Distinct Enzymic Functional Groups Are Required for the Phosphomonoesterase and Phosphodiesterase Activities of Clostridium Thermocellum Polynucleotide Kinase/Phosphatase.
From: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
The Journal of biological chemistry
- Publish Date: Jul 2006
- ISSN: 0021-9258
- Volume: 281
- Issue: 28
- Pages: 19251-9
- Medium: Print
- Language: English
- Citation (JAMA): Keppetipola Niroshika, Shuman Stewart, et al. Distinct Enzymic Functional Groups Are Required for the Phosphomonoesterase and Phosphodiesterase Activities of Clostridium Thermocellum Polynucleotide Kinase/Phosphatase.. J. Biol. Chem. Jul 2006;281:19251-9
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.
Mesh Headings (Keywords): Binding Sites, Cell Differentiation, Clostridium thermocellum, DNA Mutational Analysis, Kinetics, Manganese, Models, Molecular, Mutation, Phosphoric Diester Hydrolases, Phosphoric Monoester Hydrolases, Polynucleotide 5’-Hydroxyl-Kinase, Sodium, Substrate Specificity
Check for Full Text / PubMed Unique Identifier (PMID): 16675457
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