Abstract
Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.
Highlights
Purification and Characterizationof Indolepyruvate Decarboxylase(Received for publication, September 7, 1991, and in revised form, March 16, 1992). From the Bio Science Laboratories,M e i i Seika Kaisha, Ltd., 5-3-1, Chiyoda, Sakado-shi, Saitama350, Japan
Indolepyruvate decarboxylase,a key enzyme forin- phan aminotransferase
The enzyme exhibited decarboxylase activity with lyzed enzymatically [1,11].it seems likely that indole-3-pyruvic acid and pyruvic acid as substrates, the control of the IPyA pathway does not involve any very butnodecarboxylase activity was apparent with L- specific regulatory mechanisms at theenzymatic level
Summary
(Received for publication, September 7, 1991, and in revised form, March 16, 1992). From the Bio Science Laboratories,M e i i Seika Kaisha, Ltd., 5-3-1, Chiyoda, Sakado-shi, Saitama350, Japan. Kenten purified toapparenthomogeneityfrom Escherichia and Mann [6] suggested that IAAld is oxidized to IAA by an Homoceaoxbfsoeelpttirwaheisiscrienaeiadvemmlelidtssreeisnt,fnhorrteatchaosirmmepasbytheoaeErorrdio.nwiwdpncteihigolttdooihtoashtinapchneanhaaoegaMtcf.ettieTnin,tvhoehdaeftieonai2mcldrmeM 4elpoo0isnynScu,r0eoul+utm0ssp,v0tfeyoaho.rfrtIerseongaaptdenthchhledtiociesfdviasleieprtamnrbhtbzaeeoasyttrxetirmsenoya.nlce-a-endtiialnnhiltdeLttpelheifhltieatyIrlAnspdettrAesoaiodsnbaxudnuikcdtdnttaiihosbosiwenacrcadinotpneefanrabIpizbAalyloe,aAmuonttheftfsserct.orhoienmneTvtih~hsseemeer-ntcTaiIoonPjrnogyepdrvAIiA.tiyedpAneaoznltfdyhcbmewtaoteacoIytAeisnraAuiraegt(hpg7inree)o.svTdtoIuPthlhvcyueaeAsdst, Mg2+to the inactive monomers and dimers results in pathway, indolepyruvate decarboxylase, which catalyzes the the formation of active tetramers. These results indi- conversion of IPyA to IAAld. These results indi- conversion of IPyA to IAAld Both IPyA and IAAld are cate thatthethiamine pyrophosphate-M8+complex unstable compounds and difficult to isolate [8,9,10]. The abbreviations used are: IAA, indole-3-acetic acid IPyA, indole-3-pyruvic acid; IAAld, indole-3-acetaldehyde; L - T I ~L-,tryptophan; IPDC,indolepyruvate decarboxylase; T P P , thiamine pyrophosphate; HPLC, high performance liquid chromatography;SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. The active fractions were pooled and concentrated to 100 ml by ultrafiltration
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