Functional annotation and three-dimensional structure of an incorrectly annotated dihydroorotase from cog3964 in the amidohydrolase superfamily

Biochemistry. 2013 Jan 8;52(1):228-38. doi: 10.1021/bi301483z. Epub 2012 Dec 20.


The substrate specificities of two incorrectly annotated enzymes belonging to cog3964 from the amidohydrolase superfamily were determined. This group of enzymes are currently misannotated as either dihydroorotases or adenine deaminases. Atu3266 from Agrobacterium tumefaciens C58 and Oant2987 from Ochrobactrum anthropi ATCC 49188 were found to catalyze the hydrolysis of acetyl-(R)-mandelate and similar esters with values of k(cat)/K(m) that exceed 10(5) M(-1) s(-1). These enzymes do not catalyze the deamination of adenine or the hydrolysis of dihydroorotate. Atu3266 was crystallized and the structure determined to a resolution of 2.62 Å. The protein folds as a distorted (β/α)(8) barrel and binds two zincs in the active site. The substrate profile was determined via a combination of computational docking to the three-dimensional structure of Atu3266 and screening of a highly focused library of potential substrates. The initial weak hit was the hydrolysis of N-acetyl-D-serine (k(cat)/K(m) = 4 M(-1) s(-1)). This was followed by the progressive identification of acetyl-(R)-glycerate (k(cat)/K(m) = 4 × 10(2) M(-1) s(-1)), acetyl glycolate (k(cat)/K(m) = 1.3 × 10(4) M(-1) s(-1)), and ultimately acetyl-(R)-mandelate (k(cat)/K(m) = 2.8 × 10(5) M(-1) s(-1)).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / chemistry
  • Agrobacterium tumefaciens / enzymology*
  • Amidohydrolases / chemistry*
  • Amidohydrolases / metabolism*
  • Catalytic Domain
  • Crystallography, X-Ray
  • Dihydroorotase / chemistry*
  • Dihydroorotase / metabolism*
  • Glycine / analogs & derivatives
  • Glycine / chemistry
  • Glycine / metabolism
  • Models, Molecular
  • Ochrobactrum anthropi / chemistry
  • Ochrobactrum anthropi / enzymology*
  • Organophosphorus Compounds / chemistry
  • Organophosphorus Compounds / metabolism
  • Protein Conformation
  • Substrate Specificity


  • Organophosphorus Compounds
  • methylphosphonic acid
  • 2-phenylglycine
  • Amidohydrolases
  • Dihydroorotase
  • Glycine

Associated data

  • PDB/2OGJ