title: Functional annotation and three-dimensional structure of an incorrectly annotated dihydroorotase from cog3964 in the amidohydrolase superfamily.
abstract: 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)).
authors.affiliation: Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, TX 77842-3012, USA.
authors.forename: Argentina
authors.initials: A
authors.lastname: Ornelas
authors.forename: Magdalena
authors.initials: M
authors.lastname: Korczynska
authors.forename: Sugadev
authors.initials: S
authors.lastname: Ragumani
authors.forename: Desigan
authors.initials: D
authors.lastname: Kumaran
authors.forename: Tamari
authors.initials: T
authors.lastname: Narindoshvili
authors.forename: Brian K
authors.initials: BK
authors.lastname: Shoichet
authors.forename: Subramanyam
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D016960:Agrobacterium tumefaciens / Q000737:chemistry / Q000201:enzymology*; D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004080:Dihydroorotase / Q000737:chemistry* / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D020621:Ochrobactrum anthropi / Q000737:chemistry / Q000201:enzymology*; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D009943:Organophosphorus Compounds; C032627:methylphosphonic acid; C008852:2-phenylglycine; D000581:Amidohydrolases; D004080:Dihydroorotase; D005998:Glycine