Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase

Cheng Yang Huang, Ching Chen Hsu, Mei Chun Chen, Yuh-Shyong Yang*

*Corresponding author for this work

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

Bacterial hydantoinase possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. How the carboxylated lysine and metal binding affect the activity of hydantoinase was investigated. A significant amount of iron was always found in Agrobacterium radiobacter hydantoinase purified from unsupplemented cobalt-, manganese-, or zinc-amended Escherichia coli cell cultures. A titration curve for the reactivation of apohydantoinase with cobalt indicates that the first metal was preferentially bound but did not give any enzyme activity until the second metal was also attached to the hydantoinase. The pH profiles of the metal-reconstituted hydantoinase were dependent on the specific metal ion bound to the active site, indicating a direct involvement of metal in catalysis. Mutation of the metal binding site residues, H57A, H59A, K148A, H181A, H237A, and D313A, completely abolished hydantoinase activity but preserved about half of the metal content, except for K148A, which lost both metals in its active site. However, the activity of K148A could be chemically rescued by short-chain carboxylic acids in the presence of cobalt, indicating that the carboxylated lysine was needed to coordinate the binuclear ion within the active site of hydantoinase. The mutant D313E enzyme was also active but resulted in a pH profile different from that of wild-type hydantoinase. A mechanism for hydantoinase involving metal, carboxylated K148, and D313 was proposed.

Original languageEnglish
Pages (from-to)111-121
Number of pages11
JournalJournal of Biological Inorganic Chemistry
Volume14
Issue number1
DOIs
StatePublished - 1 Jan 2009

Keywords

  • Carboxylated lysine
  • Hydantoinase
  • Metalloenzyme
  • Site-directed mutagenesis
  • Structure-function relationship

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