STRUCTURE OF THE NIFQ GENE FROM ENTEROBACTER-AGGLOMERANS 333 AND ITS OVEREXPRESSION IN ESCHERICHIA-COLI

The nifQ gene, involved in early stages of iron-molybdenum cofactor (F eMo-co) biosynthesis, was identified downstream of the nifB and nifF g enes of Enterobacter agglomerans. This gene was cloned and its nucleot ide sequence determined. The amino acid sequence, as deduced from the nucleotide sequence, revealed an accumulation of cysteine amino acid r esidues at the C-terminal end of the protein. The cysteine cluster sho wed the following consensus sequence CYS-X4-CYS-X2-CYS-X5-Cys, which i s a typical characteristic of metal-binding proteins. Further, the nif Q gene was cloned downstream of strong transcriptional (bacteriophage lambdap(L)p(R)) and translational (atpE) signals of the expression vec tor pCYTEXP1 and expressed as an unfused, soluble protein in Escherich ia coli. The molecular mass of 19 kDa, as deduced by SDS-PAGE, is in g ood agreement with the molecular mass deduced from the nucleotide sequ ence. The availability of high-level expression clones should facilita te purification of large quantities of the recombinant NifQ protein an d elucidation of its properties.