title: Expression of spinach nitrite reductase in Escherichia coli: site-directed mutagenesis of predicted active site amino acids.
abstract: Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled [Fe4S4]-siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia. An expression system which produced enzymatically active spinach nitrite reductase (NiR) in Escherichia coli was developed in order to study the structure-function relationships of the coupled active site using site-directed mutagenesis. The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a beta-galactosidase fusion containing five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive. After optimizing growth conditions, active NiR represented 0.5-1.0% of the total protein. E. coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis. The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E. coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A278/A386 = 1.9. The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum. The specific activity for the methyl viologen-dependent reduction of nitrite of E. coli-expressed NiR was 100 U/mg and the Km determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that the E. coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D016415:Sequence Alignment; D017421:Sequence Analysis; D018724:Spinacia oleracea / Q000201:enzymology*
chemical_list: D018076:DNA, Complementary; D009572:Nitrite Reductases