Formation of active heterologous nitrate reductases between nitrate reductases A and Z of Escherichia coli

Abstract

Two nitrate reductases, NRA and NRZ, are present in Escherichia coli. These isoenzymes have the same alpha beta gamma, subunits composition and have similar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcloning the different genes and expressing them from separate transcriptional units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantly but independently can meet efficiently and associate to form active enzymes, and (ii) that the alpha subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yield an active membrane-bound heterologous enzyme. One such heterologous enzyme, alpha A beta Z gamma Z, has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heterologous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.