Functional Annotation and Three-Dimensional Structure of Dr0930 from Deinococcus radiodurans, a Close Relative of Phosphotriesterase in the Amidohydrolase Superfamily

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 Å. The protein folds as a (β/α)7β-barrel and a binuclear metal center is found at the C-terminal end of the β-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 β-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 δ- and γ-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was δ-nonanoic lactone with a kcat/Km of 1.6 × 106 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.

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily

“…This property was also observed for Dr0930 (21) and purple acid phosphatase or uteroferrin (28,29), in which the purple coloration was attributed to a charge-transfer complex between an active site tyrosine residue and an iron cation (the ␤-cation) within the binuclear metal center. In the GKL structure, the phenolic oxygen of Tyr-99 (the active site tyrosine involved in the chargetransfer complex) is 3.87 Å away from the ␤-cation, whereas in Dr0930, the conserved phenolic oxygen of the tyrosine residue in the charge-transfer complex was reported to be 3.48 Å away from its corresponding ␤-cation.…”

“…GKL also had low paraoxonase activity (k cat /K m of 4.5 M Ϫ1 s Ϫ1 for paraoxon), indicating that, like other members of the PLLs, the native substrate profile of this enzyme does not involve phosphate esters (5,21). Among the selected PLLs, GKL has a significantly higher sequence identity to an orthologous PLL from Deinococcus radiodurans (Dr0930 was reported to hydrolyze ␥-nonalactone and ␦-nonalactone efficiently but had no detectable activity against AHLs (21)) than any other reported PLLs (59% amino acid sequence identity; a matrix of pairwise sequence identities between GKL and selected members of the amidohydrolase superfamily is detailed in supplemental Table S2).…”

“…The ␣-cation, presumably occupied by Zn 2ϩ (21,23), is coordinated by His-23 and His-25 at the end of the first ␤-strand, the carboxylated Lys-145 at the end of the fourth ␤-strand, and Asp-266 (Asn 266 for the corresponding D266N GKL mutant as depicted in Fig. 2) at the end of the eighth ␤-strand.…”

“…Correspondingly, from the structure of a reported thermostable PLL from Sulfolobus solfataricus (SsoPox) with bound ligand (this structure depicts SsoPox complexed with the inhibitor N-decanoyl-L-homocysteine in an unproductive orientation (21,23)), these residues are replaced by Arg-223, Leu-72, Val-27, and Ile-261. In SsoPox, a long hydrophobic channel accommodates the aliphatic tail of the N-acylhomocysteine inhibitor and is composed of the side chains of Phe-229, Leu- The GKL monomers are related by a crystallographic 2-fold axis and form an apparent dimer in the current crystal structure (Fig.…”

Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

“…In recent years, a number of phosphotriesterase homologs have been characterized in several microorganisms and shown to proficiently hydrolyze various lactones with a weak PTE activity. These newly emerging enzymes were designated phosphotriesterase-like lactonases (PLLs) (1,16,36,53). Both BdPTE and members of the PLL group belong to the amidohydrolase superfamily (AHS), which is a family of enzymes that share a (␤/ ␣) 8 -triosephosphate isomerase (TIM) barrel fold and utilize a mononuclear or binuclear metal center for catalysis (18).…”