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Escherichia coli K-12 substr. MG1655 Enzyme: 1-deoxy-D-xylulose 5-phosphate reductoisomerase



Gene: dxr Accession Numbers: EG12715 (EcoCyc), b0173, ECK0172

Synonyms: yaeM, ispC

Regulation Summary Diagram: ?

Subunit composition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase = [Dxr]2

Summary:
1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is involved in the first committed step in the methylerythritol phosphate pathway of isoprenoid biosynthesis. Dxr catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into the dedicated MEP pathway intermediate 2-C-methyl-D-erythritol-4-phosphate (MEP). This reaction is NADPH-dependent, and required a bivalent metal cofactor [Takahashi98]. This is a two-step reaction. The first step is a non-reductive, NADPH-dependent rearrangement that generates MEP. This is followed by an NADPH-dependent reduction of this intermediate to generate the final product [Hoeffler02].

This reaction appears to depend on one or more of Dxr's histidines [Kuzuyama00]. Based on the stereospecificity of the reaction, Dxr is a class B dehydrogenase [Radykewicz00]. Different mechanisms for the DXR catalyzed reaction have been proposed and investigated [Li13a, Fox05a, Munos09].

A number of structures have been determined for Dxr. A crystal structure of the dimer to 2.5 Å resolution shows that the Dxr connective domain is responsible for dimerization and contains the bulk of the enzyme's active site [Reuter02]. A crystal structure of Dxr complexed with NADPH and a sulfur ion to 2.2 Å resolution shows that a flexible loop covers the active site [Yajima02]. Crystal structures of Dxr both unbound and bound to the inhibitor and antimalarial drug fosmidomycin show that drug binding causes a substantial conformational change [Steinbacher03, Mac05]. Crystal structures of Dxr bound to bisphonates indicate that these compounds bind the same site as fosmidomycin [Yajima04].

dxr deficient mutants required 2-C-methylerythritol, a free alcohol for MEP, for growth and survival [Takahashi98]. Analysis of dxr-deficient mutants identified Glu231 to play an important role in the conversion of DXP to MEP [Kuzuyama00].

Since the enzymes of the methylerythritol pathway are not found in humans, these enzymes, especially DXR, have attracted much interest for its potential as anti-infective drug targets. New classes of inhibitors have been extensively studied. [NguyenTrung13, Cai12, Zingle12, Jackson12] Both computational and high-throughput experimental methods have been used to attempt to identify inhibitors of Dxr [Gottlin03, Cheng04a, Merckle05].

Dxr is required for cell survival [Kuzuyama99].

Locations: cytosol

Map Position: [193,521 -> 194,717] (4.17 centisomes)
Length: 1197 bp / 398 aa

Molecular Weight of Polypeptide: 43.388 kD (from nucleotide sequence), 42.0 kD (experimental) [Takahashi98 ]

Unification Links: ASAP:ABE-0000592 , DIP:DIP-9484N , EchoBASE:EB2575 , EcoGene:EG12715 , EcoliWiki:b0173 , ModBase:P45568 , OU-Microarray:b0173 , PortEco:dxr , PR:PRO_000022494 , Pride:P45568 , Protein Model Portal:P45568 , RefSeq:NP_414715 , RegulonDB:EG12715 , SMR:P45568 , String:511145.b0173 , UniProt:P45568

Relationship Links: InterPro:IN-FAMILY:IPR003821 , InterPro:IN-FAMILY:IPR013512 , InterPro:IN-FAMILY:IPR013644 , InterPro:IN-FAMILY:IPR016040 , InterPro:IN-FAMILY:IPR026877 , Panther:IN-FAMILY:PTHR30525 , PDB:Structure:1JVS , PDB:Structure:1K5H , PDB:Structure:1ONN , PDB:Structure:1ONO , PDB:Structure:1ONP , PDB:Structure:1Q0H , PDB:Structure:1Q0L , PDB:Structure:1Q0Q , PDB:Structure:1T1R , PDB:Structure:1T1S , PDB:Structure:2EGH , PDB:Structure:3ANL , PDB:Structure:3ANM , PDB:Structure:3ANN , PDB:Structure:3R0I , Pfam:IN-FAMILY:PF02670 , Pfam:IN-FAMILY:PF08436 , Pfam:IN-FAMILY:PF13288

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006744 - ubiquinone biosynthetic process Inferred from experiment [Kuzuyama00]
GO:0008299 - isoprenoid biosynthetic process Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0016114 - terpenoid biosynthetic process Inferred by computational analysis [GOA06]
GO:0019288 - isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway Inferred by computational analysis [UniProtGOA12]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0030145 - manganese ion binding Inferred from experiment [Takahashi98]
GO:0030604 - 1-deoxy-D-xylulose-5-phosphate reductoisomerase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Kuzuyama00]
GO:0042802 - identical protein binding Inferred from experiment [Takahashi98]
GO:0046872 - metal ion binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Takahashi98]
GO:0070402 - NADPH binding Inferred from experiment Inferred by computational analysis [GOA01, Takahashi98]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

MultiFun Terms: metabolism biosynthesis of building blocks cofactors, small molecule carriers menaquinone, ubiquinone

Essentiality data for dxr knockouts: ?

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

Credits:
Curated 27-Apr-2007 by Shearer A , SRI International
Last-Curated ? 16-Aug-2013 by Kubo A , SRI International


Enzymatic reaction of: 1-deoxy-D-xylulose 5-phosphate reductoisomerase

Synonyms: 2C-methyl-D-erythritol 4-phosphate synthase, MEP synthase, DXP reductoisomerase

EC Number: 1.1.1.267

2-C-methyl-D-erythritol 4-phosphate + NADP+ <=> 1-deoxy-D-xylulose 5-phosphate + NADPH + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

This reaction is reversible. [Hoeffler02]

In Pathways: methylerythritol phosphate pathway I

Summary:
Quite a few kinetic values have been determined for Dxr, at various pH values and with varying cofactors. One analysis gives Km values of 250 µM for 1-deoxy-D-xylylose 5-phosphate (DXP) and 7.4 µM for NADPH when Mn2+ is the cofactor. When Mg2+ is the cofactor, these values are 99 µM and 18 µM, respectively. When Co2+ is the cofactor, these values are 60 µM and 8.8 µM, respectively [Kuzuyama00]. At pH 7.8, the Km for DXP also varies by cofactor, with values of 17 µM for Mn2+, 210 µM for Mg2+, and 2 µM for Co2+. At pH 6.0, the values are 520 µM for Mn2+ and 17mM for Co2+ [Mac05]. Under saturating NADPH conditions, a Km of 54 µM for DXP is reported [Fox05a].

Although this reaction is reversible, the production of 2-C-methyl-D-erythritol-4-phosphate is heavily favored in vivo [Hoeffler02].

Cofactors or Prosthetic Groups: NADPH [Kuzuyama00], Mn2+ [Mac05, Kuzuyama00, Takahashi98]

Alternative Cofactors for Mn2+: Co2+ , Mg2+

Inhibitors (Unknown Mechanism): fosmidomycin [Kuzuyama00, NguyenTrung13]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
NADPH
0.5
[Fox05a, BRENDA14]
NADPH
7.0, 9.0, 20.0, 7.4
[Kuzuyama00, BRENDA14]
1-deoxy-D-xylulose 5-phosphate
3.1
[Wong07, BRENDA14]
1-deoxy-D-xylulose 5-phosphate
60.0, 99.0, 250.0
[Kuzuyama00, BRENDA14]
1-deoxy-D-xylulose 5-phosphate
61.0
[Wong04a, BRENDA14]
1-deoxy-D-xylulose 5-phosphate
73.0, 97.0
[Hoeffler02, BRENDA14]
1-deoxy-D-xylulose 5-phosphate
17.0
[Mac05]
1-deoxy-D-xylulose 5-phosphate
45.0
29.0
[Fox05a, BRENDA14]
1-deoxy-D-xylulose 5-phosphate
81.0
33.0
[Walker05, BRENDA14]
1-deoxy-D-xylulose 5-phosphate
50.0
38.0
[Fox05, BRENDA14]

T(opt): 50 °C [BRENDA14, Kuzuyama00]

pH(opt): 7 [BRENDA14, Kuzuyama00], 7.5 [BRENDA14, Takahashi98], 7-8.5 [Kuzuyama00]


Sequence Features

Feature Class Location Citations Comment
Nucleotide-Phosphate-Binding-Region 7 -> 36
[UniProt10]
UniProt: NADP;
Mutagenesis-Variant 14
[Kuzuyama00, UniProt11a]
Alternate sequence: G → D; UniProt: Loss of solubility and activity.
Amino-Acid-Sites-That-Bind 125
[UniProt10]
UniProt: Substrate;
Metal-Binding-Site 150
[UniProt10]
UniProt: Divalent metal cation;
Metal-Binding-Site 152
[UniProt10]
UniProt: Divalent metal cation;
Mutagenesis-Variant 153
[Kuzuyama00, UniProt11a]
Alternate sequence: H → Q; UniProt: Increase in KM for substrate. Reduces activity 35-fold.
Amino-Acid-Sites-That-Bind 186
[UniProt10]
UniProt: Substrate;
Mutagenesis-Variant 209
[Kuzuyama00, UniProt11a]
Alternate sequence: H → Q; UniProt: Increase in KM for substrate. Reduces activity 5000-fold.
Amino-Acid-Sites-That-Bind 209
[UniProt10]
UniProt: Substrate;
Protein-Segment 222 -> 228
[UniProt10a]
UniProt: Binding to substrate phosphate group; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 231
[Kuzuyama00, UniProt11a]
Alternate sequence: E → K; UniProt: No effect on KM for substrate. Reduces activity by over 99.9%.
Metal-Binding-Site 231
[UniProt10]
UniProt: Divalent metal cation;
Mutagenesis-Variant 257
[Kuzuyama00, UniProt11a]
Alternate sequence: H → Q; UniProt: Strong increase in KM for substrate. Loss of activity.


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b0173 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG12715; confirmed by SwissProt match.


References

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

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

Cai12: Cai G, Deng L, Fryszczyn BG, Brown NG, Liu Z, Jiang H, Palzkill T, Song Y (2012). "Thermodynamic Investigation of Inhibitor Binding to 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase." ACS Med Chem Lett 3(6);496-500. PMID: 23050057

Cheng04a: Cheng F, Oldfield E (2004). "Inhibition of isoprene biosynthesis pathway enzymes by phosphonates, bisphosphonates, and diphosphates." J Med Chem 47(21);5149-58. PMID: 15456258

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

Fox05: Fox DT, Poulter CD (2005). "Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphoric acid analogues as alternate substrates for methylerythritol phosphate synthase." J Org Chem 70(6);1978-85. PMID: 15760175

Fox05a: Fox DT, Poulter CD (2005). "Mechanistic studies with 2-C-methyl-D-erythritol 4-phosphate synthase from Escherichia coli." Biochemistry 44(23);8360-8. PMID: 15938625

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Gottlin03: Gottlin EB, Benson RE, Conary S, Antonio B, Duke K, Payne ES, Ashraf SS, Christensen DJ (2003). "High-throughput screen for inhibitors of 1-deoxy-d-xylulose 5-phosphate reductoisomerase by surrogate ligand competition." J Biomol Screen 8(3);332-9. PMID: 12857387

Hoeffler02: Hoeffler JF, Tritsch D, Grosdemange-Billiard C, Rohmer M (2002). "Isoprenoid biosynthesis via the methylerythritol phosphate pathway. Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase." Eur J Biochem 269(18);4446-57. PMID: 12230556

Jackson12: Jackson ER, Dowd CS (2012). "Inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr): a review of the synthesis and biological evaluation of recent inhibitors." Curr Top Med Chem 12(7);706-28. PMID: 22283814

Kuzuyama00: Kuzuyama T, Takahashi S, Takagi M, Seto H (2000). "Characterization of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues." J Biol Chem 2000;275(26);19928-32. PMID: 10787409

Kuzuyama99: Kuzuyama T, Takahashi S, Seto H (1999). "Construction and characterization of Escherichia coli disruptants defective in the yaeM gene." Biosci Biotechnol Biochem 63(4);776-8. PMID: 10361694

Li13a: Li H, Tian J, Sun W, Qin W, Gao WY (2013). "Mechanistic insights into 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a key enzyme of the MEP terpenoid biosynthetic pathway." FEBS J. PMID: 24010408

Mac05: Mac Sweeney A, Lange R, Fernandes RP, Schulz H, Dale GE, Douangamath A, Proteau PJ, Oefner C (2005). "The crystal structure of E.coli 1-deoxy-D-xylulose-5-phosphate reductoisomerase in a ternary complex with the antimalarial compound fosmidomycin and NADPH reveals a tight-binding closed enzyme conformation." J Mol Biol 345(1);115-27. PMID: 15567415

Merckle05: Merckle L, de Andres-Gomez A, Dick B, Cox RJ, Godfrey CR (2005). "A fragment-based approach to understanding inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase." Chembiochem 6(10);1866-74. PMID: 16116659

Munos09: Munos JW, Pu X, Mansoorabadi SO, Kim HJ, Liu HW (2009). "A secondary kinetic isotope effect study of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase-catalyzed reaction: evidence for a retroaldol-aldol rearrangement." J Am Chem Soc 131(6);2048-9. PMID: 19159292

NguyenTrung13: Nguyen-Trung AT, Tritsch D, Grosdemange-Billiard C, Rohmer M (2013). "Synthesis of tetrazole analogues of phosphonohydroxamic acids: an attempt to improve the inhibitory activity against the DXR." Bioorg Med Chem Lett 23(6);1643-7. PMID: 23414808

Radykewicz00: Radykewicz T, Rohdich F, Wungsintaweekul J, Herz S, Kis K, Eisenreich W, Bacher A, Zenk MH, Arigoni D (2000). "Biosynthesis of terpenoids: 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Escherichia coli is a class B dehydrogenase." FEBS Lett 2000;465(2-3);157-60. PMID: 10631325

Reuter02: Reuter K, Sanderbrand S, Jomaa H, Wiesner J, Steinbrecher I, Beck E, Hintz M, Klebe G, Stubbs MT (2002). "Crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, a crucial enzyme in the non-mevalonate pathway of isoprenoid biosynthesis." J Biol Chem 277(7);5378-84. PMID: 11741911

Steinbacher03: Steinbacher S, Kaiser J, Eisenreich W, Huber R, Bacher A, Rohdich F (2003). "Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC). Implications for the catalytic mechanism and anti-malaria drug development." J Biol Chem 278(20);18401-7. PMID: 12621040

Takahashi98: Takahashi S, Kuzuyama T, Watanabe H, Seto H (1998). "A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis." Proc Natl Acad Sci U S A 1998;95(17);9879-84. PMID: 9707569

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 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 manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Walker05: Walker JR, Poulter CD (2005). "Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphate analogues as chelation-based inhibitors of methylerythritol phosphate synthase." J Org Chem 70(24);9955-9. PMID: 16292827

Wong04a: Wong A, Munos JW, Devasthali V, Johnson KA, Liu HW (2004). "Study of 1-deoxy-D-xylulose-5-phosphate reductoisomerase: synthesis and evaluation of fluorinated substrate analogues." Org Lett 6(20);3625-8. PMID: 15387564

Wong07: Wong U, Cox RJ (2007). "The chemical mechanism of D-1-deoxyxylulose-5-phosphate reductoisomerase from Escherichia coli." Angew Chem Int Ed Engl 46(26);4926-9. PMID: 17516600

Yajima02: Yajima S, Nonaka T, Kuzuyama T, Seto H, Ohsawa K (2002). "Crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase complexed with cofactors: implications of a flexible loop movement upon substrate binding." J Biochem (Tokyo) 131(3);313-7. PMID: 11872159

Yajima04: Yajima S, Hara K, Sanders JM, Yin F, Ohsawa K, Wiesner J, Jomaa H, Oldfield E (2004). "Crystallographic structures of two bisphosphonate:1-deoxyxylulose-5-phosphate reductoisomerase complexes." J Am Chem Soc 126(35);10824-5. PMID: 15339150

Zingle12: Zingle C, Kuntz L, Tritsch D, Grosdemange-Billiard C, Rohmer M (2012). "Modifications around the hydroxamic acid chelating group of fosmidomycin, an inhibitor of the metalloenzyme 1-deoxyxylulose 5-phosphate reductoisomerase (DXR)." Bioorg Med Chem Lett 22(21);6563-7. PMID: 23025997


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Thu Nov 27, 2014, BIOCYC13A.