Thenosgene cluster from gram-positive bacteriumGeobacillus thermodenitrificansNG80-2 and functional characterization of the recombinant NosZ

Abstract

The nos gene cluster encoding the activity of nitrous oxide reductase (N2OR) for the final step of the denitrification pathway has been well studied in gram-negative bacteria. Our previous study on the genome of Geobacillus thermodenitrificans NG80-2 revealed the presence of the nos gene cluster in this gram-positive bacterium. In this follow-up study, the nos gene cluster of G. thermodenitrificans NG80-2 was further analyzed and compared with those of other origins. The structural gene nosZ was heterologously expressed in Escherichia coli and the product was purified as a His-tagged fusion protein. The recombinant NosZ as prepared showed detectable N2OR activity, and the activity was enhanced by preincubation of the protein under argon and with copper compounds. The recombinant NosZ contains 2.5 atoms of copper per dimer and exhibits weak spectral features in the visible range, indicating that spontaneous incorporation of copper compounds into the NG80-2 NosZ can result in some but not full activity of the authentic NG80-2 N2OR. The enzymatic properties of the NosZ were also investigated. This is the first functional characterization of nosZ gene from gram-positive bacteria. This study indicates that the molecular mechanism for N2O reduction is conserved between gram-negative and gram-positive bacteria.