UniProt UniProt http://www.uniprot.org Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits. Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits. The Biochemical journal 1997 imex curation Hill CM., Pang SS., Duggleby RG. 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined Hill CM., Pang SS., Duggleby RG. Subunit identities were a mixture of comparison with a tagged standard, edman digestion, reconstitution of activity and a variety of methods not shown but described in great detail. None are perfect, so I've put one interaction (for the fusion protein) with a list of participant detection methods. Coomassie stained gel is so clear I think 'direct interaction' is justified. Also added the native protein complex as also shows 2 bands on the gel and enzyme activity was measured. The assay rate was monitored either by the disappearance of pyruvate or a colourmetric assay measuring the conversion of acetolactate to acetoin. Accepted 2018-JUL-16 AT 08:31 BST AT 08:31 BST by LIVIAP The Biochemical journal 1997 Only protein-protein interactions imex curation imex curation I think the tag is OK "N-terminal oligohistidine tag that was used for purification " Determination of bait for native " These results suggest that it is through the small subunit that native enzyme binds to the IMAC column, although it contains no oligohistidine sequence." - feel free to change bait/prey to unspecified if you don't agree with their evidence for this. Mock publication and experiment for abstract interactions that are not interaction evidences. Mock publication and experiment for abstract interactions that are not interaction evidences. unspecified method Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits. Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits. The Biochemical journal 1997 imex curation Hill CM., Pang SS., Duggleby RG. 09-May-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down predetermined predetermined participant predetermined Hill CM., Pang SS., Duggleby RG. Subunit identities were a mixture of comparison with a tagged standard, edman digestion, reconstitution of activity and a variety of methods not shown but described in great detail. None are perfect, so I've put one interaction (for the fusion protein) with a list of participant detection methods. Coomassie stained gel is so clear I think 'direct interaction' is justified. Also added the native protein complex as also shows 2 bands on the gel and enzyme activity was measured. Accepted 2018-JUL-16 AT 08:31 BST AT 08:31 BST by LIVIAP The Biochemical journal 1997 Only protein-protein interactions imex curation imex curation I think the tag is OK "N-terminal oligohistidine tag that was used for purification " Determination of bait for native " These results suggest that it is through the small subunit that native enzyme binds to the IMAC column, although it contains no oligohistidine sequence." - feel free to change bait/prey to unspecified if you don't agree with their evidence for this. magnesium(2+) Magnesium(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) ilvm_ecoli Acetolactate synthase isozyme 2 small subunit Acetohydroxy-acid synthase II small subunit ALS-II ilvM b3769 JW3742 protein protein ecoli Escherichia coli (strain K12) MMQHQVNVSARFNPETLERVLRVVRHRGFHVCSMNMAAASDAQNINIELTVASPRSVDLLFSQLNKLVDVAHVAICQSTTTSQQIRA CEB6B5211262E0AF pyruvate pyruvate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) ilvg_ecoli Acetolactate synthase isozyme 2 large subunit Acetohydroxy-acid synthase II large subunit ALS-II ilvG protein protein ecoli Escherichia coli (strain K12) MNGAQWVVHALRAQGVNTVFGYPGGAIMPVYDALYDGGVEHLLCRHEQGAAMAAIGYARATGKTGVCIATSGPGATNLITGLADALLDSIPVVAITGQVSAPFIGTDAFQEVDVLGLSLACTKHSFLVQSLEELPRIMAEAFDVACSGRPGPVLVDIPKDIQLASGDLEPWFTTVENEVTFPHAEVEQARQMLAKAQKPMLYVGGGVGMAQAVPALREFLAATKMPATCTLKGLGAVEADYPYYLGMLGMHGTKAANFAVQECDLLIAVGARFDDRVTGKLNTFAPHASVIHMDIDPAEMNKLRQAHVALQGDLNALLPALQQPLNQYDWQQHCAQLRDEHSWRYDHPGDAIYAPLLLKQLSDRKPADCVVTTDVGQHQMWAAQHIAHTRPENFITSSGLGTMGFGLPAAVGAQVARPNDTVVCISGDGSFMMNVQELGTVKRKQLPLKIVLLDNQRLGMVRQWQQLFFQERYSETTLTDNPDFLMLASAFGIHGQHITRKDQVEAALDTMLNSDGPYLLHVSIDELENVWPLVPPGASNSEMLEKLS DIP protein P00892 original sequence version: null A002C07DAA6476DA thiamine(1+) diphosphate(3-) thiamine(1+) diphosphate(3-) thiamine diphosphate thiamine diphosphate dianion thiamine diphosphate(2-) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) AYEKOFBPNLCAJY-UHFFFAOYSA-L AYEKOFBPNLCAJY-UHFFFAOYSA-L fad(3-) FAD(3-) FAD FAD trianion small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) IMGVNJNCCGXBHD-UYBVJOGSSA-K IMGVNJNCCGXBHD-UYBVJOGSSA-K ilvg-ilvm-1 3 5 unspecified role unspecified role unspecified role unspecified role purified purified ecoli Escherichia coli (strain K12) 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie edman degradation edman degradation predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 1, Table 1 ilvg-ilvm-2 3 5 unspecified role unspecified role unspecified role unspecified role purified purified ecoli Escherichia coli (strain K12) 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 1, Table 2 The native protein complex appeared to bind to the IMAC column via the small subunit ahas_ii_ecoli-pyruvate-1 1 6 enzyme target enzyme target substrate substrate unspecified role unspecified role 20 enzyme enzyme unspecified role unspecified role purified purified ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction Fig. 2, 4 ,5 Chlorimuron ethyl Chlorsulfuron ilvgm_ecoli 2xilvG:2xilvM IlvGM complex AHA II complex Acetohydroxyacid synthase II complex Acetolactate synthase II complex 8 cofactor cofactor coenzyme ilvm binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ilvg binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 cofactor cofactor coenzyme ilvg binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 5 enzyme enzyme ilvg binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined magnesium binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined thiamine diphosphate binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 enzyme enzyme thiamine diphosphate binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ilvm binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined fad binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined magnesium binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 4 cofactor cofactor coenzyme illvm binding region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ilvg binding region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 29 22 23 31 25 33 32 27 36 28 37 34 direct interaction direct interaction ecoli Escherichia coli (strain K12) stable complex stable complex physical interaction evidence used in manual assertion physical interaction evidence used in manual assertion IPI inferred from physical interaction Catalyzes the first step that is common to the biosynthesis of branched-chain amino acids. The reaction involves the irreversible decarboxylation of pyruvate to a bound hydroxyethyl group that then condenses with either a second pyruvate molecule to form 2-acetolactate as the first step in the biosynthesis of valine and leucine or with 2-ketobutyrate to form 2-aceto-2-hydroxybutyrate as the initial step in the biosynthesis of isoleucine. The AHAS I (CPX-3573) and AHAS III (CPX-3575) complexes are both sensitive to feedback inhibition by valine, whereas the AHAS II complex is not, enabling bacteria expressing this complex to grow in valine-rich conditions. FAD appears to be required to maintain the geometry of the active sites in the central beta‐domain of the ilvG molecule. Heterotetramer ilvG is not commonly expressed in Escherichia coli K12 due to a frameshift that disturbs the ilvG reading frame, however, occasionally the ilv02096 mutation of a 2 bp insertion exists (as shown in PMID:9581571) which causes a frameshift that restores the open reading frame, permitting the expression of this protein. Accepted 2018-JUL-13 by LIVIAP