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.
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Acknowledgments
We thank Yi-Rong Chen for purification of D313E mutant protein. This research was supported by grants from the National Science Council, Taiwan (96-2627-B-009-004).
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775_2008_428_MOESM1_ESM.pdf
The pH profiles of metal-reconstituted hydantoinase using 5-leucinyl-hydantoin as the substrate. (a) pH profile of k cat. (b) pH profile of k cat/K m. Enzymatic activity was determined using 5–100 μg metal-reconstituted hydantoinase. Closed circles denote Co-hydantoinase. Closed squares denote Mn-hydantoinase. Closed triangles denote Zn-hydantoinase. (PDF 27 kb)
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Huang, CY., Hsu, CC., Chen, MC. et al. Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase. J Biol Inorg Chem 14, 111–121 (2009). https://doi.org/10.1007/s00775-008-0428-x
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DOI: https://doi.org/10.1007/s00775-008-0428-x