Received November 30, 2001

Revised Manuscript Received March 5, 2002

Abstract:

The hyperthermophilic archeon Pyrococcus furiosus produces an extracellular -amylase that belongs to glycosyl hydrolases' family 13. This enzyme is more thermostable than its bacterial and archaeal homologues (e.g., Bacillus licheniformis TAKA-term and Pyrococcus kodakaraensis KOD1 -amylases, respectively) even without adding Ca²⁺ ions. Unlike the TAKA-therm amylase that contains no cysteine, the P. furiosus enzyme contains five cysteines (C152, C153, C165, C387, and C430), only four of which (C152, C153, C387, and C430) are conserved in the P. kodakaraensis -amylase. To test the potential function of cysteines in P. furiosus -amylase stability, these five residues were substituted with Ser or Ala-either one-by-one or in sequence-to produce eight mutant enzymes. Mutation C165S dramatically destabilized P. furiosus -amylase. At the same time, the quadruple mutant enzyme C152S/C153S/C387S/C430A (mutant SSCSA) was as thermostable as the wild-type enzyme. Mutant SSCSA and wild-type -amylases were strongly destabilized by dithiothreitol and ethylenediaminetetraacetic acid, suggesting that metal binding can be involved in this enzyme's thermostability. Inductively coupled plasma-atomic emission spectrometry showed the presence of Ca²⁺ and Zn²⁺ metal ions in P. furiosus -amylase. Although Ca²⁺ is known to contribute to -amylase's stability, the absence of two out of the three conserved Ca²⁺ ligands in the P. furiosus enzyme suggests that a different set of amino acids is involved in this enzyme's Ca²⁺ binding. We also provide evidence suggesting that Cys165 is involved in Zn²⁺ binding and that Cys165 is essential for the stability of P. furiosus -amylase at very high temperatures.

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