Arginine kinase of the sheep blowfly Lucilia cuprina: Gene identification and characterization of the native and recombinant enzyme

Arginine kinase (ATP:L-arginine omega-N-phosphotransferase, EC2.7.3.3.; AK) is an enzyme with crucial functions in the energy metabolism of insects and other invertebrates as well as some protozoa and has been proposed as a parasiticidal and pesticidal drug target. In this study we report the identification, cDNA cloning, genomic gene structure and functional expression of an AK gene from the parasitic sheep blowfly Lucilia (L) cuprina. The blowfly AK-encoding gene is devoid of introns and present in a single copy in the L cuprina genome. AK is present in L. cuprina flies as a highly expressed soluble enzyme and is more abundant in the thorax, where it represents up to 2% of the total soluble protein, compared to head or abdomen. Guanidino substrate specificity studies show that L cuprina AK is stereospecific for the L-form over the D-form of its specific substrate arginine. Furthermore, the presence of a free or esterified carboxylic group as well as an unsubstituted alpha-amine group are essential for acceptance of the L-arginine substrate while modifications in the aliphatic side chain are better tolerated. The apparent Michaelis-Menten constants K-M and the molecular size of the recombinant enzyme are virtually identical to that of the native enzyme suggesting that the gene cloned in this study encodes the highly expressed AK enzyme of the sheep blowfly. (C) 2011 Elsevier Inc. All rights reserved.