Identification Of The Nitrile Reductase Activity Of The QueF-like From Pyrobaculum Calidifontis

Archaeosine (G+) is found at position15of many archaea tRNAs. G+is thought to be important in structural stabilization of the tRNA through electrostatic interactions with the anionic phosphates in Euryarchaeota. The G+precursor,7-cyano-7-deazaguanine (preQo), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G+by Archaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G+lack ArcS homologs. Using comparative genomics approaches, two families that could functionally replace ArcS in these organisms were identified:1) GAT-QueC, a two-domain family with an N-terminal glutamine amidotransferase class-II domain fused to a domain homologous to QueC;2)QueF-like, a family homologous to the bacterial enzyme catalyzing the reduction of preQo to7-aminomethyl-7-deazaguanine.The discovery of the reduce of nitriles by biocatalysis has very important applications in industrial. It is hoped that the research of nitrile reductases will bring a forthcoming wave in biocatalysis. Thus the queF-like gene from Pyrobaculum calidifontis was selected and the gene was cloned into the pET-30Xa/LIC vector. The QueF-like protein was expressed in the E. coli strain BL21(DE3) and the QueF-like protein was purified by Ni2+-agarose affinity chromatography and concentrated by ultrafilltration. Using preQo as substrate, and N ADPH as a reductant agent, the nitrile reductase activity of the QueF-like was analysed through continuous UV-vis spectrum and reverse-phase HPLC. There produced a peak of NADP+beacuse NADPH was oxidized in UV-vis analysis, this indicate that preQo was reduced by QueF-like. It can be found that the absorption peak of preQo was disappeard in the reaction system, it also resulting in a NADP+absorption and accompanied by a significant decrease of NADPH absorption peak, this indicate that preQo was reduced by QueF-like too. All the results show that the QueF-like protein has the nitrile reductase activity, and it can reduce the preQo to preQ1. This laid the foundation for the further research on the effects of QueF-like in the synthesis of G+and its’industrial application.