Pyrococcus furiosus -Amylase Is Stabilized by Calcium and Zinc
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- 作者：Alexei Savchenko ; Claire Vieille ; Suil Kang ; and J. Gregory Zeikus
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：May 14, 2002
- 年：2002
- 卷：41
- 期：19
- 页码：6193 - 6201
- 全文大小：155K
- 年卷期：v.41,no.19(May 14, 2002)
- ISSN：1520-4995
文摘
The hyperthermophilic archeon Pyrococcus furiosus produces an extracellular 
-amylase thatbelongs to glycosyl hydrolases' family 13. This enzyme is more thermostable than its bacterial and archaealhomologues (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 potentialfunction of cysteines in P. furiosus -amylase stability, these five residues were substituted with Ser orAla-either one-by-one or in sequence-to produce eight mutant enzymes. Mutation C165S dramaticallydestabilized 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, suggestingthat metal binding can be involved in this enzyme's thermostability. Inductively coupled plasma-atomicemission spectrometry showed the presence of Ca2+ and Zn2+ metal ions in P. furiosus -amylase. AlthoughCa2+ 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'sCa2+ binding. We also provide evidence suggesting that Cys165 is involved in Zn2+ binding and thatCys165 is essential for the stability of P. furiosus -amylase at very high temperatures.
-amylase thatbelongs to glycosyl hydrolases' family 13. This enzyme is more thermostable than its bacterial and archaealhomologues (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 potentialfunction of cysteines in P. furiosus -amylase stability, these five residues were substituted with Ser orAla-either one-by-one or in sequence-to produce eight mutant enzymes. Mutation C165S dramaticallydestabilized 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, suggestingthat metal binding can be involved in this enzyme's thermostability. Inductively coupled plasma-atomicemission spectrometry showed the presence of Ca2+ and Zn2+ metal ions in P. furiosus -amylase. AlthoughCa2+ 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'sCa2+ binding. We also provide evidence suggesting that Cys165 is involved in Zn2+ binding and thatCys165 is essential for the stability of P. furiosus -amylase at very high temperatures.