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Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template.

ABSTRACT: Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure, and function. We interrogated the evolution of the phosphotriesterase from Pseudomonas diminuta (PdPTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily "young" enzyme, and it has been postulated that it has evolved from members of the phosphotriesterase-like lactonase (PLL) family that show promiscuous organophosphate-degrading activity. Starting from a weakly promiscuous PLL scaffold (Dr0930 from Deinococcus radiodurans ), we designed an extremely efficient organophosphate hydrolase (OPH) with broad substrate specificity using rational and random mutagenesis in combination with in vitro activity screening. The OPH activity for seven organophosphate substrates was simultaneously enhanced by up to 5 orders of magnitude, achieving absolute values of catalytic efficiencies up to 10(6) M(-1) s(-1). Structural and computational analyses identified the molecular basis for the enhanced OPH activity of the engineered PLL variants and demonstrated that OPH catalysis in PdPTE and the engineered PLL differ significantly in the mode of substrate binding.

SUBMITTER: Meier MM

PROVIDER: S-EPMC3786566 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template.

Meier Monika M MM Rajendran Chitra C Malisi Christoph C Fox Nicholas G NG Xu Chengfu C Schlee Sandra S Barondeau David P DP Höcker Birte B Sterner Reinhard R Raushel Frank M FM

Journal of the American Chemical Society 20130729 31

Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure, and function. We interrogated the evolution of the phosphotriesterase from Pseudomonas diminuta (PdPTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily "young" enzyme, and it has been postulated that it has evolved from members of the phos ...[more]

PMID: 23837603

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Project description:Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical ?/? fold hydrolase, was previously reported to have both ?-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classical p-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any ?-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities. IMPORTANCE:Promiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of ?/? fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies.

Dataset Information

Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template.

Publications

Molecular engineering of organophosphate hydrolysis activity from a weak promiscuous lactonase template.

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