Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis.

Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis. Research Abstract Details 

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Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis. Abstract Text:

A Shiratsuchi,S Sato,

Anthranilate synthase I (ASI) of Bacillus caldotenax, a thermophilic bacterium, was purified from a plasmid-bearing Escherichia coli and characterized. The molecular weight determination under native and denaturing conditions revealed that it was a monomeric enzyme of M(r) = 54,000. The N-terminal amino acid sequence is the same as expected from DNA sequence of trpE except that the N-terminal methionine is lacking. All four cysteines in the molecule could be titrated with 5,5'-dithiobis (2-nitrobenzoic acid) in more than 8 M urea. The purified enzyme retained its full enzymatic activity after being heated at 60 degrees C. Six mutated genes for the ASI with histidine in place of each conserved arginine, Arg321, Arg353, Arg358, Arg416, Arg429, and Arg452, were prepared by site-directed mutagenesis. All the mutated genes except one, the gene encoding an ASI mutant with histidine in place of Arg452 (R452H ASI) complemented an E. coli (trpE). The mutated ASIs were purified and compared with the wild type ASI. No distinctive differences in enzymatic properties were found between the wild type and the enzymatically active mutated ASIs. R452H ASI was enzymatically inactive, though its conformation seemed to be unchanged after the substitution based on CD spectra and the SH titration curve.

Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis. Publishing Authors By Initials

A Shiratsuchi,S Sato,

For similar investigative techniques: genetic techniques: sequence alignment research abstracts see: investigative techniques: genetic techniques: sequence alignment research

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Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 112

Page Numbers: 714-8

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Nov

YEAR: 1992

Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis. Information

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LANGUAGE: eng

NlmUniqueID: 376600

Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis. Keywords Mesh Terms:

KEYWORDS: Sequence Alignment

MESH TERMS: enzymology

Chemical & Substance for Abstract: Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis. Information

Substance Name: Anthranilate Synthase

Registry Number: EC 4.1.3.27

Grant and Affiliation Information for Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis.

AFFILIATION: Mitsubishi-Kasei Institute of Life Sciences, Tokyo.

Country: JAPAN

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MEDLINETA: J Biochem

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