Insights into the mechanism of paraoxonase-1: Comparing the reactivity of the six-bladed β-propeller hydrolases

Timothy John Grunkemeyer, David Garcia Mata, Kiran Doddapaneni, Srividya Murali, Thomas J Magliery
Biochemistry 2018 December 14
The mammalian protein paraoxonase-1 (PON1) has been explored as a promising bioscavenger treatment for organophosphorus (OP) agent poisoning, but it is not active enough to protect against many agents. Engineering is limited because PON1's catalytic mechanism is poorly understood; moreover, its native activity and substrate are unknown. PON1 is a calcium-bound six-bladed β-propeller hydrolase that shares high structural homology, a conserved metal-coordinating active site, and substrate specificity overlap with other members of a superfamily that includes squid diisopropylfluorophosphatase (DFPase), bacterial drug responsive protein 35 (Drp35), and mammalian senescence marker protein 30 (SMP30). We hypothesized that, by examining the reactivity of all four hydrolases using a common set of conservative mutations, we could gain further insight into the catalytic mechanism of PON1. We chose a set of mutations to examine conserved Asp and Glu residues in the hydrolase active sites, as well as the ligation sphere around the catalytic calcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant hydrolases were assayed against a set of lactones, aryl esters, and OPs that PON1 is known to hydrolyze. Surprisingly, some mutations of Ca2+ coordinating residues, previously thought to be essential for turnover, resulted in significant activity toward all substrate classes examined. Additionally, merely maintaining WT-like charge in the active site of PON1 was insufficient for high activity. Finally, the H115-H134 dyad does not appear to be essential for catalysis against any substrate. Therefore, previously proposed mechanisms must be re-evaluated.

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