{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T08:04:18Z","timestamp":1779177858110,"version":"3.51.4"},"reference-count":48,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2008,12,31]],"date-time":"2008-12-31T00:00:00Z","timestamp":1230681600000},"content-version":"vor","delay-in-days":3897,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[1998,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Indoleglycerol phosphate synthase catalyzes the ring closure of 1\u2010(2\u2010carboxyphenylamino)\u20101\u2010deoxyribulose 5\u2032\u2010phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from<jats:italic>Escherichia coli<\/jats:italic>have been mutated directly or randomly, to identify the catalytically essential ones. A strain of<jats:italic>E. coli<\/jats:italic>suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the<jats:italic>trp<\/jats:italic>C gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild\u2010type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady\u2010state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme\u2010bound intermediates and general acid\u2010base catalysis by K114 and E163 with the support of E53 and N184.<\/jats:p>","DOI":"10.1002\/pro.5560070518","type":"journal-article","created":{"date-parts":[[2009,2,10]],"date-time":"2009-02-10T06:28:37Z","timestamp":1234247317000},"page":"1221-1232","source":"Crossref","is-referenced-by-count":22,"title":["Mutational analysis of the active site of indoleglycerol phosphate synthase from<i>Escherichia coli<\/i>"],"prefix":"10.1002","volume":"7","author":[{"given":"Beatrice","family":"Darimont","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Catherine","family":"Stehlin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Halina","family":"Szadkoski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kasper","family":"Kirscner","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2008,12,31]]},"reference":[{"key":"e_1_2_1_2_1","doi-asserted-by":"publisher","DOI":"10.3109\/10409239009090615"},{"key":"e_1_2_1_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00593a029"},{"key":"e_1_2_1_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(76)90527-3"},{"key":"e_1_2_1_5_1","first-page":"411","volume-title":"Comprehensive organic chemistry","author":"Brown RT","year":"1979"},{"key":"e_1_2_1_6_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/18.13.4028"},{"key":"e_1_2_1_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1986.tb04605.x"},{"key":"e_1_2_1_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.86.7.2172"},{"key":"e_1_2_1_9_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00593a030"},{"key":"e_1_2_1_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/0005-2744(77)90264-9"},{"key":"e_1_2_1_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(89)80225-X"},{"key":"e_1_2_1_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00016a013"},{"key":"e_1_2_1_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(94)80799-4"},{"key":"e_1_2_1_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/0968-0004(90)90035-A"},{"key":"e_1_2_1_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(75)90154-2"},{"key":"e_1_2_1_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0969-2126(01)00267-2"},{"key":"e_1_2_1_17_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00016a014"},{"key":"e_1_2_1_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-1-4832-2734-4.50009-7"},{"key":"e_1_2_1_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(89)90359-4"},{"key":"e_1_2_1_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(87)42050-8"},{"key":"e_1_2_1_21_1","first-page":"237","article-title":"Hybrids of circular DNA single strands as intermediates in DNA cloning, nucleotide sequence analysis, and directed mutagenesis","volume":"2","author":"Kolmar H","year":"1990","journal-title":"Technique"},{"key":"e_1_2_1_22_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00332244"},{"key":"e_1_2_1_23_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2007.00901.x"},{"key":"e_1_2_1_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(78)80005-9"},{"key":"e_1_2_1_25_1","first-page":"508","article-title":"A general, PCR\u2010based method for single or combinatorial oligonucleotide\u2010directed mutagenesis on pUC\/M13 vectors","volume":"12","author":"Merino E","year":"1992","journal-title":"Biotechniques"},{"key":"e_1_2_1_26_1","volume-title":"A short course in bacterial genetics","author":"Miller JH","year":"1992"},{"key":"e_1_2_1_27_1","first-page":"2638","volume-title":"Escherichia coli and Salmonella: Cellular and molecular biology","author":"Nichols BP","year":"1996"},{"key":"e_1_2_1_28_1","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1093\/oso\/9780199636167.003.0003","volume-title":"Protein function, a practical approach","author":"Rudolph R","year":"1997"},{"key":"e_1_2_1_29_1","volume-title":"Molecular cloning: A laboratory manual","author":"Sambrook J","year":"1990"},{"key":"e_1_2_1_30_1","first-page":"404","article-title":"The \u201cmegaprimer\u201d method of site\u2010directed mutagenesis","volume":"8","author":"Sarkar G","year":"1990","journal-title":"BioTech"},{"key":"e_1_2_1_31_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.90.20.9235"},{"key":"e_1_2_1_32_1","first-page":"259","volume-title":"Protein structure, a practical approach","author":"Schmid FX","year":"1997"},{"key":"e_1_2_1_33_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.78.4.2169"},{"key":"e_1_2_1_34_1","doi-asserted-by":"crossref","first-page":"2051","DOI":"10.1016\/S0021-9258(18)69361-5","article-title":"1\u2010(o\u2010Carboxyphenylamino)\u20101\u2010deoxyribulose 5\u2010phosphate, a new intermediate in the biosynthesis of tryptophan","volume":"235","author":"Smith OH","year":"1960","journal-title":"J Biol Chemistry"},{"key":"e_1_2_1_35_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/17.12.4441"},{"key":"e_1_2_1_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(97)00066-5"},{"key":"e_1_2_1_37_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0396-329"},{"key":"e_1_2_1_38_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/18.24.7463"},{"key":"e_1_2_1_39_1","doi-asserted-by":"crossref","first-page":"4395","DOI":"10.1002\/j.1460-2075.1995.tb00118.x","article-title":"(\u03b2\u03b1)8\u2010barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima","volume":"14","author":"Sterner R","year":"1996","journal-title":"EMBO J"},{"key":"e_1_2_1_40_1","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(73)90003-X"},{"key":"e_1_2_1_41_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.76.9.4350"},{"key":"e_1_2_1_42_1","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(87)53044-0"},{"key":"e_1_2_1_43_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00102a006"},{"key":"e_1_2_1_44_1","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(92)90665-7"},{"key":"e_1_2_1_45_1","doi-asserted-by":"crossref","first-page":"1219","DOI":"10.1128\/jb.161.3.1219-1221.1985","article-title":"Site\u2010directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli","volume":"161","author":"Winans SC","year":"1985","journal-title":"J Bacterial"},{"key":"e_1_2_1_46_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.121.3135.138"},{"key":"e_1_2_1_47_1","first-page":"1453","volume-title":"Escherichia coli and Salmonella: Cellular and molecular biology","author":"Yanofsky C.","year":"1987"},{"key":"e_1_2_1_48_1","first-page":"409","article-title":"Polarity and enzyme functions in mutants of the first three genes of the tryptophan operon of","volume":"69","author":"Yanofsky C","year":"1971","journal-title":"Escherichia coli. Genetics"},{"key":"e_1_2_1_49_1","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(66)90102-1"}],"container-title":["Protein Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fpro.5560070518","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.5560070518","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,3,10]],"date-time":"2024-03-10T11:16:30Z","timestamp":1710069390000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/pro.5560070518"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,5]]},"references-count":48,"journal-issue":{"issue":"5","published-print":{"date-parts":[[1998,5]]}},"alternative-id":["10.1002\/pro.5560070518"],"URL":"https:\/\/doi.org\/10.1002\/pro.5560070518","archive":["Portico"],"relation":{},"ISSN":["0961-8368","1469-896X"],"issn-type":[{"value":"0961-8368","type":"print"},{"value":"1469-896X","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,5]]}}}