Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli.
The genes coding for histidine decarboxylase from a wild-type strain and an autoactivation mutant strain of Lactobacillus 30a have been cloned and expressed in Escherichia coli. The mutant protein, G58D, has a single Asp for Gly substitution at position 58. The cloned genes were placed under control of the beta-galactosidase promoter and the products are natural length, not fusion proteins. The enzyme kinetics of the proteins isolated from E. coli are comparable to those isolated from Lactobacillus 30a. At pH 4.8 the Km of wild-type enzyme is 0.4 mM and the kcat = 2800 min-1; the corresponding values for G58D are 0.5 mM and 2750 min-1. The wild-type and G58D have autoactivation half-times of 21 and 9 h respectively under 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. The relatively slow rate of autoactivation for purified protein and the differences in cellular and non-cellular activation rates, coupled with the fact that wild-type protein is readily activated in wild-type Lactobacillus 30a but poorly activated in E. coli, suggest that wild-type Lactobacillus 30a contains a factor, possibly an enzyme, that enhances the activation rate.
Copeland
W C
WC
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin 78712.
Vanderslice
P
P
Robertus
J D
JD
D002262:Carboxy-Lyases / Q000235:genetics*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D006640:Histidine Decarboxylase / Q000032:analysis / Q000235:genetics*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D011401:Promoter Regions, Genetic
D002262:Carboxy-Lyases; D006640:Histidine Decarboxylase