Among various snake venom components, glutaminyl cyclase (vQC) is one of the least understood protein family and none of its members has been purified or characterized. Here we confirmed the presence of vQC activity in a wide spectrum of venom species via enzymatic assay using a synthetic fluorogenic substrate. We have also cloned novel vQC cDNAs from seven species including Crotalus atrox. The results revealed more than 96% sequence similarities among vQCs and ∼75% sequence identities between vQCs and human secretory QC (hQC). The vQC glycoprotein of 43 kDa was isolated from C. atrox venom, and its N-terminal sequence was determined. The optimal pH range for vQC reaction was 7.5-8.0, and the enzymes were stable up to 50 °C. Similar to hQC, vQCs were substantially inactivated by 1 mM 1,10-phenanthroline but slightly affected by 20 mM EDTA, suggestive of a similar zinc-catalytic environment for these enzymes. Although their catalytic residues were highly conserved, vQCs were less susceptible to inhibition by synthetic imidazole derivatives which potently inhibited hQC. The 3D-models revealed that vQC and hQC structures display different surface charge distributions around the active sites, which might affect substrate and inhibitor binding affinities. The recombinant vQCs prepared from Escherichia coli displayed weaker substrate binding affinities relative to the native vQCs, possibly due to the lack of glycosylation. The present report offers new structural and functional insights into vQCs and sheds light on the specificity differences between vQCs and hQC.
Keywords: 3D models; Cloning and sequencing; Inhibition kinetics; Recombinant enzyme; Snake venom pyroglutaminyl modification.
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