PMID:23214420

Citation	Ornelas, A, Korczynska, M, Ragumani, S, Kumaran, D, Narindoshvili, T, Shoichet, BK, Swaminathan, S and Raushel, FM (2013) Functional annotation and three-dimensional structure of an incorrectly annotated dihydroorotase from cog3964 in the amidohydrolase superfamily. Biochemistry 52:228-38
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)).
Links	PubMed PMC3542638 Online version:10.1021/bi301483z
Keywords	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
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
AGRT5:Q7CS13		GO:0016788: hydrolase activity, acting on ester bonds	ECO:0000315:		F		Table 2 shoes the kinetic parameters for Atu3266. Through the testing of different substrates, it was observed that the Atu3266 gene catalytic active increased 100-fold when amide linkage was changed to an ester. The table also shows that R-enantiomer of acetylated α-hydroxyl carboxylates generated the largest rate constant.	complete
OCHA4:DEACT		GO:0052689: carboxylic ester hydrolase activity	ECO:0000314:		F		Table 2 shows kinetic parameters for Oant2987 with various substrates. The best substrate is 28. They also find that activity increases when an amide bond is changed to an ester bond.	complete CACAO 10137
OCHA4:DEACT	enables	GO:0052689: carboxylic ester hydrolase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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