Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima

Dong Hae Shin, Anne Roberts, Jaru Jancarik, Hisao Yokota, Rosalind Kim, David E. Wemmer, Sung Hou Kim

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33 Scopus citations

Abstract

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 Å resolution by selenomethionine single-wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose-6-phosphate phosphatase and sucrose-6F-phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 × 40 × 35 Å3, and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six-stranded β-sheet surrounded by four α-helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.

Original languageEnglish
Pages (from-to)1464-1472
Number of pages9
JournalProtein Science
Volume12
Issue number7
DOIs
StatePublished - 1 Jul 2003

Keywords

  • gi 4981173
  • HAD family
  • New fold
  • Phosphatase
  • Structural proteomics
  • X-ray crystallography

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