The C-terminal segment is essential for maintaining the quaternary structure and enzyme activity of the nitric oxide forming nitrite reductase from Achromobacter cycloclastes.
We have constructed and expressed a series of mutated nitrite reductase (NIR) mutants based on the sequence of NIR from Achromobacter cycloclastes. Deleting a pentapeptide, an undecapeptide, or a heptadecapeptide from the C-terminus of NIR resulted in a series of C-terminal deletion mutated proteins designated as NIR-5, NIR-11, and NIR-17, respectively. A C-terminally extended mutated protein, NIR+8, was also produced, which contains an extra octapeptide attached to the C-terminus of the wild-type NIR. An SDS-PAGE system using tris-tricine buffer could retain the native NIR in its trimeric form, thus offering a convenient method to check the quaternary structure of NIR analogs. By using this system it was found that NIR-5 was maintained as trimer and retained 72% of wild-type enzyme activity. However, both NIR-11 and NIR-17 behaved as monomers in the SDS-PAGE and lost all their enzyme activity. Although NIR+8 maintained its trimeric structure it was enzymatically inactive. These results clearly indicate that the C-terminal undecapeptide is essential for maintaining the quaternary structure as well as the full enzymatic activity, as expected from the X-ray crystallography studies.
Chang
W C
WC
Institute of Biochemical Science, National Taiwan University, Taipei, Taiwan.
Chen
J Y
JY
Chang
T
T
Liu
M Y
MY
Payne
W J
WJ
LeGall
J
J
Chang
W C
WC
D000421:Alcaligenes / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D009569:Nitric Oxide / Q000378:metabolism*; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D009569:Nitric Oxide; D009572:Nitrite Reductases