Escherichia coli K-12 substr. MG1655 Enzyme: 3-isopropylmalate dehydrogenase

Gene: leuB Accession Numbers: EG11577 (EcoCyc), b0073, ECK0075

Regulation Summary Diagram: ?

Regulation summary diagram for leuB

Subunit composition of 3-isopropylmalate dehydrogenase = [LeuB]2

3-isopropylmalate dehydrogenase (LeuB) carries out the third step in leucine biosynthesis, catalyzing the conversion of 3-isopropylmalate to 2-isopropyl-3-oxosuccinate. LeuB oxidizes 3-isopropylmalate with NAD+ to generate 2-isopropyl-3-oxosuccinate and NADH [Wallon97a, Yang74]. The activity is dependent on divalent cations, Mg2+ and Mn2+, with Mn2+ being the preferred cation [Wallon97a].

A crystal structure of 3-isopropylmalate dehydrogenase to 2.1 Å resolution reveals that it is a dimer. [Wallon97a, Wallon97]. The monomers are divided into two domains. The refolding mechanism of the homodimer was investigated by monitoring a complex time course of refolding [Graczer09].

A single amino acid repacement at glutamine 200 increased thermostability by creating an intersubunit ion pair which can join existing ion clusters [Nemeth00].

The leucine operon consists of four structural genes (leuABCD) coding for the three enzymes which are specific for leucine biosynthesis. The leuB gene codes for isopropylmalate dehydrogenase [Somers73].

In auxotrophs, starvation for leucine triggers derepression of the leu operon and increased rates of leuB mRNA transcription [Wright99].

Gene Citations: [Stratmann12]

Locations: cytosol

Map Position: [80,867 <- 81,958] (1.74 centisomes, 6°)
Length: 1092 bp / 363 aa

Molecular Weight of Polypeptide: 39.517 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0000265 , CGSC:571 , EchoBASE:EB1537 , EcoGene:EG11577 , EcoliWiki:b0073 , ModBase:P30125 , OU-Microarray:b0073 , PortEco:leuB , PR:PRO_000023082 , Pride:P30125 , Protein Model Portal:P30125 , RefSeq:NP_414615 , RegulonDB:EG11577 , SMR:P30125 , String:511145.b0073 , UniProt:P30125

Relationship Links: InterPro:IN-FAMILY:IPR001804 , InterPro:IN-FAMILY:IPR004429 , InterPro:IN-FAMILY:IPR019818 , InterPro:IN-FAMILY:IPR024084 , Panther:IN-FAMILY:PTHR11835 , PDB:Structure:1CM7 , Pfam:IN-FAMILY:PF00180 , Prosite:IN-FAMILY:PS00470

In Paralogous Gene Group: 28 (3 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for leuB

GO Terms:

Biological Process: GO:0009098 - leucine biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11, GOA06, GOA01, Somers73, Wallon97a]
GO:0034198 - cellular response to amino acid starvation Inferred from experiment [Wright99, Reimers04]
GO:0008652 - cellular amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11]
GO:0009082 - branched-chain amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment Inferred by computational analysis [GOA01, Wallon97a]
GO:0003862 - 3-isopropylmalate dehydrogenase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Wallon97a]
GO:0030145 - manganese ion binding Inferred from experiment [Wallon97a]
GO:0046872 - metal ion binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, Wallon97a]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11]
GO:0016616 - oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor Inferred by computational analysis [GOA01]
GO:0051287 - NAD binding Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, Wallon97a]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, GOA06, GOA01]

MultiFun Terms: metabolism biosynthesis of building blocks amino acids leucine

Essentiality data for leuB knockouts: ?

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 0.4% glucose No 37 Aerobic 7.2 0.27 No [Patrick07, Comment 3]
M9 medium with 1% glycerol No 37 Aerobic 7.2 0.35 No [Joyce06]
MOPS medium with 0.4% glucose Indeterminate 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
No [Feist07, Comment 4]

Curated 13-Sep-2006 by Shearer A , SRI International
Last-Curated ? 26-Sep-2013 by Kubo A , SRI International

Enzymatic reaction of: 3-isopropylmalate dehydrogenase

Synonyms: β-IPM dehydrogenase, IMDH, 3-carboxy-2-hydroxy-4-methylpentanoate: NAD+ oxidoreductase

(2R,3S)-3-isopropylmalate + NAD+ <=> (2S)-2-isopropyl-3-oxosuccinate + NADH + 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.

In Pathways: superpathway of branched amino acid biosynthesis , L-leucine biosynthesis

The kMs listed below were determined at 40° C, as was the kcat value of 69/s [Wallon97a].

Cofactors or Prosthetic Groups: Mg2+ [Wallon97a], Mn2+ [Wallon97a]

Kinetic Parameters:

Km (μM)
[Wallon97a, BRENDA14]

T(opt): 70 °C [BRENDA14, Wallon97a]

pH(opt): 7.6 [BRENDA14, Wallon97a]

Sequence Features

Protein sequence of LeuB with features indicated

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Link97, UniProt11]
UniProt: Removed.
Chain 2 -> 363
UniProt: 3-isopropylmalate dehydrogenase;
Nucleotide-Phosphate-Binding-Region 78 -> 91
UniProt: NAD; Non-Experimental Qualifier: by similarity;
Conserved-Region 90 -> 100
substrate recognition region
Amino-Acid-Sites-That-Bind 99
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 109
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 138
UniProt: Substrate; Non-Experimental Qualifier: by similarity;
Amino-Acid-Site 145
UniProt: Important for catalysis; Sequence Annotation Type: site; Non-Experimental Qualifier: by similarity;
Amino-Acid-Site 195
UniProt: Important for catalysis; Sequence Annotation Type: site; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 227
UniProt: Magnesium or manganese; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 251
UniProt: Magnesium or manganese; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 255
UniProt: Magnesium or manganese; Non-Experimental Qualifier: by similarity;
Nucleotide-Phosphate-Binding-Region 285 -> 297
UniProt: NAD; Non-Experimental Qualifier: by similarity;

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram


Peter D. Karp on Wed Jan 18, 2006:
Gene right-end position adjusted based on analysis performed in the 2005 E. coli annotation update [Riley06 ].
10/20/97 Gene b0073 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11577; confirmed by SwissProt match.


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."

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

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

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

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."

Graczer09: Graczer E, Varga A, Melnik B, Semisotnov G, Zavodszky P, Vas M (2009). "Symmetrical refolding of protein domains and subunits: example of the dimeric two-domain 3-isopropylmalate dehydrogenases." Biochemistry 48(5);1123-34. PMID: 19154118

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

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

Link97: Link AJ, Robison K, Church GM (1997). "Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12." Electrophoresis 18(8);1259-313. PMID: 9298646

Nemeth00: Nemeth A, Svingor A, Pocsik M, Dobo J, Magyar C, Szilagyi A, Gal P, Zavodszky P (2000). "Mirror image mutations reveal the significance of an intersubunit ion cluster in the stability of 3-isopropylmalate dehydrogenase." FEBS Lett 468(1);48-52. PMID: 10683439

Patrick07: Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007). "Multicopy suppression underpins metabolic evolvability." Mol Biol Evol 24(12);2716-22. PMID: 17884825

Reimers04: Reimers JM, Schmidt KH, Longacre A, Reschke DK, Wright BE (2004). "Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs." Microbiology 150(Pt 5);1457-66. PMID: 15133107

Riley06: Riley M, Abe T, Arnaud MB, Berlyn MK, Blattner FR, Chaudhuri RR, Glasner JD, Horiuchi T, Keseler IM, Kosuge T, Mori H, Perna NT, Plunkett G, Rudd KE, Serres MH, Thomas GH, Thomson NR, Wishart D, Wanner BL (2006). "Escherichia coli K-12: a cooperatively developed annotation snapshot--2005." Nucleic Acids Res 34(1);1-9. PMID: 16397293

Somers73: Somers JM, Amzallag A, Middleton RB (1973). "Genetic fine structure of the leucine operon of Escherichia coli K-12." J Bacteriol 113(3);1268-72. PMID: 4570778

Stratmann12: Stratmann T, Pul Ü, Wurm R, Wagner R, Schnetz K (2012). "RcsB-BglJ activates the Escherichia coli leuO gene, encoding an H-NS antagonist and pleiotropic regulator of virulence determinants." Mol Microbiol 83(6);1109-23. PMID: 22295907

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11: 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."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

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

Vorobieva14: Vorobieva AA, Khan MS, Soumillion P (2014). "Escherichia coli D-Malate Dehydrogenase: a Generalist Enzyme Active in the Leucine Biosynthesis Pathway." J Biol Chem. PMID: 25160617

Wallon97: Wallon G, Kryger G, Lovett ST, Oshima T, Ringe D, Petsko GA (1997). "Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus." J Mol Biol 266(5);1016-31. PMID: 9086278

Wallon97a: Wallon G, Yamamoto K, Kirino H, Yamagishi A, Lovett ST, Petsko GA, Oshima T (1997). "Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli." Biochim Biophys Acta 1337(1);105-12. PMID: 9003442

Wright99: Wright BE, Longacre A, Reimers JM (1999). "Hypermutation in derepressed operons of Escherichia coli K12." Proc Natl Acad Sci U S A 96(9);5089-94. PMID: 10220423

Yang74: Yang HL, Kessler DP (1974). "Genetic analysis of the leucine region in Escherichia coli B-r: gene-enzyme assignments." J Bacteriol 117(1);63-72. PMID: 4587614

Other References Related to Gene Regulation

Chen01: Chen CC, Fang M, Majumder A, Wu HY (2001). "A 72-base pair AT-rich DNA sequence element functions as a bacterial gene silencer." J Biol Chem 276(12);9478-85. PMID: 11121424

Gemmill83: Gemmill RM, Jones JW, Haughn GW, Calvo JM (1983). "Transcription initiation sites of the leucine operons of Salmonella typhimurium and Escherichia coli." J Mol Biol 170(1);39-59. PMID: 6195343

Landgraf99: Landgraf JR, Boxer JA, Calvo JM (1999). "Escherichia coli Lrp (leucine-responsive regulatory protein) does not directly regulate expression of the leu operon promoter." J Bacteriol 181(20);6547-51. PMID: 10515950

Lin92: Lin R, D'Ari R, Newman EB (1992). "Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine." J Bacteriol 1992;174(6);1948-55. PMID: 1532173

Vartak91: Vartak NB, Liu L, Wang BM, Berg CM (1991). "A functional leuABCD operon is required for leucine synthesis by the tyrosine-repressible transaminase in Escherichia coli K-12." J Bacteriol 173(12);3864-71. PMID: 1646790

Wessler81: Wessler SR, Calvo JM (1981). "Control of leu operon expression in Escherichia coli by a transcription attenuation mechanism." J Mol Biol 149(4);579-97. PMID: 6171647

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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 19.0 on Thu Oct 8, 2015, biocyc13.