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Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2006 Jul 14; Vol. 281 (28), pp. 19251-9. Date of Electronic Publication: 2006 May 04. - Publication Year :
- 2006
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Abstract
- The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp-392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu.
- Subjects :
- Binding Sites
Cell Differentiation
DNA Mutational Analysis
Kinetics
Manganese chemistry
Models, Molecular
Mutation
Polynucleotide 5'-Hydroxyl-Kinase chemistry
Sodium chemistry
Substrate Specificity
Clostridium thermocellum enzymology
Phosphoric Diester Hydrolases metabolism
Phosphoric Monoester Hydrolases metabolism
Polynucleotide 5'-Hydroxyl-Kinase physiology
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 281
- Issue :
- 28
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 16675457
- Full Text :
- https://doi.org/10.1074/jbc.M602549200