title: Probing the roles of conserved residues in uridyltransferase domain of Escherichia coli K12 GlmU by site-directed mutagenesis.
abstract: N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme that catalyzes both acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthesis pathway. Our previous study demonstrated that the uridyltransferase domain of GlmU (tGlmU) exhibited a flexible substrate specificity, which could be further applied in unnatural sugar nucleotides preparation. However, the structural basis of tolerating variant substrates is still not clear. Herein, we further investigated the roles of several highly conserved amino acid residues involved in substrate binding and recognition by structure- and sequence-guided site-directed mutagenesis. Out of total 16 mutants designed, tGlmU Q76E mutant which had a novel catalytic activity to convert CTP and GlcNAc-1P into unnatural sugar nucleotide CDP-GlcNAc was identified. Furthermore, tGlmU Y103F and N169R mutants were also investigated to have enhanced uridyltransferase activities compared with wide-type tGlmU.
authors.affiliation: National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, People's Republic of China; The State Key Laboratory of Microbial Technology and School of Life Sciences, Shandong University, Jinan, Shandong 250100, People's Republic of China.
authors.forename: Shuaishuai
authors.initials: S
authors.lastname: Wang
authors.affiliation: National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, People's Republic of China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Fu
authors.affiliation: The State Key Laboratory of Microbial Technology and School of Life Sciences, Shandong University, Jinan, Shandong 250100, People's Republic of China; Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA.
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Liu
authors.affiliation: National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, People's Republic of China.
authors.forename: Xian-wei
authors.initials: XW
authors.lastname: Liu
authors.affiliation: Department of Food Science and Engineering, Nanshan University, Yantai, Shandong 265713, People's Republic of China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, People's Republic of China. Electronic address: fangjunqiang@sdu.edu.cn.
authors.forename: Junqiang
authors.initials: J
authors.lastname: Fang
authors.affiliation: National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, People's Republic of China; Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA.
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D017124:Conserved Sequence; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; C520333:GlmU protein, E coli; D009097:Multienzyme Complexes; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase; D000117:Acetylglucosamine
keywords: Site-directed mutagenesis; Substrate specificity; UDP-GlcNAc; Uridyltransferase