Functional annotation and structural characterization of a novel lactonase hydrolyzing D-xylono-1,4-lactone-5-phosphate and L-arabino-1,4-lactone-5-phosphate

Biochemistry. 2014 Jul 22;53(28):4727-38. doi: 10.1021/bi500595c. Epub 2014 Jul 9.


A novel lactonase from Mycoplasma synoviae 53 (MS53_0025) and Mycoplasma agalactiae PG2 (MAG_6390) was characterized by protein structure determination, molecular docking, gene context analysis, and library screening. The crystal structure of MS53_0025 was determined to a resolution of 2.06 Å. This protein adopts a typical amidohydrolase (β/α)8-fold and contains a binuclear zinc center located at the C-terminal end of the β-barrel. A phosphate molecule was bound in the active site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278. Both docking and gene context analysis were used to narrow the theoretical substrate profile of the enzyme, thus directing empirical screening to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of d-xylono-1,4-lactone-5-phosphate (2) with kcat/Km values of 4.7 × 10(4) and 5.7 × 10(4) M(-1) s(-1) and l-arabino-1,4-lactone-5-phosphate (7) with kcat/Km values of 1.3 × 10(4) and 2.2 × 10(4) M(-1) s(-1), respectively. The identification of the substrate profile of these two phospho-furanose lactonases emerged only when all methods were integrated and therefore provides a blueprint for future substrate identification of highly related amidohydrolase superfamily members.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amidohydrolases / chemistry*
  • Amidohydrolases / genetics
  • Amidohydrolases / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Crystallography, X-Ray
  • Lactones / chemistry*
  • Lactones / metabolism
  • Molecular Docking Simulation*
  • Mycoplasma synoviae / enzymology*
  • Mycoplasma synoviae / genetics
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Sugar Phosphates / chemistry*
  • Sugar Phosphates / genetics
  • Sugar Phosphates / metabolism


  • Bacterial Proteins
  • Lactones
  • Sugar Phosphates
  • arabinonolactone-5-phosphate
  • Amidohydrolases

Associated data

  • PDB/3MSR
  • PDB/3OVG