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The hyperthermophilic archeon Pyrococcus furiosus produces an extracellular alpha-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 alpha-amylases, respectively) even without adding Ca(2+) 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 alpha-amylase. To test the potential function of cysteines in P. furiosus alpha-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 alpha-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 alpha-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 Ca(2+) and Zn(2+) metal ions in P. furiosus alpha-amylase. Although Ca(2+) is known to contribute to alpha-amylase's stability, the absence of two out of the three conserved Ca(2+) ligands in the P. furiosus enzyme suggests that a different set of amino acids is involved in this enzyme's Ca(2+) binding. We also provide evidence suggesting that Cys165 is involved in Zn(2+) binding and that Cys165 is essential for the stability of P. furiosus alpha-amylase at very high temperatures.

Backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A) were investigated at 30 degrees C using (15)N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange. With kinetic parameters identical to those of Escherichia coli AK (ECAK) at 30 degrees C, TNAK is a unique hyperthermophilic enzyme. These catalytic properties make TNAK an interesting and novel model to study the interplay between protein rigidity, stability, and activity. Comparison of fast time scale dynamics (picosecond to nanosecond) in the open and closed states of TNAK and ECAK at 30 degrees C reveals a uniformly higher rigidity across all domains of TNAK. Within this framework of a rigid TNAK structure, several residues located in the AMP-binding domain and in the core-lid hinge regions display high picosecond to nanosecond time scale flexibility. Together with the recent comparison of ECAK dynamics with those of hyperthermophilic Aquifex aeolicus AK (AAAK), our results provide strong evidence for the role of picosecond to nanosecond time scale fluctuations in both stability and activity. In the slow time scales, TNAK's increased rigidity is not uniform but localized in the AMP-binding and lid domains. The core domain amides of ECAK and TNAK in the open and closed states show comparable protection against exchange. Significantly, the hinges framing the lid domain show similar exchange data in ECAK and TNAK open and closed forms. Our NMR relaxation and hydrogen-deuterium exchange studies therefore suggest that TNAK maintains high activity at 30 degrees C by localizing flexibility to the hinge regions that are key to facilitating conformational changes.