title: Substrate promiscuity of N-acetylhexosamine 1-kinases.
abstract: N-Acetylhexosamine 1-kinase (NahK) catalyzes the direct addition of a phosphate from adenosine 5'-triphosphate (ATP) to the anomeric position of N-acetylhexosamine and shows similar activity towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Herein we report the cloning, characterization, and substrate specificity studies of two NahKs from Bifidobacterium infantis ATCC15697 and Bifidobacterium longum ATCC55813, respectively. A new capillary electrophoresis assay method has been developed for enzyme activity assays. Both enzymes have a good expression level in E. coli (180-185 mg/L culture) and can tolerate diverse modifications at C2 of GlcNAc and GalNAc. Various GlcNAc derivatives with C6, both C2 and C6, as well as both C2 and C3 modifications are tolerable substrates for the newly cloned NahKs. Quite interestingly, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C2, C4, and C6 derivatives. These NahKs are excellent catalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA. yhzli@ucdavis.edu
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Li
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Kam
authors.initials: K
authors.lastname: Lau
authors.forename: Li
authors.initials: L
authors.lastname: Cai
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Cao
authors.forename: Vinod Kumar
authors.initials: VK
authors.lastname: Tiwari
authors.forename: Jingyao
authors.initials: J
authors.lastname: Qu
authors.forename: Vireak
authors.initials: V
authors.lastname: Thon
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
mesh_terms: D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D002236:Carbohydrate Conformation; D003001:Cloning, Molecular; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014169:Transformation, Bacterial
chemical_list: D007527:Isoenzymes; D010710:Phosphates; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D005947:Glucose; D000116:Acetylgalactosamine; D008358:Mannose; D000117:Acetylglucosamine; D005690:Galactose