Identification of Amino Acid Residues Involved in Feedback Inhibition of the Anthranilate Synthase in Escherichia coli

Joon Hyeok Kwak, Kwang Won Hong, Sung Haeng Lee, Jin Han Hong, Se Yong Lee

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

The first step of the branch pathway in tryptophan biosynthesis is catalyzed by anthranilate synthase, which is subjected to feedback inhibition by the end product of the pathway. The trpEFBR gene from a mutant Escherichia coli strain coding for anthranilate synthase that was insensitive to feedback inhibition by tryptophan has been cloned. To identify the amino acid changes involved in the feedback regulation of anthranilate synthase, the nucleotide sequence of the mutant trpEFBR gene was determined. Sequence analysis of the trpEFBR gene revealed that four bases were changed in the structural gene while alteration was not found in the 5′ control region. Among these base changes, only two base substitutions caused the alterations in amino acid sequences. From the results of restriction fragment exchange mapping, the 61st nucleotide, C to A substitution, that changed Pro21→Ser was identified as the cause of the desensitization to feedback inhibition by tryptophan. Additional feedback-resistant enzymes of the E. coli anthranilate syntheses were constructed by site-directed mutagenesis to examine the effect of the Ser40→Arg40 change found in the trpEFBR gene of Brevibacterium lactofermentum. From the feedback inhibition analysis, the Pro21→Ser and Ser40-→Arg mutants maintained about 50% and 90% of their maximal activities, respectively, even at the extreme concentration of 10 mM tryptophan. From these results, we suggest that the Pro21 and Ser40 residues are involved in the tryptophan binding in the E. coli enzyme.

Original languageEnglish
Pages (from-to)20-24
Number of pages5
JournalJournal of Biochemistry and Molecular Biology
Volume32
Issue number1
StatePublished - 31 Jan 1999

Keywords

  • Anthranilate synthase
  • Feedback inhibition
  • Site-directed mutagenesis

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