The cloning, expression and protein engineering studies on nitrite reductase of Achromobacter cycloclastes

• Main Author: 張為超
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/53695234096594953857

博士 === 國立臺灣大學 === 機械工程學研究所 === 87 === The nitrite reductase (NIR) gene has been cloned and characterized from Achromobacter cycloclastes. NIR gene encodes a protein of 378 amino acid residues including a putative signal peptide of 38 residues. The DNA-derived amino acid sequence of NIR is in complete agreement with that from Edman degradation. NIR gene contains its own FNR box in the 5’ upstream region and a TA-rich region which could be the transcription start site. NIR is expressed in E. coli. The recombinant NIR could be recognized by rabbit antisera and demonstrated full enzyme activity.The tris-tricine SDS-PAGE has been used to analyze the quaternary structure of NIR protein. NIR migrates as trimer and monomer in this system while it behaves as dimer in HPLC gel-filtration or tris-glycine SDS-PAGE. The dissociation of trimer to monomer is effected by heating, suggesting that the molecular interaction between monomer is heat-labile. We have constructed and expressed a series of mutants with deletion of a pentapeptide, an undecapeptide, or a heptadecapeptide from the C-terminus of NIR designated as ENIRC-5, ENIRC-11 and ENIRC-17, respectively. A C-terminally extended mutated protein, ENIRC+8, is also produced, which contains an extra octapeptide attached to the C-terminus of the wide-type NIR. ENIRC-5 maintains as trimer and retained 72% of the wile-type NIR. However, ENIRC-11 and ENIRC-17 behave as monomers in the tris-tricine SDS-PAGE and lose all their enzyme activity. Although ENIRC+8 maintains its trimer stucture it is enzymatially inactive. These results clearly indicate that the C-terminal undecapeptide is essential for maintaining the quaternary structure as well as the full enzymatic activity. We propose a “solvent channel” model for intramolecular electron transfer between type I and type II copper sites. Water molecule bound in NIR protein may be involved in electron transfer. A NIR mutant, I257E, loses its enzyme activity, indicating that the I257 is important in the electron transfer pathway.