PMID:19159332

Citation	Xiang, DF, Kolb, P, Fedorov, AA, Meier, MM, Fedorov, LV, Nguyen, TT, Sterner, R, Almo, SC, Shoichet, BK and Raushel, FM (2009) Functional annotation and three-dimensional structure of Dr0930 from Deinococcus radiodurans, a close relative of phosphotriesterase in the amidohydrolase superfamily. Biochemistry 48:2237-47
Abstract	Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3121, and the structure was determined to a resolution of 2.1 A. The protein folds as a (beta/alpha)7beta-barrel, and a binuclear metal center is found at the C-terminal end of the beta-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the beta-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of delta- and gamma-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was delta-nonanoic lactone with a kcat/Km of 1.6 x 10(6) M-1 s-1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of kcat and kcat/Km of 0.07 min-1 and 0.8 M-1 s-1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is 30%. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a kcat of 1.14 min-1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.
Links	PubMed PMC3176505 Online version:10.1021/bi802274f
Keywords	Amidohydrolases/chemistry; Amidohydrolases/genetics; Amidohydrolases/metabolism; Amino Acid Sequence; Amino Acid Substitution/physiology; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Deinococcus/enzymology; Deinococcus/genetics; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Kinetics; Lactones/chemistry; Metals, Heavy/chemistry; Models, Molecular; Molecular Sequence Data; Phosphoric Triester Hydrolases/chemistry; Phosphoric Triester Hydrolases/genetics; Phosphoric Triester Hydrolases/metabolism; Protein Conformation; Protein Multimerization; Pseudomonas/enzymology; Pseudomonas/genetics; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Spectrophotometry; Static Electricity; Structural Homology, Protein; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
DEIRA:Q9RVU2		GO:0046573: lactonohydrolase activity	ECO:0000314:		F		Table-2 shows kinetic parameters of the different forms of the enzyme with variuos substrates. The best substrates are identified to be gamma and delta lactones	complete CACAO 10138
DEIRA:Q9RVU2	enables	GO:0046573: lactonohydrolase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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