Insect carboxylesterases from the alphaEsterase gene cluster, such as alphaE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcalphaE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of alpha-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcalphaE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical alpha/beta-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal alpha-helix that serves as a membrane anchor. Soaking of LcalphaE7 crystals in OPs led to the capture of a crystallographic snapshot of LcalphaE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcalphaE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants ( approximately 10(6) M(-1) s(-1)) indicative of a natural substrate.
Title: Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester Fujii R, Utsunomiya Y, Hiratake J, Sogabe A, Sakata K Ref: Biochimica & Biophysica Acta, 1631:197, 2003 : PubMed
The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.
