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 five orders of magnitude, achieving absolute values of catalytic efficiencies up to 106 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.

The nature of chemical innovation: new enzymes by evolution

“…Experiments lend credence to the proposal that promiscuity underlies much of enzyme innovation. Numerous studies have shown that an enzyme optimized for one reaction can adapt under laboratory mutation and screening/selection to take on the function of a different member of the superfamily, sometimes with efficiency that rivals nature's solutions (Aharoni et al 2005;Gerlt & Babbitt, 2009;Meier et al 2013).…”

Enzyme promiscuity: using the dark side of enzyme specificity in white biotechnology

“…However, as Nobeli et al [11] had pointed out, such results lay the foundation of subsequent efforts using protein engineering/ directed evolution to improve upon these catalytic activities. Large number of successful results which prove this are already available in the literature [12,[71][72][73].…”

Recent advances in enzyme promiscuity

“…We characterized several such mutants with broader stereo-specificity and also with different leaving groups, which could be used for the in vivo detoxification of real nerve agents. These in vivo prophylactic activity of evolved variants and the newly developed screens provide the foundation for designing PON1 and other OP degrading enzymes for prophylaxis against other G-type agents [3,43].…”