Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli |
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Authors: | Copeland William C; Vanderslice Peter; Robertus Jon D |
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Affiliation: | Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas Austin, TX 78712, USA |
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Abstract: | The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the Km of wild-type enzymeis 0.4 mM and the kcat = 2800 min1; the correspondingvalues for G58D are 0.5 mM and 2750 min1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K+ and pH 7.0.At pH 7.6 and 0.8 M K+ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate. |
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Keywords: | autoactivation/ gene expression/ histidine decarboxylase |
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