THE NAPEDABC GENE-CLUSTER ENCODING THE PERIPLASMIC NITRATE REDUCTASE SYSTEM OF THIOSPHAERA-PANTOTROPHA

The napEDABC locus coding for the periplasmic nitrate reductase of Thi osphaera pantotropha has been cloned and sequenced. The large and smal l subunits of the enzyme are coded by napA and napB. The sequence of N apA indicates that this protein binds the GMP-conjugated form of the m olybdopterin cofactor. Cysteine-181 is proposed to ligate the molybden um atom. It is inferred that the active site of the periplasmic nitrat e reductase is structurally related to those of the molybdenum-depende nt formate dehydrogenases and bacterial assimilatory nitrate reductase s, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [ 4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histi dine ligation. napC is predicted to code for a membrane-anchored tetra haem c-type cytochrome that shows sequence similarity to the NirT cyto chrome c family. NapC may be the direct electron donor to the NapAB co mplex. napD is predicted to encode a soluble cytoplasmic protein and n apE a monotopic integral membrane protein. napDABC genes can be discer ned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that th is operon encodes a periplasmic nitrate reductase system, while napD a nd napC are identified adjacent to the napAB genes of Alcaligenes eutr ophus H16.