[Active sites of N-acetylhexosamine 1-kinase from Bifidobacterium longum].
OBJECTIVE
To study the active sites of N-acetylhexosamine 1-kinase (NahK) from Bifidobacterium longum JCM12 17.


METHODS
We obtained expression strains of 10 single-mutants at 4 sites of NahK by site-directed mutagenesis, and expressed and purified both wild-type (WT) and mutant enzymes. Then, their optimum pH and optimum concentration of Mg²⁺ were determined by DNS assay and NADH-coupled microplate photometric assay, and their kinetic parameters were measured.


RESULTS
Four mutants (D208A, D208N, D208E and I24A) lost most part of the catalytic activity. The optimum pH of mutants H31A, H31V, F247A and I24V switched from pH 7.5 (for WT) to pH 7.0, and the optimum concentration of Mg²⁺ of mutants H31A and F247A increased to 10 mmol/L from 5 mmol/L (for WT). The kinetic parameters of WT and mutants indicate that mutant F247Y had higher enzymatic activity toward GlcNAc, GalNAc and ATP than WT.


CONCLUSIONS
The key amino acids that affect the catalytic activity of NahK were determined by site-directed mutagenesis, and together with the mutant that has higher catalytic efficiency, this has laid a foundation for further modification and evolution of NahK.
Wanyi
W
Guan
Jing
J
Bai
Tianhui
T
Zhou
Baohua
B
Zhao
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001644:Bifidobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004795:Enzyme Stability; D006632:Histamine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C021606:N-acetylhistamine; D006632:Histamine; D010770:Phosphotransferases