title: Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.
abstract: The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent kcatvalue. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides.
  
  
  
  It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent kcatvalues. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity.
authors.affiliation: Laboratory for Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.|International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, Fukuoka, Japan.
authors.forename: Yuki
authors.identifier: http://orcid.org/0000-0002-6342-1924
authors.initials: Y
authors.lastname: Honda
authors.affiliation: Laboratory for Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zang
authors.affiliation: Laboratory for Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shimizu
authors.affiliation: Laboratory for Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Dadashipour
authors.affiliation: State Key Laboratory of Marine Environmental Science, Institute of Marine Microbes and Ecospheres, Xiamen University, Xiamen, People's Republic of China.
authors.forename: Zilian
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Laboratory for Functional Genomics of Extremophiles, Faculty of Agriculture, Kyushu University, Fukuoka, Japan kawarabayasi.yutaka@aist.go.jp.|National Institute of Advanced Industrial Science and Technology (AIST), Bioproduction Research Institute, Ibaraki, Japan.
authors.forename: Yutaka
authors.identifier: http://orcid.org/0000-0001-6410-4050
authors.initials: Y
authors.lastname: Kawarabayasi
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016944:Sulfolobus / Q000235:genetics* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D050505:Mutant Proteins
keywords: Sulfolobus tokodaii; double mutants; site saturation mutagenesis; site-directed mutagenesis; sugar-1-phosphate nucleotidylyltransferase; the ST0452 protein; thermostable enzyme