 – 237

12

Beta strand

241 – 247

7

Helix

248 – 250

3

Helix

253 – 255

3

Turn

256 – 258

3

Beta strand

260 – 266

7

Helix

272 – 284

13

Beta strand

289 – 292

4

Beta strand

300 – 302

3

Helix

306 – 308

3

Sequences

	Sequence		Length	Mass (Da)
	P62623 [UniParc]. Last modified July 19, 2004. Version 1. Checksum: 0E7B378BD49AB771	FASTA	316	34,775
	10 20 30 40 50 60 MQILLANPRG FCAGVDRAIS IVENALAIYG APIYVRHEVV HNRYVVDSLR ERGAIFIEQI 70 80 90 100 110 120 SEVPDGAILI FSAHGVSQAV RNEAKSRDLT VFDATCPLVT KVHMEVARAS RRGEESILIG 130 140 150 160 170 180 HAGHPEVEGT MGQYSNPEGG MYLVESPDDV WKLTVKNEEK LSFMTQTTLS VDDTSDVIDA 190 200 210 220 230 240 LRKRFPKIVG PRKDDICYAT TNRQEAVRAL AEQAEVVLVV GSKNSSNSNR LAELAQRMGK 250 260 270 280 290 300 RAFLIDDAKD IQEEWVKEVK CVGVTAGASA PDILVQNVVA RLQQLGGGEA IPLEGREENI 310 VFEVPKELRV DIREVD

References

	« Hide 'large scale' referencesShow 'large scale' references »
[1]	"Studies on the nonmevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein." Rohdich F., Hecht S., Gaertner K., Adam P., Krieger C., Amslinger S., Arigoni D., Bacher A., Eisenreich W. Proc. Natl. Acad. Sci. U.S.A. 99:1158-1163(2002) [PubMed] [Europe PMC] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], CHARACTERIZATION.
[2]	"Nucleotide sequence of the lsp-dapB interval in Escherichia coli." Bouvier J., Stragier P. Nucleic Acids Res. 19:180-180(1991) [PubMed] [Europe PMC] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA]. Strain: K12.
[3]	"Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region." Yura T., Mori H., Nagai H., Nagata T., Ishihama A., Fujita N., Isono K., Mizobuchi K., Nakata A. Nucleic Acids Res. 20:3305-3308(1992) [PubMed] [Europe PMC] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. Strain: K12.
[4]	"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] [Europe PMC] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. Strain: K12 / MG1655 / ATCC 47076.
[5]	"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] [Europe PMC] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. Strain: K12 / W3110 / ATCC 27325 / DSM 5911.
[6]	"Identification of the Escherichia coli lytB gene, which is involved in penicillin tolerance and control of the stringent response." Gustafson C.E., Kaul S., Ishiguro E.E. J. Bacteriol. 175:1203-1205(1993) [PubMed] [Europe PMC] [Abstract] Cited for: IDENTIFICATION.
[7]	"LytB, a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli." Altincicek B., Kollas A.-K., Eberl M., Wiesner J., Sanderbrand S., Hintz M., Beck E., Jomaa H. FEBS Lett. 499:37-40(2001) [PubMed] [Europe PMC] [Abstract] Cited for: PATHWAY. Strain: K12 / ATCC 23716 / DSM 498.
[8]	"The lytB gene of Escherichia coli is essential and specifies a product needed for isoprenoid biosynthesis." McAteer S., Coulson A., McLennan N., Masters M. J. Bacteriol. 183:7403-7407(2001) [PubMed] [Europe PMC] [Abstract] Cited for: PATHWAY.
[9]	"Biosynthesis of terpenes: studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase." Adam P., Hecht S., Eisenreich W., Kaiser J., Graewert T., Arigoni D., Bacher A., Rohdich F. Proc. Natl. Acad. Sci. U.S.A. 99:12108-12113(2002) [PubMed] [Europe PMC] [Abstract] Cited for: CATALYTIC ACTIVITY.
[10]	"Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe-4S] protein." Wolff M., Seemann M., Tse Sum Bui B., Frapart Y., Tritsch D., Garcia Estrabot A., Rodriguez-Concepcion M., Boronat A., Marquet A., Rohmer M. FEBS Lett. 541:115-120(2003) [PubMed] [Europe PMC] [Abstract] Cited for: IRON-SULFUR CLUSTER, SUBUNIT.
[11]	"The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein." Rohdich F., Zepeck F., Adam P., Hecht S., Kaiser J., Laupitz R., Graewert T., Amslinger S., Eisenreich W., Bacher A., Arigoni D. Proc. Natl. Acad. Sci. U.S.A. 100:1586-1591(2003) [PubMed] [Europe PMC] [Abstract] Cited for: CATALYTIC ACTIVITY.
[12]	"IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis." Graewert T., Kaiser J., Zepeck F., Laupitz R., Hecht S., Amslinger S., Schramek N., Schleicher E., Weber S., Haslbeck M., Buchner J., Rieder C., Arigoni D., Bacher A., Eisenreich W., Rohdich F. J. Am. Chem. Soc. 126:12847-12855(2004) [PubMed] [Europe PMC] [Abstract] Cited for: PRESENCE OF A 3FE-4S IRON-SULFUR CLUSTER, ENZYME REGULATION, KINETIC PARAMETERS, MUTAGENESIS OF CYS-12; CYS-96 AND CYS-197.
[13]	"Structure of active IspH enzyme from Escherichia coli provides mechanistic insights into substrate reduction." Graewert T., Rohdich F., Span I., Bacher A., Eisenreich W., Eppinger J., Groll M. Angew. Chem. Int. Ed. Engl. 48:5756-5759(2009) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.80 ANGSTROMS) IN COMPLEX WITH IRON-SULFUR (3FE-4S), FUNCTION, COFACTOR, SUBUNIT, MUTAGENESIS OF HIS-41; HIS-74; VAL-99; HIS-124; GLU-126; THR-167; SER-225 AND ASN-227.
[14]	"Probing the reaction mechanism of IspH protein by x-ray structure analysis." Graewert T., Span I., Eisenreich W., Rohdich F., Eppinger J., Bacher A., Groll M. Proc. Natl. Acad. Sci. U.S.A. 107:1077-1081(2010) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.70 ANGSTROMS) IN COMPLEX WITH SUBSTRATE AND IRON-SULFUR (3FE-4S), FUNCTION, COFACTOR, SUBUNIT, MUTAGENESIS OF CYS-12; CYS-96 AND CYS-197.
[15]	"Crystal structures of mutant IspH proteins reveal a rotation of the substrate's hydroxymethyl group during catalysis." Span I., Graewert T., Bacher A., Eisenreich W., Groll M. J. Mol. Biol. 416:1-9(2012) [PubMed] [Europe PMC] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (1.40 ANGSTROMS) IN COMPLEX WITH SUBSTRATE AND IRON-SULFUR (4FE-4S), FUNCTION, COFACTOR, SUBUNIT, MUTAGENESIS OF GLU-126 AND THR-167.
+	Additional computationally mapped references.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ

AY062212 Genomic DNA. Translation: AAL38655.1.
X54945 Genomic DNA. Translation: CAA38707.1.
U00096 Genomic DNA. Translation: AAC73140.1.
AP009048 Genomic DNA. Translation: BAB96598.1.

PIR

JE0403.

RefSeq

NP_414570.1. NC_000913.2.
YP_488335.1. NC_007779.1.

3D structure databases

PDBe
RCSB PDB
PDBj

Entry	Method	Resolution (Å)	Chain	Positions	PDBsum
3F7T	X-ray	1.80	A/B	1-316	[»]
3KE8	X-ray	1.70	A/B	1-316	[»]
3KE9	X-ray	1.90	A/B	1-316	[»]
3KEF	X-ray	1.70	A/B	1-316	[»]
3KEL	X-ray	1.80	A/B	1-316	[»]
3KEM	X-ray	2.00	A/B	1-316	[»]
3SZL	X-ray	1.60	A/B	1-316	[»]
3SZO	X-ray	1.60	A/B	1-316	[»]
3SZU	X-ray	1.40	A/B	1-316	[»]
3T0F	X-ray	1.90	A/B	1-316	[»]
3T0G	X-ray	2.10	A/B	1-316	[»]
3URK	X-ray	1.50	A/B	1-316	[»]
3UTC	X-ray	1.90	A/B	1-316	[»]
3UTD	X-ray	1.70	A/B	1-316	[»]
3UV3	X-ray	1.60	A/B	1-316	[»]
3UV6	X-ray	1.70	A/B	1-316	[»]
3UV7	X-ray	1.60	A/B	1-316	[»]
3UWM	X-ray	1.80	A/B	1-316	[»]
3ZGL	X-ray	1.68	A/B	1-316	[»]
3ZGN	X-ray	1.95	A/B	1-316	[»]
4EB3	X-ray	1.90	A/B	1-315	[»]
4H4C	X-ray	1.80	A/B	1-315	[»]
4H4D	X-ray	1.35	A/B	1-315	[»]
4H4E	X-ray	1.70	A/B	1-315	[»]

ProteinModelPortal

P62623.

SMR

P62623. Positions 1-310.

ModBase

Search...

Protein-protein interaction databases

DIP

DIP-35808N.

IntAct

P62623. 5 interactions.

STRING

511145.b0029.

Proteomic databases

PaxDb

P62623.

PRIDE

P62623.

Protocols and materials databases

StructuralBiologyKnowledgebase

Search...

Genome annotation databases

EnsemblBacteria

AAC73140; AAC73140; b0029.
BAB96598; BAB96598; BAB96598.

GeneID

12933923.
944777.

KEGG

ecj:Y75_p0029.
eco:b0029.

PATRIC

32115149. VBIEscCol129921_0026.

Organism-specific databases

EchoBASE

EB1073.

EcoGene

EG11081. ispH.

Phylogenomic databases

eggNOG

COG0761.

HOGENOM

HOG000220192.

KO

K03527.

OMA

QDAMFSL.

ProtClustDB

PRK01045.

Enzyme and pathway databases

BioCyc

EcoCyc:EG11081-MONOMER.
ECOL316407:JW0027-MONOMER.
MetaCyc:EG11081-MONOMER.

BRENDA

1.17.1.2. 2026.

UniPathway

UPA00056; UER00097.
UPA00059; UER00105.

Gene expression databases

Genevestigator

P62623.

Family and domain databases

HAMAP

MF_00191. IspH.

InterPro

IPR003451. LytB.
[Graphical view]

Pfam

PF02401. LYTB. 1 hit.
[Graphical view]

TIGRFAMs

TIGR00216. ispH_lytB. 1 hit.

ProtoNet

Search...

Other

EvolutionaryTrace

P62623.

Entry information

Entry name

ISPH_ECOLI

Accession

Primary (citable) accession number: P62623
Secondary accession number(s): P22565

Entry history

Integrated into UniProtKB/Swiss-Prot:	July 19, 2004
Last sequence update:	July 19, 2004
Last modified:	May 1, 2013
This is version 83 of the entry and version 1 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation program

Prokaryotic Protein Annotation Program