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Noda Institute for Scientiﬁc Research GRANT
2011 Young Investigator Research Grant

Identiﬁcation of the function of a putative sugar kinase from
halophilic archaea
Takaaki SATO
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University
Research aims
According to the phylogenetic tree based on 16S rRNA
sequences, organisms are classiﬁed into three domains, Eucarya, Bacteria and Archaea. Archaea possess unique metabolisms different from those of Eucarya and Bacteria. I
have previously identiﬁed an archaeal pathway involved inAMP metabolism. Two novel enzymes, AMP phosphorylase and ribose-1,5-bisphosphate isomerase (R15Pi), andRubisco, whose physiological function in Archaea has been
unknown, constitute the pathway (Fig. 1)1).
Interestingly, 4 of 17 halophilic archaea whose genome
analyses have been completed harbor only an R15Pi homolog (R15Pih) but not homologs of AMPpase and Rubisco. This raised the possibility that R15Pihs catalyze anunknown reaction distinct from the general R15Pi reaction.
Most of R15Pihs form an operon with sugar kinase and uridine phosphorylase catalyzing reactions similar to those ofthe enzymes in the AMP metabolic pathway (Fig. 1). Thissuggests that these genes function cooperatively in an unknown metabolic pathway. In order to clarify the metabolic
pathway composed of the three genes, I here aimed to identify the function of the sugar kinase.

Kinase activity measurement
Kinase activity was measured by determining theamounts of ADP (NDP) produced from ATP (NTP) in thekinase reaction using pyruvate kinase (PK) and lactate dehydrogenase (LDH). In this assay, the kinase, PK, and LDH
reactions are coupled. As LDH consumes NADH, the decrease in absorbance at 340 nm deriving from the consumption of NADH was determined. As this method detects
ADP (NDP) formation, and not the phosphorylated substrate, various sugars can be tested as substrate.
Results
Preparation of recombinant RbsK proteinsI tried preparing recombinant RbsK proteins fromhalophilic archaea that harbor both sugar kinase andR15Pih, Halobacterium salinarum, Halorubrum lacusprofundi, Haloterrigena turkmenica, and Halopiger xanaduensis (Hs-RbsK, Hl-RbsK, Ht-RbsK, and Hx-RbsK, respec-Methods
Preparation of recombinant RbsK proteins
I carried out overexpression of the rbsK genes from
halophilic archaea whose codons were changed suitable forexpression in the bacterium Escherichia coli. The cells
were then sonicated and the supernatant was subjected to ananion exchange column, Resource Q, and subsequently to agel ﬁltration column, Superdex 200.

Fig. 1.

Fig. 2.

Puriﬁed Hx-RbsK

Archaeal AMP metabolic pathway and predicted metabolism in haloarchaea

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Fig. 3.

Substrate speciﬁcity of Hx-RbsK (A). Phosphate donor speciﬁcity of Hx-RbsK (B)tively).
As a result, Hl-RbsK and Ht-RbsK could not be expressed in E. coli. Although Hs-RbsK protein was detectedin E. coli, the protein formed inclusion body. On the other
hand, RbsK from Halopiger xanaduensis (Hx-RbsK) wassuccessfully produced in E. coli in a soluble form after sonication of the cells. I then carried out the puriﬁcation of theHx-RbsK by anion exchange and gel ﬁltration chromatography (Fig. 2).
Kinase activity measurement of Hx-RbsK
I investigated the substrate speciﬁcity of Hx-RbsK utilizing various sugars, applying monosaccharides, disaccharides, sugar phosphates, amino sugars, nucleosides as substrates. As the phosphate donor speciﬁcity of the protein
was unclear, a mixture of ATP, CTP, UTP, and GTP was utilized as the phosphate donor. As a result, the protein exhibited a signiﬁcant kinase activity toward ribulose 5-phos-phate (Ru5P) (Fig. 3A). In addition, it turned out that theprotein utilized only CTP as phosphate donor (Fig. 3B).
ConclusionIn this study, it is suggested that Hx-RbsK catalyzed
phosphorylation of Ru5P. There is the possibility that product is ribulose 1,5-bisphosphate (RuBP), the substrate forRubisco. It is interesting that halophilic archaea without
Rubisco may produce the substrate for Rubisco, suggestinga novel pathway and/or enzymes. I plan to investigate
whether the Hx-RbsK actually produces RuBP, and if so,
how RuBP is metabolized.
Reference
1)

Sato,T., Atomi, H., and Imanaka, T. (2007) Archaeal type III
RuBisCOs function in a pathway for AMP metabolism. Science 315:1003–1006.