Increasing in archaeal GlcNAc-1-P uridyltransferase activity by targeted mutagenesis while retaining its extreme thermostability.
UDP-GlcNAc, an activated and essential form of GlcNAc which is an important component in the polysaccharide structure of most organisms, is synthesized from GlcNAc-1-P and UTP by GlcNAc-1-P UTase. We previously reported the identification of the extremely thermostable ST0452 protein, which has dual sugar-1-P NTase activities (Glc-1-P TTase and GlcNAc-1-P UTase activities) from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Detailed analyses of the protein indicated that the activity is slightly lower than that of bacteria. For industrial applications, activity needs to be increased without decreasing thermostability. Therefore, to enhance this activity, we introduced mutations into the amino acid residues located within the predicted reaction centre by targeted mutagenesis. All 12 mutant ST0452 proteins showed no decrease in thermostability. Among them, six mutant proteins were found to have increased GlcNAc-1-P UTase activity under optimal reaction conditions with sufficient substrates or an appropriate metal ion. Our results indicate that targeted mutagenesis is a powerful technique for in vitro production of a thermostable enzyme with enhanced activity. The results of this study also indicate that the space for the metal ion is important for selecting the type of metal ion and also affects the rate of the reaction.
Zhang
Zilian
Z
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki, Japan.
Akutsu
Jun-Ichi
J
Tsujimura
Masanari
M
Kawarabayasi
Yutaka
Y
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D016944:Sulfolobus / Q000201:enzymology*
D019843:Archaeal Proteins; D008670:Metals; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase