title: Identification and characterization of an archaeal kojibiose catabolic pathway in the hyperthermophilic Pyrococcus sp. strain ST04.
abstract: A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and β-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and β-glucose-1-phosphate (β-G1P). The Km values for kojibiose and phosphate were determined to be 2.53 ± 0.21 mM and 1.34 ± 0.04 mM, respectively. PsPGM then converts β-G1P into G6P in the presence of 6 mM MgCl2. Conversion activity from β-G1P to G6P was 46.81 ± 3.66 U/mg, and reverse conversion activity from G6P to β-G1P was 3.51 ± 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90°C for PsKP and 95°C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea.
authors.affiliation: Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin, Republic of Korea.
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Jung
authors.forename: Dong-Ho
authors.initials: DH
authors.lastname: Seo
authors.forename: James F
authors.initials: JF
authors.lastname: Holden
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal / Q000502:physiology*; D008969:Molecular Sequence Data; D019711:Pyrococcus / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D004187:Disaccharides; C008237:kojibiose